Physiol Rev
84: 1341–1379, 2004; 10.1152/physrev.00046.2003.
Role of Caveolae and Caveolins in Health and Disease
ALEX W. COHEN, ROBERT HNASKO, WILLIAM SCHUBERT, AND MICHAEL P. LISANTI
Department of Molecular Pharmacology and the Albert Einstein Cancer Center,
Albert Einstein College of Medicine, Bronx, New York
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I. Perspective
II. Caveolae
A. Discovery and morphology
B. Tissue distribution
C. Biochemical properties
III. The Caveolins
A. Initial discovery
B. The caveolin gene family
C. Caveolin protein characterization
IV. Functional Roles of Caveolae/Caveolins
A. Vesicular transport
B. Cholesterol homeostasis
C. Signal transduction
D. Proteomics
V. Caveolins and Human Disease: Knockout Mouse Models
A. Caveolin-1
B. Caveolin-2
C. Caveolin-3
D. Caveolin-1/3
VI. Conclusions and Future Directions
Cohen, Alex W., Robert Hnasko, William Schubert, and Michael P. Lisanti. Role of Caveolae and Caveolins
in Health and Disease. Physiol Rev 84: 1341–1379, 2004; 10.1152/physrev.00046.2003.—Although they were
discovered more than 50 years ago, caveolae have remained enigmatic plasmalemmal organelles. With their
characteristic “flasklike” shape and virtually ubiquitous tissue distribution, these interesting structures have
been implicated in a wide range of cellular functions. Similar to clathrin-coated pits, caveolae function as
macromolecular vesicular transporters, while their unique lipid composition classifies them as plasma membrane lipid rafts, structures enriched in a variety of signaling molecules. The caveolin proteins (caveolin-1, -2,
and -3) serve as the structural components of caveolae, while also functioning as scaffolding proteins, capable
of recruiting numerous signaling molecules to caveolae, as well as regulating their activity. That so many
signaling molecules and signaling cascades are regulated by an interaction with the caveolins provides a
paradigm by which numerous disease processes may be affected by ablation or mutation of these proteins.
Indeed, studies in caveolin-deficient mice have implicated these structures in a host of human diseases,
including diabetes, cancer, cardiovascular disease, atherosclerosis, pulmonary fibrosis, and a variety of degenerative muscular dystrophies. In this review, we provide an in depth summary regarding the mechanisms by
which caveolae and caveolins participate in human disease processes.
I. PERSPECTIVE
Since their initial discovery in the early 1950s, caveolae,
with their unique flask-shaped morphology, have provoked a
multitude of conjecture as to their functional significance.
Although detailed morphological examinations have provided some insight into their function, it was not until the
discovery of the caveolin coat proteins (caveolin-1, -2, -3)
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that the true nature and importance of these organelles was
realized. Since that time, the exponential growth of the
caveolae field has provided numerous clues as to the physiological functions of both the caveolae organelle and the
caveolin coat proteins. The recent generation of mice deficient in the various caveolin genes has provided new in vivo
animal models with which to elucidate the exact physiological significance of a given caveolin gene product.
0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society
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sion and the development/progression of certain human
diseases.
II. CAVEOLAE
A. Discovery and Morphology
Indeed, evidence is accumulating that implicates the
caveolin gene family in the pathogenesis of numerous
human diseases, including cancer, muscular dystrophy,
and type II diabetes. This review describes the 1) biochemical properties of the caveolin protein products, 2)
the physiological consequences of caveolin gene ablation
in mice, and 3) the relationship between caveolin expres-
FIG. 2. Ultrastructural analysis of
purified caveolae. A, top: caveolae purified from mouse lung appear as ⬃100-nm
vesicles (arrowheads) and as curved or
U-shaped structures (arrows) by transmission electron microscopy. Bar, 0.1
␮m. Bottom: these domains were fixed
with paraformaldehyde and immunolabeled with anti-caveolin-1 IgG and 10-nm
gold-conjugated secondary antibodies.
Note that these domains contain an
abundance of caveolin-1. To preserve immunoreactivity, it was necessary to exclude fixation with glutaraldehyde and
OsO4. [Modified from Lisanti et al. (131).]
B, top: isolated mouse lung caveolae as
seen by low-angle platinum shadowing.
These domains are ⬃50 –100 nm in diameter and possess a distinctive granular
appearance. Bar, 0.1 ␮m. Bottom: staining with anti-caveolin-1 IgG and 10-nm
gold-conjugated secondary antibodies reveals caveolin-1 in these domains (magnification is twice that of the top panel).
[Modified from Lisanti et al. (130).]
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FIG. 1. Stylized view of the cell depicting morphological variants of
caveolae and select subcellular compartments. These include 1) fenstra,
2) a transcellular channel, 3) traditional caveolae, 4) plasmalemmal
vesicles (fully invaginated, static caveolae), 5) a vesiculo-vacuolar organelle (a grapelike cluster of interconnected caveolae and vacuoles), 6)
cavicles (mobile, internalized caveolae not associated with the plasma
membrane), and 7) a caveosome (a slow moving, irregularly shaped,
cytoplasmic organelle). Golgi, dark blue; endoplasmic reticulum, yellow.
Caveolae are morphologically identifiable plasma
membrane invaginations that are distinct from the larger
electron-dense clathrin-coated pits. Originally identified
in the 1950s by electron microscopists investigating cellular ultrastructure, caveolae appear as “smooth” uncoated pits or vesicles at the plasma membrane, typically
observed using conventional resin-embedded techniques.
In general, caveolae are 50- to 100-nm flask-shaped
invaginations of the plasma membrane that can be singular or found in detached grapelike clusters (rosette formation) and long tubular structures thought to evolve
from the fusion of individual caveolae (Fig. 1). Isolated
caveolae appear as vesicular or curved U-shaped structures possessing a distinctive granular appearance using
techniques such as transmission electron microscopy and
low-angle platinum shadowing (Fig. 2). While the overall
function of the prototypical caveolae organelle has been
an area of intense exploration, little attention has focused
COHEN, HNASKO, SCHUBERT, AND LISANTI
on the specialized function, if any, of the various morphological subsets of caveolae. Consequently, the functional
significance of these caveolae-related organelles remains
unknown.
B. Tissue Distribution
C. Biochemical Properties
Unlike earlier views of the plasma membrane as a
“fluid mosaic” (235), where integral membrane proteins
were thought to float and diffuse freely through a sea of
homogeneous lipids, a more contemporary view of the
plasma membrane is that proteins are much more heterogeneously distributed and can be found clustered within
specialized microdomains, termed lipid rafts. These lipid
rafts are thought to form via the aggregation of glycosphingolipids and sphingomyelin in the Golgi apparatus
(held together by transient and weak molecular interactions) and are then delivered to the plasma membrane as
concentrated units (263, 264). These lipid rafts are also
enriched in cholesterol and several resident proteins, including glycophosphatidylinositol (GPI)-linked proteins.
Relative to the plasma membrane proper, which contains
an abundance of cis-unsaturated phospholipids, the
sphingolipids in lipid rafts contain primarily saturated
fatty acyl chains, allowing tighter molecular packing that
results in a higher melting temperature (Tm ⬃41°C vs. Tm
⬍0°C for phospholipids) (223). The high cholesterol and
sphingolipid content of lipid rafts imparts a resistance to
extraction in nonionic detergents such as Triton X-100 at
4°C and a light buoyant-density in sucrose gradients,
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properties instrumental for their purification and biochemical characterization. It should be mentioned that
the exact nature and defining characteristics of lipid rafts,
as well as the techniques involved in isolating them, are
now quite well-developed (for recent reviews of this subject, see Refs. 90, 165, 187).
Caveolae represent a morphologically identifiable
subset of lipid rafts. They contain the coat protein caveolin, which is essential for the invagination of the plasma
membrane through a largely unknown process, giving
them their characteristic flasklike appearance. While the
overall biochemical composition of lipid rafts and caveolae is thought to overlap, these microdomains are not
completely equivalent. In addition to the caveolins, several proteins have been shown to preferentially localize to
either caveolae or lipid rafts, respectively (134).
III. THE CAVEOLINS
A. Initial Discovery
In the late 1980s, investigators attempting to identify
possible targets for phosphorylation-mediated cellular
transformation used a then recently produced antiphosphotyrosine antibody (PY-20) to affinity purify several
phosphotyrosine-containing proteins from Rous sarcomatransformed chicken embryonic fibroblast (77). One of
the four major proteins identified was found to be resistant to extraction with nonionic detergents and demonstrated a staining pattern that was simultaneously punctate, concentrated at cell margins, and focused in parallel
arrays along actin stress fibers. The localization of this
22-kDa protein was also shown to change after cellular
transformation; indeed, the tyrosine phosphorylation of
this protein was dependent on transformation with v-Src,
suggesting a role for this protein in oncogenesis (76). This
22-kDa protein was localized to caveolae by immunoelectron microscopy, and ultrastructural analysis revealed
that numerous striated filaments coat the cytoplasmic
surface of these organelles. In searching for the protein
components of these striations, it was found that antibodies generated against the 22-kDa protein stained the striated filaments, and thus the 22-kDa protein was termed
caveolin (now called caveolin-1). Furthermore, the caveolin filaments were found to be resistant to extraction with
high salt, and treatment of cells with cholesterol-chelating
agents resulted in the flattening of membrane caveolae,
with disassociation of the striated coat (203). Later, using
oligonucleotides predicted from its peptide sequence, the
caveolin gene was cloned. Overexpression of the caveolin-1 cDNA in fibroblasts resulted in caveolin-1 localization to caveolae, confirming that it is the major resident
protein of this organelle (75).
Working on a completely different aspect of cell biology, Kurzchalia et al. (115) identified a set of CHAPS-
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Caveolae were simultaneously identified in capillary
endothelial cells and epithelial cells from the mouse gall
bladder (181, 285). Since then, caveolae have been identified in a wide variety of tissues and cell types. While no
all-encompassing ultrastructural study has been undertaken, a review of published literature reveals that caveolae are present to some degree in most differentiated cell
types. Particularly, caveolae have been well-described in
adipocytes, where they are extremely abundant, endothelial cells, type I pneumocytes of the lung, and striated and
smooth muscle cells. Because of their relative abundance
in endothelial cells and type I pneumocytes, the two
major constituents of lung alveoli, the lung stands out as
one of the most abundant sources of identifiable caveolae,
second only to adipocytes. Ultrastructural analysis of adipocytes has shown that as much as 20% of the total
plasma membrane is occupied by caveolae (53). Thus
caveolae can greatly increase the surface area of numerous cell types, an observation that lends credence to the
original speculation that caveolae are involved in macromolecular transport and mechanotransduction events.
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insoluble proteins, one of which they termed VIP21 (vesicular integral-membrane protein of 21 kDa). The cDNA
for this protein was cloned and transiently expressed in
mammalian cells. By immunofluorescence microscopy,
VIP21 was found to localize to the Golgi apparatus, the
plasma membrane, and membrane-bound vesicles (115).
In later work, Glenney (75) showed that the sequences for
VIP21 and caveolin were identical and thus these two
research groups had independently identified the same
protein by distinct biochemical methods.
B. The Caveolin Gene Family
FIG. 3. Schematic depiction of the caveolin gene family. Colorcoded boxes indicate the exon arrangement of each caveolin family
member. The numbers within each box refer to the number of nucleotides in each exon.
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C. Caveolin Protein Characterization
As the prototypical caveolin family member, caveolin-1
has been the primary focus of many biochemical studies and
provides the basis of our knowledge regarding the interaction of these proteins with their subcellular environment.
Therefore, the attributes of caveolin-1 are considered here;
however, it can be inferred that the majority of these characteristic are valid for the other caveolin family members.
Caveolin-1 is a 22-kDa protein of 178 amino acids with
adipocytes, endothelial cells, fibroblasts, and type I pneumocytes having the highest levels of expression. It is now
known that caveolin-1 expression is sufficient and necessary
to drive the formation of morphologically identifiable caveolae (128, 197). In addition, caveolin-1 expression is necessary
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To date, three members of the caveolin (CAV) gene
family have been identified (Fig. 3). Caveolin (later
termed caveolin-1; Ref. 216) was the first gene discovered
and is composed of three exons that are highly conserved
in sequence and structure across species. Caveolin-2 was
discovered when micro-sequencing of purified adipocyte
caveolae membrane domains revealed a strikingly similar
peptide sequence to that of caveolin-1, differing in several
key conserved caveolin-1 residues. Cross-referencing this
new peptide sequence with known expressed sequence
tags (EST) revealed an EST encoding a caveolin-1-like
protein of 162 amino acids, subsequently termed caveolin-2 (216). Caveolin-3 was cloned from a cDNA library
using a caveolin-1-related sequence found immediately
downstream of the rat oxytocin receptor (256).
In addition to these two new members of the caveolin
gene family, it was also found that both caveolin-1 and -2
have multiple isoforms. Caveolin-1 has two isoforms,
termed ␣ and ␤, with the ␣-isoform consisting of residues
1–178 and the ␤-isoform containing residues 32–178, resulting in a protein ⬃3 kDa smaller in size (128, 217). The
␤-isoform is thought to derive from an alternate translation initiation site occurring at a methionine in position
32. When expressed in Sf21 insect cells, which lack endogenous caveolae, both isoforms of caveolin-1 (Cav-1␣
and Cav-1␤) are capable of driving caveolae formation
(128). In addition, analysis of the size distribution of
caveolae in this setting revealed a mean of 80 ⫾ 14.8 nm
with 95% of caveolae being between 50 –105 nm (Fig. 4).
Furthermore, whole-mount electron microscopic evaluation of isolated recombinant caveolae revealed that both
the ␣- and ␤-isoforms of caveolin-1 generate morphologically similar cup-shaped caveolae (Fig. 4). While the exact functional significance of these distinct isoforms remains unclear, studies have suggested that the caveolin-1␣ isoform is localized predominantly to deeply
invaginated caveolae and can more efficiently drive the
formation of caveolae than the ␤-isoform (65, 217). Caveolin-2 has three identified isoforms, the full-length caveolin2␣, and two truncated variants, termed caveolin-2␤ and
-2␥. The ␤-isoform is thought to be an alternate splice
variant, with a distinct subcellular distribution from fulllength caveolin-2␣ (113). Little is known about the functional significance, if any, of the Cav-2␤ and -2␥ isoforms.
The various caveolin genes and proteins share significant homology. Human caveolin-2 is ⬃38% identical and
⬃58% similar to human caveolin-1, while caveolin-3 is
⬃65% identical and ⬃85% similar to caveolin-1. In addition, the caveolin proteins are highly conserved throughout evolution (Fig. 5). Moreover, a short stretch of eight
amino acids has been identified (FEDVIAEP) that constitutes the “caveolin signature sequence,” a motif that is
identical between all three caveolin proteins.
The Cav-1 and Cav-2 genes have a relatively ubiquitous distribution pattern, being coexpressed in most differentiated cells types, with the notable exception of skeletal muscle fibers and cardiac myocytes (215). Initial
examination of murine tissue by Northern blot showed
that Cav-3 expression is limited to skeletal muscle, the
diaphragm, and the heart (256, 272). Additionally, the
expression of Cav-3 has been shown to be dependent on
the degree of muscle cell differentiation, as Cav-3 mRNA
is only detectable in differentiated, nonproliferating
C2C12 myoblasts versus the proliferating precursor myoblast cell type in which both Cav-1 and Cav-2 are highly
expressed (256, 272).
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for the stable expression and membrane localization of
caveolin-2. Indeed, caveolin-2 alone is insufficient to induce
caveolae biogenesis (199). The Cav-3 protein is muscle specific and, like Cav-1, is sufficient to drive the formation of
caveolae (67).
1. Membrane topology and attachment
Initial studies into the subcellular localization of
caveolin-1 revealed that it is an integral membrane pro-
tein, as it was found to be resistant to extraction with
sodium carbonate and high salt concentrations (115, 203,
212). In addition, several lines of evidence have contributed to the generally accepted view that both the NH2 and
COOH termini of caveolin-1 face the cytoplasm, with an
intervening hydrophobic domain inserted into the membrane (Fig. 6). Preliminary findings indicative of this unusual topology were suggested by Dupree et al. (42) after
antibodies directed against either the NH2- or COOHFIG. 5. Protein sequence alignment
of caveolin-1 across several species. Sequences are presented in decreasing order of similarity to human caveolin-1.
Identical residues are not shown in the
bovine, mouse, chick, and fugu sequences, while changed amino acids are
indicated. A ⫺ indicates a space inserted
to maximize homology among species.
Colors indicate proline (purple, P); aspartic or glutamic acid (red, D and E); arginine or lysine (blue, R and K); phenylalanine, isoleucine, methionine, valine,
tryptophan, or tyrosine (green, F, I, L, M,
V, W, and Y); and all other amino acids
alanine, cysteine, glycine, histidine, asparagine, serine, and threonine (black, A,
C, G, H, N, Q, S, and T). The single red
box outlines the oligomerization domain,
the double red box outlines the scaffolding domain, and the blue box outlines the
membrane spanning domain. The SwissProt accession numbers are as follows:
human (P49817), bovine (P79132), mouse
(P49817), chick (P35431), and fugu
(Q9YGM8).
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FIG. 4. Caveolin-1 expression generates
caveolae organelles. Expression of mammalian caveolin-1 isoforms (␣ and ␤) in Sf21
insect cells. A: Western blot analysis of baculovirus-based recombinant expression of
caveolin-1␣ and caveolin-1␤ in insect cells reveals that the ␣-isoform is ⬃3 kDa larger that
the ␤-isoform. B: transmission electron microscopy of Sf21 insect cells expressing
caveolin-1␣. Note the accumulation of caveolae-sized vesicles, similar in size and shape to
those seen in mammalian cells (⬃50 –100 nm
in diameter). Comparable results were obtained with caveolin-1␤ expressing cells. Bar,
100 nm. C: quantitation of the size distribution
of reconstituted caveolae in Sf21 insect cells.
Measurements of over 100 vesicular profiles
revealed a mean of 80.3 ⫾ 14.8 (SD) nm in
diameter. Ninety-five percent of the caveolaesized vesicles were 50 –105 nm in diameter (2
SD). Identical results were obtained with the
expression of caveolin-1␣ and -1␤. D: purified
recombinant caveolae isolated from Sf21 insect cells have a similar morphological appearance to those isolated from mammalian
cells. Note that these caveolae appear as cupshaped vesicular (⬃50 –100 nm) structures
(arrows). Identical results were obtained with
caveolin-1␣ and -1␤. Bar, 100 nm. [Modified
from Li et al. (128).]
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
terminal domains of caveolin-1 were unable to label the
protein in the absence of cellular permeablization. Additional studies demonstrated that the COOH terminus of
caveolin-1 is palmitoylated and the NH2 terminus is tyrosine phosphorylated, two posttranslational modifications that require both ends of the protein to remain
cytoplasmic (39, 127). Furthermore, surface biotinylation
studies failed to label caveolin-1, further supporting the
idea that no portion of the caveolin-1 protein is extracellular (211). The membrane insertion of caveolin-1 occurs
via the classical endoplasmic reticulum (ER) machinery,
resulting in an unusually short transmembrane domain of
32 hydrophobic amino acids (residues 102–134) thought
to form a unique hair-pin loop configuration that prevents
caveolin-1 from completely spanning the plasma membrane in a traditional double-pass fashion (Fig. 6) (159).
Mutational analysis and domain mapping experiments have demonstrated the importance of two other
regions of the caveolin-1 protein for membrane attachment. Interestingly, the postulated membrane-spanning
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region (residues 102–134) was not found to be essential
for this function. Rather, two adjacent regions that flank
this central hydrophobic domain, residues 82–101 and
residues 135–150, were found to bind to membranes with
high affinity (2, 145, 220, 221). These regions are now
referred to as the NH2-terminal membrane attachment
domain (N-MAD) and COOH-terminal membrane attachment domain (C-MAD), respectively. The C-MAD contains
a Golgi-targeting sequence dissimilar from other known
Golgi-associated sequences and was found to localize an
otherwise cytoplasmic green fluorescent protein (GFP) to
the cis-Golgi when expressed as a fusion protein (145,
220). When GFP was fused to the C-MAD of caveolin-1,
the resultant protein was resistance to extraction in both
Triton X-100 at 4°C and alkaline carbonate buffer, two
properties consistent with an integral membrane protein
(220). Several critical experiments reveal the importance
of caveolin-1 residues 82–101 (N-MAD) in membrane attachment (221). Deletion mutagenesis showed that a
caveolin-1 mutant containing only residues 1–101 re-
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FIG. 6. Caveolin-1 membrane topology and protein
domains. In this view, caveolin-1 is depicted as a homodimer for simplicity. Mutational analysis has shown
that the C-MAD (blue; residues 135–150) and N-MAD
(yellow; residues 82–101) are important for membrane
attachment, while the transmembrane domain (red; residues 102–134) is thought to insert into the membrane.
Oligomerization is mediated by residues 61–101 (hashed
pink). The scaffolding domain (yellow; residues 82–101)
recognizes a hydrophobic caveolin-binding motif
present within many signaling molecules. Caveolin-1 is
also palmitoylated on three conserved cysteine residues
(green; 133, 143, and 156). Note that caveolin-1 is not a
conventional transmembrane protein. It is thought to
have a unique hairpin topology, with no exposure to the
extracellular environment.
COHEN, HNASKO, SCHUBERT, AND LISANTI
mained associated with membranes, while another mutant composed of residues 1– 81 localized to the cytoplasmic compartment (221). Like the C-MAD, the N-MAD-GFP
fusion protein was also targeted to membranes. However,
while the C-MAD-GFP fusion protein showed a predominant Golgi-like distribution, the N-MAD-GFP fusion protein localized to plasma membrane caveolae (145, 220).
Further mutational analysis of the 20-amino acid N-MAD
domain identified a short membrane attachment sequence
(KYWFYR) that was sufficient to confer membrane localization to a GFP fusion protein (280). Although the KYWFYR motif targets GFP to the membrane, the entire 20amino acid N-MAD is necessary for caveolar targeting.
Caveolin-1 isoforms form high-molecular-mass oligomers of ⬃400 kDa, as demonstrated using velocity
gradient centrifugation (159, 211). Interestingly, deletion
mutagenesis studies showed that caveolin-1 contains an
oligomerization domain mapping to residues 61–101,
which mediates the homo-oligomerization of 14 –16 individual caveolin-1 molecules (Fig. 6) (211). These caveolin-1 oligomers are thought to undergo a second stage of
oligomerization during transport from the trans-Golgi to
plasma membrane caveolae, whereby several oligomers
self-associate via COOH-terminal interactions, forming a
large network of caveolin (243). Sargiacomo et al. (211)
examined the ultrastructure of purified caveolin-1 homooligomers derived from rat lung tissue and found that
these individual homo-oligomers appear as globular particles of 4 – 6 nm in the presence of the nonionic detergent
octyl glucoside (Fig. 7). After removal of this detergent,
caveolin-1 homo-oligomers associated into larger, nonlinear polymers of ⬃25 nm in an apparent side-by-side fash-
FIG. 7. Low-angle platinum shadowing of purified
caveolin-1 homo-oligomers. Caveolin-1 was purified by
velocity gradient centrifugation from isolated murine
lung caveolae. A: caveolin-1 homo-oligomers appear as
4- to 6-nm individual globular particles in the presence
of the nonionic detergent octyl glucoside. B: after removal of octyl glucoside, individual caveolin homooligomers self-associate into ⬃25-nm nonlinear structures, reminiscent of intact caveolae. The boxed area is
shown at higher magnification to illustrate the side-byside packing of caveolin oligomers. Bar, 0.05 ␮m.
[Adapted from Sargiacomo et al. (211).]
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2. Oligomerization domain
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3. The caveolin scaffolding domain
The caveolin-1 scaffolding domain (CSD) was originally defined by a series of experiments, including deletion mutagenensis of Cav-1-GST fusion proteins, which
identified a region within caveolin-1 (residues 82–101)
capable of mediating protein-protein interactions (33–35,
104, 124, 243). Using a GST-fusion protein containing the
caveolin-1 scaffolding domain as a receptor to select peptide ligands from a bacteriophage display library, two
related but distinct caveolin binding motifs (CBM) were
identified in most proteins shown to interact with caveolin-1 (CBM; ⌽XXXX⌽XX⌽ and ⌽X⌽XXXX⌽, where ⌽
represents an aromatic amino acid) (33). The scaffolding
domain has since been shown to serve a dual role, acting
both as an anchor holding various proteins within caveolae as well as a regulatory element capable of either
inhibiting or enhancing a given protein’s signaling activity.
IV. FUNCTIONAL ROLES
OF CAVEOLAE/CAVEOLINS
A. Vesicular Transport
Based purely on their ultrastructural appearance as
plasma membrane invaginations, caveolae were originally
thought to function as macromolecular transport vesicles.
Initially, their proposed function was limited to the process of pinocytosis or “cellular drinking.” However, with
the advent of new tools to investigate their function, their
role as vesicular transporters has expanded to include
transcytosis and endocytosis.
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1. Transcytosis
The term transcytosis was first used by Simionescu
et al. (233) to describe the movement of macromolecules
from the luminal side of capillary endothelial cells to the
interstitial space via membrane-bound vesicles. This early
research involved ultrastructural quantification of probes,
such as gold-labeled albumin and peroxidase-labeled
small heme derivatives, in what appeared to be transendothelial channels formed by the coalescence of caveolae
(74, 233). While these reports demonstrate an accumulation of labeled probes in both abluminal caveolae and the
interstitial space, indicative of transcytosis, the possibility
remained that these tracers were subject to paracellular
transport (between cells). Furthermore, their presence in
abluminal caveolae could also have been attributed to
retrograde filling, i.e., paracellular transport followed by
entry into caveolae.
It was not until two major advancements in the field,
1) the introduction of novel caveolae-specific tracer molecules and 2) the development of caveolin knockout
mice, that the role of caveolae in transcytosis could be
resolved. Recently, Schnitzer and co-workers (151) used a
novel approach that employed antibodies generated
against antigens present only on the luminal surface of
caveolae. Using these antibodies, this group demonstrated that caveolae are capable of macromolecular
transcytosis, as they found an ⬃50-fold increase in the
interstitial accumulation of caveolae-specific antibodies,
compared with control antibodies (151). Because their
specific probe accumulates in tissue, while a control
probe of similar structure does not, these authors conclude that nonspecific pathways, such as paracellular
transport, cannot account for the interstitial accumulation.
Measurements of transcytosis in caveolin-1 knockout
mice, which lack detectable caveolae in cell types such as
endothelial and epithelial cells, show no interstitial accumulation of gold-labeled albumin in knockout, but significant accumulation in wild-type mice (224, 225). Similarly,
when incubated with radio-iodinated albumin, isolated
aortic rings from caveolin knockout mice showed no
uptake of this tracer, while aortic rings from wild-type
animals showed temperature- and time-dependent albumin uptake (225). In contrast to these findings however,
Drab et al. (40) found no change in the apparent transcytosis of albumin into the cerebrospinal fluid (CSF) of their
independently generated Cav-1 null mice. Here, these authors assessed albumin transcytosis across endothelial
cells by measuring the concentration of albumin in the
CSF of wild-type and Cav-1 null mice using SDS-PAGE.
These authors conclude that either 1) caveolae are not
involved in endothelial cell transcytosis or 2) an unidentified compensatory mechanism exists. To further clarify
this issue, Schubert et al. (225) examined the microvas-
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ion, further supporting the notion that this may be the
mechanism by which individual homo-oligomers associate into larger multimeric caveolar complexes in vivo.
In contrast, caveolin-2 is incapable of forming highmolecular-mass homo-oligomeric complexes (216), a
property that may contribute to its inability to form caveolae by itself. However, caveolin-2 does form hetero-oligomeric complexes with caveolin-1 in the ER, an association
that serves to both stabilize caveolin-2 against proteasomal degradation and allow its transport from the Golgi to
the plasma membrane (160, 185, 198, 214).
Caveolin-3 is known to form high-molecular-mass
homo-oligomeric complexes in situ, in a fashion similar to
that of caveolin-1 (239, 256). In addition, while it has long
been thought that caveolin-3 does not bind to caveolin-2,
recent findings in isolated neonatal cardiac myocytes and
smooth muscle cells suggest otherwise (206, 278). In
these systems, it was found that caveolin-2 coimmunoprecipitates with caveolin-3 and cofractionates with caveolin-3 in sucrose density gradient centrifugation (206, 278).
1349
COHEN, HNASKO, SCHUBERT, AND LISANTI
cular permeability of wild-type and Cav-1 null mice, finding that indeed Cav-1 null mice demonstrate significant
increases in the extravascular deposition of radioiodinated albumin. Additional experiments led us to conclude
that defective transcytosis of albumin is compensated for
by increased paracellular transport between endothelial
cells, a process mediated by increases in plasma nitric
oxide levels. Thus with these studies it now seems clear
that caveolae do indeed mediate transcytosis (i.e., transcellular transport) of specific macromolecules in endothelial cells.
2. Endocytosis
Physiol Rev • VOL
1. Pathogens linked to caveolar internalization
and/or signaling
TABLE
Pathogens
Viruses
Simian virus 40
Respiratory syncytial virus
Japanese encephalitis virus
Human immunodeficiency virus
Echovirus 7
Enterovirus 70
Bacteria
Campylobacter jejuni
Escherichia coli K1
FimH-expressing E. coli
Dr/afa E. coli family members
Chlamydia trachomatis
Mycobacterium bovis
Mycobacterium kansasii
Fungi
Pneumocystis carinii
Parasites
Toxoplasma gondii
Plasmodium falciparum
Toxins
Vibrio cholerae toxin (cholera toxin)
Clostridium septicum (␣-toxin)
Enterobacterial lipopolysaccharide
Aeromonas hydrophila toxin (Aerolysin)
Helicobacter pylori toxin (VacA toxin)
Prion
Scrapie prion protein (PrPsc)
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Reference Nos.
169
273
129
16, 166
271
108
13, 281
250
3
229
228
73
193
167, 230
161
118
177
80
270
1
202
267, 268
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While clathrin-mediated endocytosis stands as the
prototypical pathway for the internalization of many extracellular substances, alternative mechanisms also exist,
including those mediated by caveolae. Caveolar transport
may overlap with those events mediated by coated pits,
but caveolae may serve selective transport functions as
well.
This alternative caveolar pathway was first recognized by the observation that cholera toxin was preferentially bound and internalized via caveolae (177). Additional observations suggesting a role for caveolae in the
process of endocytosis include 1) the identification of
several molecules involved in vesicle docking and fusion
(i.e., SNARE proteins) shared by clathrin-coated pits
(CCPs) and 2) the detection of the small GTPase dynamin, which is known to be involved in the internalization of CCPs, in the necks of caveolae following specific
stimuli (91, 173, 222). Taken together, these data have
helped solidify the idea that caveolae are indeed functional endocytic vesicles. Dynamin seems to play a homologous role in the internalization of both CCPs and
caveolae, as overexpression of a dominant negative dynamin mutant inhibits endocytosis by both pathways
(173). Although the exact process mediating caveolar endocytosis remains unknown, several recent findings suggest that the activation of tyrosine kinase-dependent signaling is an important step (190). Indeed, treating cells
with phosphatase inhibitors increases endocytosis via
caveolae. Similarly, ligands that are known to be internalized via caveolae, including several pathogenic agents,
activate tyrosine kinase signaling pathways upon binding
to their cellular receptors (21, 170, 171, 188, 192).
Perhaps the most well-documented infectious agent
that selectively uses caveolae to enter cells is simian virus
40 (SV40). It has been shown that SV40 is internalized
specifically by caveolae by several means, including that
the overexpression of a dominant negative epidermal
growth factor (EGF) receptor pathway mutant (Eps15)
will selectively inhibit clathrin-mediated endocytosis
without effects on SV40 internalization (192). Additionally, the overexpression of a dominant negative caveolin
mutant, which inhibits caveolar-mediated endocytosis, resulted in little or no SV40 infection (191, 204). Following
its initial binding, SV40 accumulates in what has now
been termed a “caveosome,” an endocytic vesicle containing caveolin-1 and devoid of markers of clathrin-mediated
endocytosis (191). After accumulation, SV40 is delivered
to the ER, completing a mechanism that bypasses the
lysosomal compartment thereby preventing SV40 inactivation (171, 191). Although SV40 is the only virus shown
unequivocally to enter cells via caveolae en route to the
ER, this may prove prototypical for many viruses (170). It
is unclear if SV40 is an infectious agent to humans or a
mediator of disease, but its exploitation of caveolae to
gain entry into cells and avoid degradation may prove a
robust strategy co-opted by many pathogens. It should
also be noted that viral endocytosis via caveolae may be
a relatively slow process and that caveolae, on the whole,
are thought to be relatively stable structures (190, 260);
however, this remains a hotly debated topic (164).
A growing list of pathogens, including viruses, bacteria and their associated toxins, fungi, and even prions,
can interact with caveolae membrane domains (Table 1).
The intracellular trafficking of these agents via caveolae
differs dramatically from the usual route of ligands internalized by clathrin-medited endocytosis. The use of
caveolae for cellular entry allows the pathogen to avoid
classical endosome-lysosome trafficking and, consequently, avoid such degradative compartments in the cell.
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
B. Cholesterol Homeostasis
The interrelationship between cholesterol and caveolin has a wide and varied history. Rothberg et al. (203) first
recognized the importance of cellular cholesterol balance
on caveolar structure when they found that treating cells
with cholesterol binding agents, such as filipin and nystatin, resulted in the flattening of morphologically identifiable caveolae and the disruption of the striated caveolin
protein coat. Additionally, cholesterol regulates caveolin-1 expression at the level of transcription through two
steroid regulatory binding elements in the caveolin-1 promoter. This results in decreased caveolin-1 mRNA levels
during periods of cholesterol depletion, while cholesterol
loading has the opposite effect (8, 58). Additionally, the
oxidation of cholesterol into cholesterone by cholesterol
oxidase functionally disrupts cellular cholesterol balance
and results in the internalization of caveolin-1 from the
plasma membrane with its translocation to the ER and the
Golgi apparatus (237). Thus caveolin-1 expression and its
intracellular distribution are clearly dependent upon cellular cholesterol levels.
In an attempt to determine the interrelationship between caveolin-1 and the cellular cholesterol transport
machinery, Frank et al. (63) identified a direct role for
cholesterol in the stabilization of caveolin-1. These authors found that the expression of endogenous caveolin-1
increased ⬃18-fold when human embryonic kidney
(HEK) 293 cells were transfected with the scavenger receptor B1 (SR-B1) cDNA, but not with a cDNA encoding
Physiol Rev • VOL
CD36 (63). Because SR-B1, but not CD36, results in the
accumulation of intracellular cholesterol, these authors
postulated that cholesterol, not SR-B1 itself, was responsible for the increase in caveolin-1 expression. Indeed,
when HEK 293 cells were loaded with cholesterol, caveolin-1 protein expression increased dramatically by Western blot analysis. In addition, the distribution of caveolin-1 expression was also affected, with the intracellular
clustering of caveolin-1 in cholesterol-loaded COS-7 cells.
Thus, in addition to positive transcriptional regulation,
cholesterol also directly modulates caveolin-1 expression
via stabilization of the protein itself.
This interrelationship between caveolin-1 and cholesterol is further complicated by recent evidence suggesting that caveolin-1 can regulate cholesterol levels
by modulating its cellular influx and efflux. Caveolin-1
has been shown to transport newly synthesized cholesterol from the ER to membrane caveolae, where it is
delivered to plasma high-density lipoproteins (HDL).
Furthermore, while extracellular cholesterol primarily
enters cells via clathrin-mediated endocytosis of lowdensity lipoproteins (LDL), a secondary pathway involving caveolae may mediate the influx of cholesterol
from HDL particles by way of the scavenger receptor
B1 (SR-B1), which has recently been shown to colocalize with caveolin-1 in caveolae (81). Therefore, caveolae may be the principal location whereby plasma membrane cholesterol is exchanged between HDL and the
cell membrane.
C. Signal Transduction
Perhaps one of the most important realizations concerning caveolae and caveolins was that these elements
play an important functional role in the modulation of cell
signal transduction. For a more in-depth review of caveolae and caveolins in signal transduction, the reader is
referred to References 117, 200, 219, 234. Lisanti and
colleagues (131, 212) were the first to recognize this property when they made the surprising discovery that biochemically purified caveolae microdomains contained an
abundance of signaling molecules, such as Src-like tyrosine kinases and heterotrimeric G proteins (131, 212).
Such initial discoveries led these authors to propose that
caveolin-1 may serve to compartmentalize certain signaling molecules within caveolae, thereby concentrating and
localizing these elements within the cell with the prospect
of rapidly and selectively modulating cell signaling events
(130). Thus caveolae may serve as docking points for
numerous cell surface receptors, which when activated
by ligand binding are recruited to caveolae (Fig. 8). Here,
activated receptors interact with their respective caveolae-associated signaling components, leading to their
rapid and selective activation in the confines of a specific
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The mechanism of pathogen binding usually involves the
utilization of one or more of the glycolipid or GPI-anchored moieties that cluster within the caveolae of host
cells. It appears that disparate pathogens can utilize common binding sites localized within caveolae (170, 231,
232). The numbers of currently known pathogens that can
use caveolae for cellular invasion probably represents a
small fraction that will ultimately be shown to use caveolae-mediated cell entry (Table 1). Interestingly, the Marburg and Ebola viruses, which result in lethal hemorrhagic fever, have been shown to utilize the folate receptor ␣ as a cofactor to gain entry into cells (20).
Consequently, these pathogens may exploit caveolar potocytosis as a mechanism for cellular invasion. In the
same auspice, strategies aimed to exploit the folate receptor for therapeutic gene transfer may prove useful for
the treatment of human disease (37, 236). Importantly, as
pathogens exploit caveolae as a common mechanism to
gain entry into cells and avoid degradative membrane
compartments, this may benefit pharmaceutical strategies
aimed at reengineering common pathogen moieties to
facilitate entry, translocation, and delivery of therapeutic
agents.
COHEN, HNASKO, SCHUBERT, AND LISANTI
1351
lin-1 as both a regulator of signal transduction as well as
a scaffolding protein, sequestering agents within caveolae. Such experimental findings led to the proposal of the
“caveolae signaling hypothesis,” which attributes the regulation of numerous signaling molecules to interactions
with caveolae organelles and the caveolin proteins (130,
176). It is important to note that while caveolin seems to
be a negative regulator of the vast majority of signaling
proteins with which it interacts, at least one protein, the
insulin receptor, is positively regulated by an interaction
with caveolin-1 (27, 30, 172, 286).
D. Proteomics
8. Signaling through caveolae. In this view, two separate
receptors are shown docking with a cholesterol and caveolin-enriched
caveola organelle, following ligand-mediated stimulation. The ␤-adrenergic receptor (␤-AR; blue) is a conventional G protein-coupled receptor
with seven membrane-spanning domains. When stimulated, this receptor initiates a signaling cascade conveyed through several caveolaelocalized proteins, beginning with the activation of Gs subunits. This, in
turn, leads to the activation of adenylyl cyclase, which increases intracellular cAMP concentrations, resulting in the activation of protein
kinase A (PKA). On the right, an activated epidermal growth factor
receptor (EGF-R) is also shown docking with the caveola, leading to the
activation of a proliferative pathway involving several caveolae-associated proteins of the p42/44 mitogen-activated protein kinase cascade
(Ras/Raf/MEK/ERK).
subcellular environment. This hypothesis is strengthened
by studies showing that the GTPase activity of G protein
␣-subunits could be suppressed by a peptide derived from
the NH2 terminus of caveolin-1, the caveolin scaffolding
domain, demonstrating the interdependence of these proteins for functional activity (126).
Caveolins are now thought to act as scaffolding proteins, concentrating specific signaling molecules within
caveolae via an interaction between the CSD (caveolin-1
residues 82–101) and an aromatic amino acid-based
caveolin-binding domain (CBD; described above) usually
found in the active catalytic domain of a given caveolaeassociated protein. Following these early findings, a rapid
succession of studies showed that an enormity of signaling molecules copurify with caveolae by detergent-resistant extraction and sucrose gradient centrifugation (reviewed in Ref. 200). Furthermore, many of these proteins
have now been shown to be regulated by an interaction
with caveolin-1, including H-Ras, Src-family kinases, and
endothelial nitric oxide synthase (eNOS). Caveolin-1, for
instance, binds to wild-type H-Ras with high affinity in
vitro but does not bind to mutationally activated H-Ras
(G12V) (241). Furthermore, a nonfarnesylated mutant of
H-Ras (C186S), which normally remains cytoplasmic, is
recruited to caveolae membranes by overexpression of
the caveolin-1 cDNA (241). Thus this prototypical set of
experimental data demonstrates the dual role of caveoPhysiol Rev • VOL
Proteomic analysis of purified caveolae has identified
a wide assortment of proteins that are localized to these
structures. The first detailed proteomic analysis of caveolae was carried out in 1994. Based on their buoyancy and
resistance to detergent solubilization, Lisanti et al. (131)
used sucrose density ultracentrifugation to purify caveolae-rich membrane domains from murine lung tissue. This
procedure allowed for the exclusion of ⱖ98% of an integral plasma membrane protein marker, while retaining
⬃85% of total caveolin and ⬃55% of GPI-linked marker
proteins. When these purified caveolae were analyzed by
protein microsequencing and Western blotting, these authors provided the first glimpse into resident caveolar
protein components, identifying a preponderance of cytoplasmically oriented signaling molecules (Src-like kinases
and heterotrimeric G protein subunits), including the
small GTPases Rap 1, Rap 2, and TC 21, and cytoskeletal
elements, such as monomeric G-actin and myosin regulatory light chain (Fig. 9A). In addition, several transmembrane proteins were identified, including CD36 and
RAGE, as well as an abundance of GPI-linked proteins
(131). This initial proteomic analysis of caveolae allowed
for the first large-scale characterization of caveolae-enriched protein constituents and provided the basis for
many follow-up investigations into the functional significance that caveolar localization may confer upon these
proteins. Importantly, extremely similar results were obtained during the proteomic analysis of purified adipocyte
caveolae (Table 2).
To identify proteins that are tightly associated with
caveolin-1, purified lung caveolae were first dissociated in
octyl-glucoside and then allowed to reassociate after removal of the detergent, and subjected to a second round
of purification (217). These reassembled caveolae were
then analyzed by protein microsequencing (Fig. 9B). With
the use of this caveolar assembly/disassembly approach,
it was observed that caveolin-1 forms a tight complex
with a number of proteins, including a transmembrane
protein (CD36), four GPI-linked proteins (membrane
dipeptidase, 5⬘-nucleotidase, ceruloplasmin, carbonic an-
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FIG.
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
hydrase IV), actin, a calcium-binding protein (calsequestrin), cytoplasmically oriented signaling molecules (Lyn
tyrosine kinase; Gi-2␣), and two novel 45-kDa proteins
(now known as flotillin-1/2) (217).
More recently, Sprenger et al. (247) explored the proteomic composition of endothelial cell caveolae isolated by
two distinct methodologies. In their first approach, these
authors coated the outer plasma membrane of cultured human endothelial cells with positively charged silica, allowing
subfractionation of luminal membrane components. Homogenization of the silica-bound membranes in cold Triton
X-100 allows for the release of caveolar vesicles, which can
be subsequently purified by sucrose density gradient centrifugation. However, when the resultant fractions were analyzed by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization (MALDI), it was found
that this methodology fails to purify caveolae, as almost no
known caveolar resident proteins were identified. Furthermore, it seems that this methodology provides for an enrichment of ER proteins, as these were the main resident proteins identified (247).
Physiol Rev • VOL
When these authors next used a procedure to isolate
caveolae based on their resistance to extraction in cold
Triton X-100 and their propensity to float in a sucrose
density gradient, they were able to confirm the validity of
this methodology, showing an abundance of known
caveolar resident proteins, such as caveolin-1 and flotillin-1. Subsequent two-dimensional gel and MALDI analysis of these isolated microdomains provided insight into
their protein components, identifying a host of proteins
similar to those described above. In addition, these authors recognized, for the first time, the existence and
caveolar localization of several proteins whose identity
had only been postulated based on analysis of the human
genome (247). These proteins include a stomatin-like protein (SLP-2), a glucose-regulated-like protein (SGRP58),
as well as the X-linked retinitis pigmentosa 2 protein
(XRP-2). Because at least one of these genes has been
identified as causing human disease (XRP-2), its localization to caveolae provides a new avenue by which its
molecular function may be addressed. Taken together,
proteomic studies such as those described above provide
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FIG. 9. Proteomic analysis of purified caveolae
and caveolin-associated proteins. A: caveolae proteomics. Proteins copurifying with caveolae organelles were
further partitioned into hydrophobic and hydrophilic
fractions with the detergent Triton X-114, resolved by
SDS-PAGE, and stained with Coomassie blue. Note
that few proteins segregate into the hydrophilic aqueous portion (Aq), while the vast majority of proteins
partition into the hydrophobic detergent fraction
(Det). Several prominent bands were subjected to microsequencing, and their assigned identities are indicated. [Modified from Lisanti et al. (131).] B: proteomics of caveolin-associated proteins. Proteins copurifying with caveolin-1, after a cycle of disassembly and
reassembly, were resolved by SDS-PAGE and visualized by Ponceau S. These proteins were subjected to
microsequencing analysis to determine their identity,
as indicated. [Modified from Scherer et al. (217).]
1353
COHEN, HNASKO, SCHUBERT, AND LISANTI
TABLE
2.
Internal microsequencing of purified murine adipocyte caveolae
Molecular Mass, kDa
Identity
Sequence
Reported
Apparent
88
90
31
35
24
21–24
21
22
36
36
40
37
45
45
45
43
18
18
50–60
50–65
68
70–75
Transmembrane
CD36
Plasma membrane porin
Caveolin-1
Caveolin-2
Cytoplasmically oriented signalling molecules
Annexin II
KSLYYYIQQDTK
KTPAQYDASEL
XTERXVXHLQK
Cytoskeletal elements
Actin
KEITALAPSXM
KIIAPPERK
KEITALAPSTMK
KDLYANNVMSGGTTMYPQIADRMQ
KYPIEXGIITNXDDME
KIXHHTFYNELRVAPEEH
KEAFTVIDQNRDXII
Myosin regulatory (DTNB) light chain 2
Luminal content molecules/endocytic ligands?
Calsequestrin
KLKPESMYETWE
KAYEDAAEEFHPYIRFFAT
KAVAQDNXENPDLSIIXI
KAYEDAAXXF
KXXAQDNTE
KEYEATLEEXXAKDDPHAXYSTVFD
KYNGVFQEXXQAEDK
KLVVSTQTALA
KVLTSSARQRLRXASIQK
KEXXHGDLLEXADDRADLAK
KLVNELTEFAK
Albumin
powerful tools by which new caveolar protein components can be identified and may present a basis for the
further implication of these structures in human disease.
V. CAVEOLINS AND HUMAN DISEASE:
KNOCKOUT MOUSE MODELS
The recent generation of caveolin (⫺/⫺) null mice
has made it possible to evaluate the significance of each
caveolin protein in the context of whole animal physiology, while yielding valuable new models for the study of
human disease. Perhaps one of the more surprising findings regarding these mice was that all of the caveolindeficient mouse models generated (Cav-1 null, Cav-2 null,
Cav-3 null, and Cav-1/3 double knockout mice) are viable
Physiol Rev • VOL
and fertile. This is remarkable given the diverse attributes
of the caveolin proteins and their widespread tissue distributions. Furthermore, ultrastructural analysis of Cav1/3 double knockout mice revealed a complete loss of
caveolae in both nonmuscle and striated muscle tissues,
respectively, thus confirming the essential role of these
proteins in caveolar biogenesis. However, these results
contrast with those observed in Cav-2 null mice, which
retain morphologically identifiable caveolae. While these
mice have, thus far, provided unique animal models by
which to study the physiological roles of the caveolin
proteins, their potential as empirical models of human
disease is only now being fully realized. In the sections
below, we outline our current understanding regarding
the phenotypic characteristic of each caveolin null mouse
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KPVYISLP
KTGTTVYRQFXIFDVQNXDD
KRPYIVPILXLXETGTIXD
KIEALK
KELLXGYK
KSARDVFTK
KLTFDSSFSPNTGK
KXNTDNTLGTEITVEDQLAR
KHLNDDVVK
KYVDSEGHLYTVPIRENGNIYK
KEIDLVNRDPK
KIFSNIRISTQK
KLGFEDLIAEPEXTH
1354
ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
(Cav-1, Cav-2, Cav-3 single-knockouts, and the Cav-1/3
double-knockout) and how these changes relate to physiological processes and the pathogenesis of human disease.
A. Caveolin-1
1. Cellular transformation and tumorigenesis
Caveolin-1 was initially identified as a major substrate for tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryonic fibroblasts, suggestPhysiol Rev • VOL
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Cav-1 null mice were generated and initially characterized by two independent groups simultaneously (40,
198). As reported by both groups, these mice have a
complete absence of morphologically identifiable caveolae in all tissues and cell types that normally express
caveolin-1 (i.e., endothelia and adipocytes), while retaining identifiable caveolae in tissues that normally express
caveolin-3 (i.e., skeletal and cardiac myocytes). Indeed,
the Cav-1 null mouse unequivocally demonstrates the essential role of caveolin-1 in caveolar biogenesis in nonmuscle cells. An interesting caveat of caveolin-1 ablation
is a consequential loss of the caveolin-2 protein by ⬃90%,
in all caveolin-1-expressing tissues. This finding was not
completely unexpected considering the known role of
caveolin-1 in its hetero-oligomerization with caveolin-2,
and the ability of caveolin-1 to recruit caveolin-2 from the
Golgi to the plasma membrane (125, 160, 185). This reduction in caveolin-2 protein levels is the result of caveolin-2 destabilization and subsequent proteasomal degradation, rather than altered transcriptional regulation. Indeed, the treatment of mouse embryonic fibroblasts
(MEFs) derived from Cav-1 null animals with the proteasomal inhibitor MG-132 is sufficient to rescue the caveolin-2 protein expression (197). These data were supported
by the observation that, in untreated Cav-1 null MEFs,
caveolin-2 remained trapped in the Golgi apparatus, apparently unable to transit to plasmalemmal caveolae in
the absence of its molecular chaperone, caveolin-1. In
MEFs treated with MG-132, caveolin-2 expression increased to levels near those in wild-type cells; however,
the protein remained confined to the Golgi (197). Thus
Cav-1 null mice are essentially caveolin-2 deficient as
well, and this must be considered in the phenotypic characterization of Cav-1-deficient mice. In support of this
notion, our group has shown that the restrictive lung
phenotype initially described in Cav-1 null animals is actually due to a selective loss of caveolin-2, as determined
through the generation and characterization of Cav-2 null
mice (199).
The sections below detail the various phenotypes
identified to date in Cav-1 null mice and their relation to
human disease.
ing that it may be a target for inactivation during oncogenesis (76). Thus it has been proposed that caveolin-1
acts as a tumor suppressor protein, inhibiting the functional signaling activity of several protooncogenes and
consequently disrupting the process of cellular transformation (48 –51, 66, 70, 114, 123, 209).
Numerous follow-up studies designed to test this
hypothesis have contributed a myriad of evidence suggesting that caveolin-1 may indeed possess tumor suppressor capabilities. For instance, caveolin-1 mRNA and
protein expression are downregulated in NIH-3T3 cells
transformed with several activated oncogenes, such as
v-Abl, Bcr-Abl, and H-Ras (G12V) (48, 114). The ability of
these transformed cells to grow in soft agar, a hallmark of
cellular transformation, was found to inversely correlate
with caveolin-1 protein levels. The reintroduction of
caveolin-1 under the control of an inducible promoter was
sufficient to inhibit the anchorage-independent growth of
these cells, thus reverting their transformed phenotype
(48). In addition, some studies have identified similar
reductions in caveolin-1 protein and mRNA levels in
breast cancer cell lines (47, 51, 60, 94, 123, 292).
In further support of a role of caveolin-1 as a tumor
suppressor, it has been shown that targeted downregulation of caveolin-1 using a vector-based anti-sense approach resulted in the transformation of NIH-3T3 cells,
enhanced their anchorage independent growth, and hyperactivated the Ras-p42/44 mitogen-activated protein
(MAP) kinase cascade (70). When injected into nude
mice, these NIH-3T3 cells expressing the caveolin-1 antisense construct were capable of forming large tumors,
compared with matched NIH-3T3 cells lacking the caveolin-1 anti-sense vector.
Importantly, these results were reversible, as loss of
the caveolin-1 anti-sense vector, and thus reexpression of
caveolin-1, reverted the transformed phenotype. Although
it is generally accepted that caveolin-1 is indeed targeted
for downregulation during oncogenesis, the mechanisms
required for this reduction in mRNA levels have, for the
most part, remained enigmatic (52). The identification of
c-Myc-repressive and p53-responsive elements in the
caveolin-1 promoter may provide common targets by
which various oncogenes regulate caveolin-1 gene expression (183, 196).
Genetic evidence supporting the role of caveolin-1 as
a tumor suppressor has emerged from gene mapping studies, which revealed that the human CAV-1 gene maps to
the long arm of human chromosome 7 (7q31.1). This
region, the D7S522 locus, encompasses a known fragile
site (FRA7G) and is often associated with loss of heterozygosity (LOH) in various cancers, including breast,
prostate, ovarian, and renal carcinomas (105, 110, 289 –
291). The deletion of this region and its association with
the pathogenesis of several different types of cancers
lends credible support to the presence of a tumor sup-
COHEN, HNASKO, SCHUBERT, AND LISANTI
FIG. 10. Genomic organization of human chromosome 7 (7q31.1)
relative to the D7S522 locus, CAV-1, and CAV-2. Colored boxes represent exons and their corresponding number of nucleotides. Open boxes
represent introns and their corresponding size in kilobases (kb).
Physiol Rev • VOL
spontaneous tumors. However, the characterization of
the proliferative capacity of cells derived from these mice
did reveal their hyperproliferation. For instance, cultured
primary mouse embryonic fibroblasts (MEFs) derived
from Cav-1 null mice show a marked increase in growth
rate, compared with matched control wild-type MEFs
(197). In addition, the lungs of Cav-1 null animals were
found to be hypercellular, indicative of altered cell cycle
regulation. Although Cav-1 null mice do not show any
increased incidence of spontaneous mammary tumor formation, they develop significant mammary epithelial cell
hyperplasia as early as 6 wk of age, in the nulliparous
state. In 9-mo-old Cav-1 null mice, this mammary epithelial cell hyperplasia is also accompanied by an increase in
lobular development, with acini formation and fibrosis
(121).
Because these results indicate that loss of caveolin-1
may be a contributing factor in oncogenesis, Capozza et
al. (18) subjected the skin of Cav-1 null mice to the
carcinogen 7,12-dimethylbenzanthracene (DMBA). Here,
it was shown that repeated applications of this compound
resulted in a 10-fold increase in tumor incidence, a 15-fold
increase in tumor multiplicity, and a 35-fold increase in
tumor area per mouse, compared with wild-type mice
treated identically (18). In addition, before the formation
of overt skin tumors, Cav-1 null mice showed severe
epidermal hyperplasia, accompanied by increases in cyclin D1 expression and the hyperactivation of the p42/44
MAP kinase cascade. These findings suggest that loss of
caveolin-1 sensitizes mouse keratinocytes to oncogenic
transformation and that caveolin-1 does indeed function
as a tumor suppressor gene.
Following a similar rationale, Cav-1 null mice were
interbred with a tumor-prone transgenic model of breast
cancer (MMTV-PyMT). MMTV-PyMT mice develop spontaneous mammary tumors by ⬃8 wk of age involving the
entire mammary (146). When crossed with MMTV-PyMT
mice, Cav-1 null mice develop multifocal dysplastic mammary lesions at a much earlier age (3 wk) than MMTVPyMT mice alone (274). At this early age, there is an
approximately twofold increase in the number of lesions
per mouse, as well as an approximately sixfold increase in
the area occupied by these lesions in PyMT/Cav-1 (⫺/⫺)
mice compared with PyMT/Cav-1 (⫹/⫹) mice. Furthermore, PyMT/Cav-1 (⫺/⫺) lesions were of a higher histological grade and contained many more papillary projections than those of PyMT/Cav-1 (⫹/⫹) mice. Western blot
analysis of these dysplatic lesions revealed a dramatic
elevation in cyclin D1 protein expression, thus providing
a potential mechanism by which loss of caveolin-1 accelerates the appearance of these lesions.
Thus the loss of caveolin-1 alone appears insufficient
to induce cell transformation in vivo, but loss of caveolin-1 potentiates this process when combined with a transforming agent (a carcinogen or tumor-prone genetic back-
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pressor gene within this genetic locus. While no genes
have been directly mapped to the D7S522 locus, the closest genes to this region encode caveolin-2 and caveolin-1
(Fig. 10) (50, 51). The CAV-2 gene is found ⬃67 kb downstream of the microsatellite repeat marker D7S522, while
the CAV-1 gene is located ⬃86 kb downstream. Furthermore, the CAV-1 promoter has been reported to be hypermethylated in several cancer cell lines, including those
derived from breast and prostate, suggesting that transcriptional silencing may abrogate caveolin-1 expression
in human cancers (36, 51). As others have not shown
significant changes in the methylation status of the caveolin-1 gene in human tumor cell lines, a result which may
reflect technical differences, the transcriptional downregulation of caveolin-1 by promoter silencing will require
further clarification (99).
If caveolin-1 truly possesses tumor suppressor characteristics, then by what means does it inhibit cell growth
and transformation? Because caveolin-1 is a known scaffolding protein, interacting with and regulating a variety
of signaling pathways, it is possible that this protein may
similarly regulate protooncogenes. Indeed, overexpression of caveolin-1 in cultured cells is sufficient to inhibit
signaling from several proliferative pathways. For instance, it has been shown that key components of the
Ras-p42/44 MAP kinase cascade (MEK and ERK) reside
within caveolae and that these and other members of this
signaling cascade are negatively regulated by a direct
interaction with caveolin-1 (46, 70, 137, 139, 238, 241). It
has been shown that transient transfection of caveolin-1
dramatically inhibits signaling along the Raf-1/MEK/ERK
pathway, and the kinase activity of MEK-1 and ERK-2 are
inhibited by incubation with caveolin-1 scaffolding domain-based peptides in vitro (46). Furthermore, a reciprocal relationship exists between caveolin-1 expression
and the activation of p42/44 MAP kinase, suggesting that
caveolin-1 normally functions to inhibit signaling through
this cascade (48, 52, 70, 114).
With so much data indicating that caveolin-1 possesses important tumor suppressor properties, it was
somewhat surprising that Cav-1 null mice do not form
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
Physiol Rev • VOL
text of this apparent dichotomy. Perhaps additional celltype or tissue-specific alterations, in combination with the
dysregulation of caveolin-1 expression, are necessary to
promote the pathogenesis of these cancers (122).
C) PROSTATE CANCER. Although no specific mutations
have been identified in the caveolin-1 gene in either prostate cancers or prostate cancer-derived cell lines, caveolin-1 is upregulated in many primary prostate tumors.
Additionally, caveolin-1 expression is even further upregulated in metastatic prostate cells (288). This observation is supported by the detection of a significant number
of lymph node metastases in both human and mouse
prostate cancers that expresses caveolin-1. Interestingly,
normal prostate epithelial cells, which are the likely precursors of malignant cells, have undetectable levels of
caveolin-1.
During the process of prostate cancer metastasis,
focal areas in the primary tumor upregulate caveolin-1, a
process that may be androgen dependent. Indeed, testosterone transcriptionally activates caveolin-1 expression
via an androgen receptor-dependent mechanism (282).
Furthermore, when stably transfected with caveolin-1 antisense cDNA, metastatic prostate cancer cell lines result in
tumors that are 10% smaller in intact mice, but 40% smaller
in castrated animals, compared with vector alone (259).
Additionally, both intact and castrated mice showed a 17%
decrease in incidence of metastasis and 52% reduction in
tumor volume. These data suggest that caveolin-1 expression in androgen-responsive mouse prostate cancer cells
suppresses apoptosis in vitro and in vivo.
Although the expression of caveolin-1 appears to be
associated with the development of metastatic prostate
cancer, the role caveolin-1 plays in malignant progression
remains controversial. Indeed, caveolin-1 is a suspected
tumor suppressor, so how can caveolin-1 contribute to
the metastatic progression of prostate cancer via antiapoptotic actions and act as a tumor suppressor role in
other cells? The capacity of this alternating function of
growth suppression and growth promotion for caveolin-1
has not yet been resolved.
Caveolin-1 has been detected in the serum of patients
with prostate cancer, an observation supported by detectable caveolin-1 in mouse and human prostate cancer cell
conditioned media. Furthermore, secreted caveolin-1 is
bioactive, capable of stimulating cell viability and clonal
growth of a prostate cancer cell line, a process inhibited
by neutralizing caveolin-1 antibodies (162). The phosphorylation of caveolin-1 on serine-80 within the ER converts
caveolin-1 from an integral membrane protein to a luminal
secretory protein (136, 218).
Thus conversion of caveolin-1 to a secreted protein
provides yet another mechanism to subvert the tumor
suppressor effects of caveolin-1 by altering its membrane
topology. For example, phosphorylation at this site results in the regulated secretion of caveolin-1 from human
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ground) (18, 274). This is not surprising, as the process
of cell transformation and the development of cancer in
vivo is a multistep process that involves the selective and
progressive loss of several tumor suppressors (such
as p53, INK4a, and Rb), as well as the mutational activation or upregulation of certain key protooncogenes
[Ras(G12V), c-Myc, and c-Neu/ErbB2]. Indeed, the etiology of most cancers does not reflect alterations in a single
gene, but rather the functional loss or induction of a
series of key regulatory proteins that, in combination,
disrupts the normal regulation of the cell cycle and subsequently leads to uncontrolled cell growth (140).
A) BREAST CANCER. Fueled by evidence suggesting a role
for caveolin-1 in tumorigenesis, many clinically oriented
studies have been undertaken to examine the regulation
of caveolin-1 in a variety of human cancers. Analysis of
human breast cancer samples revealed that up to 16% of
these cancers have a CAV-1 gene point mutation (P132L),
with the majority of the mutations being found in invasive
carcinomas (88). Following up on this finding, Lee et al.
(121) examined the behavior of the Cav-1 (P132L) mutant
in a mammary cell culture model and found that it is
mislocalized, being retained in the Golgi apparatus. Furthermore, coexpression of the P132L mutant along with
the wild-type caveolin-1 cDNA resulted in the retention of
the wild-type caveolin-1 protein in the Golgi, indicating
that the P132L mutant acts in a dominant negative manner, promoting the retention of wild-type caveolin-1. This
suggests that a single allelic adulteration of the CAV-1
gene, the P132L mutation, is sufficient to render caveolin-1 inactive in cells.
B) GASTROINTESTINAL CANCER. In gastrointestinal cancers,
the role of caveolin-1 seems less clear. For instance, the
expression of both the caveolin-1 mRNA and protein in
human colon carcinoma cell lines is reduced (4). In contrast to these findings, several studies have shown elevations in caveolin-1 expression in colonic tumor tissue. In
comparing normal colonic mucosa to that of an adenocarcinoma, it was found that caveolin-1 levels were increased in ⬃78% of adenocarcinomas, compared with 6%
of normal epithelial tissue samples (59). In a similar study,
it was found that up to 45% of patients with squamous cell
carcinoma of the esophagus had elevated levels of caveolin-1 expression (109). Furthermore, caveolin-1 expression negatively correlated with patient long-term survival
and prognosis.
Yet, in an examination of frank colonic tumors, it was
shown that ⬃67% of these colon cancers had reduced
levels of caveolin-1 expression. When a cDNA encoding
full-length caveolin-1 was transfected into several colon
cancer cell lines, the ability of these cells to form tumors
in nude mice was decreased drastically (4).
Thus it is difficult to draw any definitive conclusions
about the contribution of caveolin-1 expression levels to
the pathogenesis of gastrointestinal cancers in the con-
COHEN, HNASKO, SCHUBERT, AND LISANTI
Physiol Rev • VOL
pathways and, consequently, a plethora of downstream
events. In this model, the combined loss of caveolin-1,
with that of loss of a tumor suppressor or addition of an
activated oncogene, may be sufficient to 1) induce cell
transformation and/or 2) potentiate the transformed phenotype, by disrupting associated downstream signaling
events involved in cell cycle regulation.
2. Glucose metabolism, insulin signaling,
and diabetes
Type II diabetes is a genetically heterogeneous metabolic disease characterized by chronic hyperglycemia
and abnormalities in lipid metabolism afflicting nearly 5%
of the Western world. While a definitive understanding of
the molecular mechanisms involved in the pathogenesis
of this disease has remained elusive, numerous genetic
candidates have been identified that, when dysfunctional,
result in defective peripheral insulin action. Shortly after
their initial discovery, caveolins were implicated in insulin signaling and have thus become an interesting candidate disease genes in type II diabetes.
Initial interest in caveolae as sites for insulin signaling came from ultrastructural studies of rat adipocytes,
which showed that gold-labeled insulin ligand clustered
within plasmalemmal caveolae (78, 79). Further studies
showed that caveolae are highly enriched in adipocytes
and that both caveolin-1 mRNA and protein levels increase over 25-fold during the differentiation of mouse
3T3-L1 fibroblasts into adipocytes (an insulin-dependent
phenomenon), also suggestive of a role for these microdomains in insulin signaling (215). Additional experimentation revealed that insulin treatment of these cells results
in the association of the glucose transporter GLUT4 with
caveolin-enriched membrane microdomains, as well as
the tyrosine phosphorylation of caveolin-1 on residue 14
(22, 31, 32, 112, 122, 215). Intriguingly, in adipocytes,
caveolin-1 tyrosine phosphorylation is specific for insulin,
as EGF, platelet derived growth factor (PDGF), tumor
necrosis factor (TNF)-␣, or interleukin (IL)-6 did not result in any measurable caveolin-1 phosphorylation (122).
In addition to these findings, several investigators
have used biochemical means to assess the contribution
of caveolin/caveolae to insulin signaling. Because caveolae are highly enriched in cholesterol, agents such as
␤-methyl-cyclodextrin (␤MCD), which bind and sequester
membrane cholesterol, can be used to reversibly flatten
caveolae and disrupt signaling through these microdomains. With the use of this methodology, it has been
shown that ␤MCD-treated rat adipocytes have a significantly reduced response to insulin stimulation, as both
ATP citrate lyase phosphorylation and glucose uptake
were greatly decreased (84). Replenishment of plasma
membrane cholesterol resulted in morphologically identifiable caveolae and a reversal of this phenotype, data
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prostate cancer cells that, in turn, can stimulate cancer
cell growth in an autocrine/paracrine manner (255, 282).
Caveolin-1 expression is also associated with the
metastasis of lung and pancreatic adenocarcinomas and
negatively correlates with patient survival (95, 252).
D) MULTIDRUG RESISTANT CANCERS. The development of
resistance to a variety of chemotherapeutic agents is often the primary cause for treatment failure in cancer
patients. In vitro studies have shown that cells that develop resistance to one type of drug often have resistance
to a multitude of other structurally and functionally distinct compounds, a phenomenon termed multidrug resistance (MDR). A variety of cellular changes have been
found to accompany the development of the MDR phenotype, including the “classic” activation of P-glycoprotein,
an ATP-dependent drug efflux pump (12). Additionally,
increases in plasma membrane sphingolipid and cholesterol content, the components of lipid rafts and caveolae,
often accompany the development of MDR. Such changes
are thought to influence the action of P-glycoprotein,
which has been shown to associate with lipid rafts and
caveolae, by altering the availability of drugs to the cellular efflux machinery (120). In addition, caveolin-1 has
been identified as an upregulated gene in a number of
MDR human cancer cell lines derived from a variety of
primary tumor sources, including breast, colon, ovary,
and lung (119, 287). Many of these cell lines also show
increased expression of caveolin-2, as well as an increase
in the number of identifiable surface caveolae. In MDR,
increases in caveolin-1 expression may serve to accelerate the efflux of drugs by facilitating the transport of these
substances from intracellular compartments to plasma
membrane efflux pumps, via routing through the intracellular cholesterol transport pathway (133).
Although increased expression of caveolin-1 may enhance the MDR phenotype, it may also serve to decrease
the transformed phenotype of the same cells by promoting cell adhesion and senescence. As mentioned above,
several proproliferative pathways are negatively regulated by caveolin-1, including cyclin D1 expression and
Ras-p42/44 MAP kinase cascade activation, a process that
would promote cell senescence. In addition, it has been
reported that MDR cells undergo a “reverse transformation,” demonstrating a reduced ability to form secondary
tumors in athymic mice, with a concomitant decrease in
anchorage-independent growth (7). Thus the acquisition
of MDR may allow cells to survive high concentrations of
cytotoxic agents, but it conversely impedes their transformed phenotype.
This process of sequential loss/induction of regulatory genes is well documented during the process of
cancer metastasis (56). The widespread association of
caveolin-1 and caveolae with multiple signal transduction
pathways suggests that the loss of caveolin-1 serves as a
common point that functionally regulates many signaling
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
based on their detergent resistance, it was found that the
insulin receptor no longer cofractionated with caveolin-1,
suggesting that caveolar resident proteins may have varied degrees of detergent resistance.
Perhaps the most convincing data regarding the role
of caveolin-1 in insulin signaling and diabetes came with
the development of the Cav-1 null mouse. Although this
mouse was not found to be overtly diabetic, as might be
predicted from previous experiments, Cav-1 null mice do
develop an interesting metabolic phenotype. Preliminary
analysis of this phenotype showed that Cav-1 null mice
are resistant to diet-induced obesity and developed progressive adipose tissue atrophy (197). As seen in Figure
11, a variant of diet-induced obesity, i.e., interbreeding
Cav-1 null mice with LepR (db/db) mice, leads to a very
pronounced phenotype whereby LepR (db/db)/Cav-1
(⫺/⫺) null mice weigh approximately half that of LepR
(db/db) Cav-1 (⫹/⫹) wild-type mice at 1 year of age [95.01
⫾ 2.08 vs. 48.5 ⫾ 1.04 (SE) g; P ⬍ 0.00005].
Metabolically, these abnormalities in Cav-1 (⫺/⫺)
mice are characterized by elevated free fatty acid and
triglyceride levels, decreased leptin and Acrp30 levels,
and no changes in plasma insulin or glucose levels (197).
However, when these mice were later placed on a high-fat
diet for 9 mo, they were found to develop postprandial
hyperinsulinemia (30). Additionally, when young Cav-1
null mice were challenged with an insulin tolerance test
(ITT), they showed markedly decreased glucose uptake
compared with wild-type control animals. These metabolic derangements are similar to those seen in prediabetic individuals in the human population, suggesting that
caveolin-1 does indeed play a critical role in insulin signaling in vivo.
FIG. 11. Loss of caveolin-1 rescues
the phenotype of LepR (db/db) mice.
Cav-1 null mice were interbred with LepR
(db/db) mice to produce the corresponding two cohorts: LepR (db/db)/Cav-1
(⫹/⫹) and LepR (db/db)/Cav-1 (⫺/⫺). The
mass of 1-year-old male mice that have
been fed a standard Chow diet are represented for each cohort, respectively
[95.01 ⫾ 2.08 vs. 48.5 ⫾ 1.04 (SE) g; P ⬍
0.00005].
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suggesting that caveolae/caveolin are involved in insulin
signaling. Further studies corroborated these findings,
demonstrating that cholesterol depletion resulted in decreased activation of downstream insulin responsive elements, such as insulin receptor substrate (IRS)-1 and
protein kinase B (PKB)/Akt (186).
Using cell fractionation schemes to biochemically
purify caveolae and their resident proteins, several groups
have investigated the caveolar localization of the insulin
receptor with mixed results. Mastick and colleagues (31, 84,
163, 286) used two properties of caveolae and lipid rafts to
isolate these domains from insulin stimulated 3T3-L1 adipocytes: 1) their relative resistance to extraction in Triton
X-100 at 4°C and 2) their propensity to “float” on a discontinuous sucrose gradient. These authors found that while up
to 2% of the total phosphorylated insulin receptor is insoluble in cold Triton X-100, only a very small percentage (0.3%)
cosediments with caveolar resident proteins such as caveolin-1, suggesting that the insulin receptor does not localize to
caveolae (31). In support of these findings, Muller et al. (163)
prepared cellular extracts using either ice-cold Triton X-100
or sodium carbonate, followed by sucrose density gradient
centrifugation. Western blot analysis of the subsequent fraction revealed that the majority of the insulin receptor was
excluded from the “floating” caveolar pool (163).
While these results are indicative of the separation of
insulin receptor from caveolae, at least two studies have
presented clear contrary evidence (84, 286). In one of
these studies, caveolar resident proteins were isolated by
a detergent-free method followed by sucrose gradient
centrifugation. Interestingly, this methodology yielded an
⬃36-fold enrichment of the insulin receptor in the caveolar fraction, while excluding noncaveolar proteins (i.e.,
clathrin) (84). In addition, when caveolae were isolated
COHEN, HNASKO, SCHUBERT, AND LISANTI
resistance despite a significant reduction in the insulinresponsiveness of isolated adipocytes.
Also of note are findings that a subset of patients with
severe insulin resistance were found to have mutations in
the caveolin-binding motif of the insulin receptor (Table
3). These mutations led to the rapid degradation of the
insulin receptor when expressed in cultured cells, similar
to the degradation observed in Cav-1 null animals (100,
101, 103, 157, 158). Taken together, these findings further
support the idea that caveolin-1 stabilizes the insulin receptor against degradation in the human population and
that perhaps some diabetic patients exist with caveolin-1
mutations.
3. Modulation of lipid storage and breakdown
Recently, it has been shown that caveolin-1 and -2
can be directed to lipid droplets, in a variety of cell types
(64, 178, 195). Fujimoto et al. (64) found that the ␤-isoform of caveolin-2, an endogenous NH2-terminal truncation of the caveolin-2 molecule, is constitutively localized
to the surface of lipid droplets, by immunofluorescence
and immunoelectron microscopy. In addition, these authors found that disruption of vesicular transport with
brefeldin-A caused the further accumulation of caveolin-2, as well as caveolin-1, in lipid droplets (64). A similar
report found that caveolin-1 accumulated in lipid droplets
when it was modified by the addition of an ER retrieval
sequence, thus forcing its retention in the ER (178). Furthermore, truncation mutants of all three caveolin iso-
FIG. 12. Recombinant expression of fulllength caveolin-1 in Cav-1 (⫺/⫺) null MEFs rescues insulin receptor protein expression. A: Western blot analysis of lysates from wild-type (WT)
and Cav-1 (⫺/⫺) null mouse embryonic fibroblasts (MEFs) reveals that WT cells contain significantly more insulin receptor than those of the
Cav-1 null cells. B and C: transient transfection of
the full-length caveolin-1 cDNA (⫹), but not vector alone (⫺), into Cav-1 null MEFs (B) or HEK
293 cells (C) significantly increases the expression of the insulin receptor, as seen by Western
blot. D: transfection of the full-length caveolin-1
cDNA rescues insulin receptor expression in
Cav-1 null MEFs, as assessed by immunofluorescence microscopy. Under phase contrast, four different Cav-1 null MEFs can be seen, of which only
one received the caveolin-1 cDNA (arrow). Immunofluorescence microscopy of these cells following colabeling with anti-caveolin-1 IgG (green)
and anti-insulin receptor IgG (red) revealed that
the cell that was transfected with the caveolin-1
cDNA (arrow) showed an increase in insulin receptor protein expression, while the other three
cells (arrowheads) did not. [Adapted from Cohen
et al. (30).]
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Further analysis of these physiological abnormalities
revealed that Cav-1 null mice have an ⬃90% decrease in
detectable insulin receptor levels, selectively in adipose
tissue (30). Furthermore, insulin stimulation of these mice
did not cause measurable activation of downstream targets, such as PKB/Akt and GSK-3␤, indicating that caveolin-1 is necessary for insulin’s action in adipocytes. In
Cav-1 null MEFs, transfection of the full-length caveolin-1
cDNA restored insulin receptor levels, as seen by both
Western blot and immunofluorescence microscopy (Fig.
12). This effect was attributed to a stabilizing effect of the
caveolin-1 scaffolding domain, rescuing the insulin receptor from proteasomal degradation, thus providing a molecular mechanism to explain these findings (30).
Next, we directly investigated the role of the caveolin-1 scaffolding domain on insulin receptor stability by
incubating cells with a plasma membrane-permeable derivative of the scaffolding domain peptide. When Cav-1
null MEFs were treated with the caveolin-scaffolding domain, expression levels of the insulin receptor were dramatically increased, as assessed by Western blot (27).
Thus the scaffolding domain alone is sufficient to functionally stabilize the insulin receptor.
In light of current data, caveolin-1 can be thought of
as a major player in insulin signaling in tissues where it is
expressed; however, loss of caveolin-1 is not sufficient to
produce fulminant diabetes. This is consistent with other
mouse models, specifically the adipose tissue selective
insulin receptor knockout mouse (FIRKO) (9). These
mice phenotypically have relatively mild full-body insulin
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
3. Mutations of the caveolin-binding motif within
the human insulin receptor are associated with insulin
resistance and insulin receptor instability
TABLE
Reference Nos.
00
Mutations and Associated Disease
12
11
93
Insulin Receptor
Caveolin
Binding Motif
WSFGVVLW
LSFGVVLW
WSFGVVLS
Wild type; normal
W1193L; severe insulin resistance 100, 101, 103, 213
W1200S; severe insulin resistance 157, 158
[Adapted from Cohen et al. (30).]
FIG. 13. Immunofluorescence microscopy
of lipid droplets in a 3T3-L1 adipocyte. Fully
differentiated 3T3-L1 adipocytes were fixed in
paraformaldehyde, permeablized with 0.02% Triton X-100, and colabeled with anti-perilipin IgG
(red) and the neutral lipid-specific stain Bodipy
495/503 (green). Note that perilipin (red) forms
a ring around the lipid droplets (green) in these
cells. Original magnification, ⫻60.
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forms have been localized to lipid droplets (195). More
recently, a proteomics screen of isolated lipid droplets
derived from Chinese hamster ovary (CHO)-K2 cells identified that caveolin-1 is normally found as a component of
lipid droplet membranes. Furthermore, the amount of
caveolin-1 present in these droplets increased with lipid
loading (138).
A latter study identified a short hydrophobic stretch
of amino acids (residues 101–134) within caveolin-1 that
was required for targeting to lipid droplets. Furthermore,
mutational insertion of select leucine residues within this
hydrophobic domain blocked functional lipid droplet targeting of caveolin-1 (179). While these studies suggest
that caveolin-1 may modulate lipid droplet structure or
function, further work is necessary to elucidate the contribution of caveolin-1 to this important process.
The abnormalities in lipid homeostasis presented by
Cav-1 null mice suggest relevance to the pathogenesis of
metabolic human diseases. Indeed, the metabolic derangements found in Cav-1 null mice are characterized by
postprandial hypertriglyceridemia, elevated serum free
fatty acids (FFA), and elevations in serum very-low-density lipoproteins (VLDL)/chylomicrons. These phenotypic
characteristics parallel many of those associated with
type II diabetes, suggesting the importance of caveolin-1
in normal lipid metabolism.
In a direct physiological assessment of this proposed
function, our group explored the role of caveolin-1 in lipid
droplet metabolism, i.e., formation and breakdown, using
the Cav-1 null mouse as a model organism. Lipid breakdown, or lipolysis, occurs through stimulation of ␤-adrenergic receptors by any number of agonists, which essentially all lead to the activation of protein kinase A (PKA).
The two major downstream lipolytic targets in the adipocyte of PKA-mediated phosphorylation are the neutral
lipid lipase, hormone sensitive lipase (HSL), and the lipid
droplet coat protein perilipin (85, 107, 143, 147, 226, 257).
Upon PKA-mediated phosphorylation, HSL translocates from the cytoplasm to the lipid droplet, via an
interaction with perilipin, where it acts upon stored triglycerides (15, 24 –26, 43, 85, 249, 253, 254). However, the
activation of lipolysis is also dependent on PKA-mediated
phosphorylation of perilipin-A (143, 144). It is thought that
perilipin-A functions as a protective coat (Fig. 13), surrounding the lipid droplet until phosphorylated by PKA,
whereupon it undergoes a conformational change, leaving
the lipid droplet an open target for HSL and breakdown of
stored triglycerides (24 –26, 143, 254).
In our recent study on this subject, we found that
Cav-1 null mice have a severely blunted response to both
COHEN, HNASKO, SCHUBERT, AND LISANTI
sary for lipid accumulation and lipid droplet stabilization, as Cav-1 null Peri-MEFs contained significantly
less lipid than wild-type Peri-MEFs. Furthermore, the
lipid droplets in these cells were generally smaller and
significantly less abundant (Fig. 14A). In addition, we
noticed an electron-dense area surrounding the lipid
droplets of wild-type, but not Cav-1 null Peri-MEFs
(Fig. 14B). Ultrastructural analysis of wild-type and
Cav-1 null perigonadal fat pads, using a relatively new
technique involving high-pressure freeze substitution,
revealed a morphological correlate of the physiological
differences we found in lipolysis and lipid droplet formation in the absence of caveolin-1. In wild-type but
not Cav-1 null adipocytes, we observed an electrondense band surrounding the lipid droplet that most
likely represents a conglomeration of various proteins,
similar to that observed in wild-type Peri-MEFs, but
much more pronounced (29). Indeed, many recent studies have identified several proteins that specifically
target to the lipid droplet, including TIP47, adipophilin,
perilipin, and S3–12 (14, 82, 142, 156, 174, 258, 275, 276).
These proteins all have certain structural similarities
and are thought to be involved in modulating lipid
FIG. 14. Ultrastructural analysis of perilipin-A expressing mouse embryonic fibroblasts (Peri-MEFs) after
lipid loading. A: as seen by transmission electron microscopy, lipid droplets (arrows) of wild-type Peri-MEFs
appear larger and more abundant than those of Cav-1
(⫺/⫺) null Peri-MEFs following incubation with oleic
acid for 24 h (to promote lipid accumulation). Bar, 500
nm. B: densitometric quantification of the area directly
adjacent to the lipid droplet reveals a region of significant electron density in wild-type Peri-MEFs, but not in
Cav-1 null Peri-MEFs. This area most likely represents a
conglomeration of lipid droplet structural proteins (i.e.,
perilipin, TIP47, and S3–12) as well as many unidentified
proteins. Bar, 200 nm.
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pharmacological and physiological lipolytic agonists (29).
Furthermore, isolated perigonadal adipocytes derived
from Cav-1 null mice responded poorly to lipolytic stimuli
compared with wild-type cells. Further studies revealed
that PKA activity was dramatically increased in Cav-1 null
fat pads; however, this increased activity did not translate
into perilipin phosphorylation. In addition, we found that
␤-adrenergic receptor stimulation of 3T3-L1 adipocytes
resulted in coimmunoprecipitation of perilipin, caveolin-1, and the catalytic subunit of PKA. This ligand-dependent complex formation between perilipin and PKA was
clearly defective in Cav-1 null adipocytes. These results
suggest that perilipin phosphorylation, and thus functional lipolysis, is dependent on complex formation between perilipin and the catalytic subunit of PKA, and that
caveolin-1 facilitates this interaction.
In this same study, we also identified a role for
caveolin-1 in de novo lipid accumulation and lipid droplet stabilization. Wild-type and Cav-1 null MEFs, into
which the perilipin-A cDNA had been stably transfected
(Peri-MEFs), were loaded for 24 h with oleic acid to
promote lipid storage (29). Subsequent analysis of
these cells revealed that caveolin-1 is normally neces-
1361
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
storage. Some of these proteins, along with other yet
unidentified proteins, may be components of the electron-dense band that we observe in wild-type adipocytes.
4. Vascular abnormalities: hypertrophic
cardiomyopathy
Physiol Rev • VOL
5. Vascular abnormalities: increased nitric
oxide production
Initial reports of Cav-1 null mice demonstrated the
unequivocal role of this protein in the negative regulation
of nitric oxide (NO) production. Experiments performed
in isolated aortic rings, which measured both vasoconshowed and vasorelaxation responses of the tissue,
showed that caveolin-1 is essential for regulating vascular
tone via modulation of eNOS activity.
Isolated aortic rings from Cav-1 null animals were
unable to establish a steady-state contractile tone, oscillating at a frequency of once per minute (40). When
acetylcholine was added to the media to stimulate vasorelaxation, the aortic rings from Cav-1 null mice showed a
significantly greater (maximum 100%) relaxation response
than wild-type control rings (maximum 45%) (198). Additionally, tension measurements showed that Cav-1 null aortic
rings failed to contract in response to the ␣1-adrenergic
agonist phenylephrine to the same extent as wild-type aortic
rings (198). These effects could be abrogated by the addition
of the potent eNOS inhibitor NG-nitro-L-arginine methyl ester
(L-NAME), thus implicating increased NO production in this
phenotypic characteristic (198).
6. Vascular abnormalities: impaired angiogenesis
Angiogenesis is a process that describes the formation of new blood vessels from the preexisting vasculature, through the proliferation of endothelial and smooth
muscle cells. Angiogenesis is a normally quiescent process that is activated during such conditions as wound
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Hypertrophic cardiomyopathy (HCM) affects nearly
1 in 500 individuals in the general population resulting in
significant morbidity and mortality. A wide variety of
genetic mutations have been identified that result in a
heterogeneous clinical presentation and patient course.
However, the one commonality that exists among most of
the mutated genes is that they encode cardiac myocyte
contractile proteins. Yet, regardless of the gene involved,
hallmark findings in patients with HCM include cardiomyocyte hypertrophy, interstitial inflammation and fibrosis, myocyte degeneration, and cardiac dysfunction.
Because caveolin-1 expression is limited to the supporting cell types of the heart (i.e., fibroblasts and endothelial cells), it was somewhat surprising that Cav-1 null
mice develop cardiac disease similar to HCM of humans.
To date, two independent studies examining the effects of
caveolin-1 gene ablation on cardiac structure and function have been published, with somewhat disparate results (28, 293). In examining our Cav-1 null mice at three
time points, 2, 4, and 12 mo of age, by cardiac gated
magnetic resonance imagining (MRI) and transthoracic
echocardiography (TEE), our group found that loss of
caveolin-1 leads to progressive concentric left ventricular
hypertrophy and severe right ventricular dilation (28).
Together, MRI and TEE produce the most reliable measure currently possible of several cardiac parameters,
including end diastolic and systolic diameters, relative
anterior, posterior and septal wall thicknesses, as well as
fractional shortening (28).
In a similar study of this cardiac phenotype, Zhao et al.
(293) used TEE to observe that their independently generated Cav-1 null mice developed a dilated cardiomyopathy,
evident at 5 mo of age. The different phenotypes reported by
these two groups may be due to several factors including the
genetic background of the mice, the choice of anesthetic
used, as well as the limitations of a single methodology
(TEE) used by the later group for evaluating cardiac wall
thickness. In our studies, we found that Cav-1 null mice are
exquisitely susceptible to commonly used inhaled anesthetics, resulting in bradychardia and artificial chamber dilation
at dosages that do not produce these effects in wild-type
mice (unpublished observation). Because of this, we used
the anesthetic chloral hydrate, which has very limited cardiac effects and produced data corroborating that obtained
by MRI (28). It is unclear which anesthetic was used by Zhao
et al. (293), but we suspect that differences in the anesthetic
used may explain these apparently conflicting results (293).
The molecular mechanism responsible for the cardiac phenotype observed in Cav-1 null mice involves a
reversion to a state of early/fetal gene transcription, as is
often the case in cardiomyopathies. Both groups noted a
marked increase in atrial natriuretic factor (ANF) in ventricular tissue from Cav-1 null mice (28, 293). In addition,
histopathological examination of Cav-1 null mouse hearts
revealed areas of myocyte necrosis and foci of interstitial
inflammation, as well as prevalent fibrotic lesions. Mechanistically, these pathological abnormalities were accompanied by hyperactivation of the p42/44 MAP kinase cascade, specifically in areas of interstitial fibrosis (28). Isolation of primary cardiac fibroblasts demonstrated that
the loss of caveolin-1 in this cell type leads to hyperactivation of the p42/424 MAP kinase cascade. It is conceivable that, consistent with the literature, elevations in the
p42/44 MAP kinase cascade in the cardiac fibroblast result
in the secretion of growth factor molecules, such as endothelin-1 and transforming growth factor-␤, which then
promote the hypertrophic response in neighboring cardiac myocytes (28).
These and other cardiovascular phenotypes in caveolin-knockout mice are summarized in Table 4.
COHEN, HNASKO, SCHUBERT, AND LISANTI
TABLE
4.
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Cardiovascular phenotypes in caveolin-knockout mice
Expression
Cardiovascular Phenotypes/Abnormalities in Cav-Knockout (KO) Mice
Cav-1
Endothelia, smooth muscle
cells, macrophages, and
cardiac fibroblasts
Cav-2
Same as Cav-1
Cav-3
Smooth muscle cells and
cardiac myocytes
Cav-1 KO: lung hypercellularity; pulmonary hypertension/fibrosis; increased microvascular
permeability; decreased angiogenesis capacity; athero-protection; increased neointimal
hyperplasia (smooth muscle cell proliferation); arterial hyper-relaxation/decreased
vascular tone; hypertrophic cardiomyopathy due to the hyperproliferation of cardiac
fibroblasts; reductions in life span; hyperactivation of eNOS and Ras-p42/44 MAP kinase
signaling cascades in endothelial cells and cardiac fibroblasts, respectively.
Cav-2 KO: lung hypercellularity; pulmonary hypertension/fibrosis without hypertrophic
cardiomyopathy.
Cav-3 KO: hypertrophic cardiomyopathy and hyperactivation of the Ras-p42/44 MAP
kinase signaling cascade in cardiac myocytes. Also, a human Cav-3 (T63S) mutation
has been discovered in patients with familial hypertrophic cardiomyopathy (FHCM)
(inherited) (see Ref. 89).
Cav-1/3 dKO: severe hypertrophic cardiomyopathy in double-KO mice due to loss of
caveolins in both cardiac fibroblasts (Cav-1) and cardiac myocytes (Cav-3).
See text for details and specific references.
healing or changes in female reproductive tissues, associated with the preparation and maintenance of pregnancy. In addition, angiogenesis represents a hallmark
feature of tumorigenesis, an essential component for tumor growth. Previous in vitro studies have implicated
caveolin-1 in this process, demonstrating that caveolin-1
expression positively correlates with capillary tubule formation (83, 135). In accordance with these findings,
Woodman et al. (277) demonstrated that Cav-1 null mice
implanted with Matrigel plugs (a complex mixture of
extracellular matrix components) combined with the angiogenic agent fibroblast growth factor-2 show a significant reduction in blood vessel infiltration and vessel density compared with wild-type mice (277). In addition,
these authors found that the subcutaneous injection of
the melanoma cell line B16-F10 resulted in significantly
fewer and smaller primary tumors in Cav-1 null mice than
in their wild-type counterparts. Histopathological and ultrastructural examination of these tumors showed that
there was a decrease in blood vessel density and an
incomplete formation of capillaries, also lacking identifiable endothelial cell caveolae. These data provide the first
in vivo evidence indicating that caveolin-1-deficient endothelial cells have a diminished response to angiogenic
growth factors and support the notion that loss of caveolin-1 can retard tumor growth via a diminished angiogenic
response (277).
7. Vascular abnormalities: atheroprotection
Atherosclerosis is the most prevalent contributing
factor to coronary heart disease, the leading cause of
death in the Western world. The pathogenesis of atherosclerosis is most often initiated by injury to the vascular
endothelium resulting in the accumulation of macrophages and LDL at the site of injury. Recruited macrophages will engulf and oxidize the accumulating lipids,
resulting in 1) foam cell formation, 2) the release of
Physiol Rev • VOL
various degradative enzymes and cytotoxic substances, 3)
the subsequent denudation of the overlying endothelium,
and 4) the accumulation of platelets that combine to form
a plaque (168). Over time, this plaque increases in size and
can eventually rupture producing vaso-occulsion and local ischemia, leading to a myocardial infarction or a cerebral vascular accident (CVA; stroke).
Caveolin-1 and endothelial cell caveolae have been
postulated to play an important role in the development of
atherosclerotic lesions, based on studies showing that
oxidized LDL particles are taken up by caveolae and that
the LDL scavenger receptor CD36 localizes to caveolae
and interacts with caveolin-1 (63, 111, 132, 265). To directly address this issue, Cav-1 null mice were interbred
with the atherosclerosis-prone apolipoprotein E null mice
(194) to generate ApoE/Cav-1 double-knockout (dKO)
mice. While Cav-1 null mice exhibit normal baseline
plasma cholesterol levels and elevations in plasma triglyceride levels, interbreeding with ApoE null mice results in
an approximately twofold increase in plasma cholesterol
levels (62, 197). However, in spite of this normally
“proatherogenic” hypercholesterolemia, loss of caveolin-1
conferred dramatic protection against atheroma formation (62). Gross examination of aortic preparations demonstrated that ApoE/Cav-1 dKO mice have an ⬃70% reduction in lesion area, compared with ApoE null mice
(Fig. 15). Mechanistically, these changes were associated
with alterations in several proatherogenic endothelial cell
molecules, such as CD36 and vascular cell adhesion molecule (VCAM)-1 (62). These alterations may affect the
recruitment and migration of monocytes/macrophages
into areas of endothelial lesions, as well as decrease the
uptake and deposition of certain lipoproteins.
Cav-1 null mice also show other vascular defects,
such as increased neointimal hyperplasia (smooth muscle
cell proliferation) in response to vascular injury of the
carotid artery (87). Interestingly, this phenotype appears
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Genes
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
FIG. 15. Loss of caveolin-1 is protective against
atherosclerosis. Whole-mount en face visualization of
Sudan IV-stained aortas demonstrates a significant reduction (⬃65%) in the gross appearance of atheromatous lesions (red) in 6-mo-old male ApoE/Cav-1 dKO
mice compared with matched ApoE KO mice, both fed
a Western-type diet for 5 mo. [Adapted from Frank et al.
(62).]
8. Abnormalities of the urogenital system
Cav-1 null male mice have recently been shown to
exhibit severely compromised renal calcium reabsorption, leading to hypercalciuria and urolithiasis (17). Cao et
al. (17) found that, by 5 mo of age, 67% of male Cav-1 null
mice have developed noticeable early urinary bladder
calculi, compared with 19% of wild-type male mice. At this
same age, frank urinary bladder stone formation was
observed in as many as 13% of Cav-1 null male mice, while
no stones were detected in wild-type male mice of the
same age. Additionally, neither stones nor calculi were
observed in female Cav-1 and wild-type mice, indicating a
multigenetic gender-specific cause potentiating stone formation (17). Investigating the mechanisms behind this
phenomenon, it was shown that male Cav-1 null mice
excrete significantly higher amounts of calcium in their
urine, compared with wild-type male mice. Immunohistochemical analysis of the kidney revealed that caveolin-1 is
normally expressed in the epithelial cells of the distal
convoluted tubule and that a lack of caveolin-1 expression in these cells resulted in mislocalization of an important calcium transporter. Further analysis indicated that
Cav-1 null mice show compromised renal calcium reabsorption, while maintaining otherwise normal kidney
function. Thus this study illustrates the essential role of
caveolin-1 in normally regulating urine calcium homeostasis (in males) and suggests that impaired caveolin-1 function may contribute to human calculi or stone formation.
Loss of caveolin-1 in male mice has also been found
to lead to a syndrome similar to that of lower urinary tract
dysfunction (LUTD) in elderly adult male humans. Interestingly, only the bladders of male Cav-1 null mice, but
not Cav-2, -3, or -1/3 null mice, show pathological changes,
despite the fact that the bladder is composed primarily of
Physiol Rev • VOL
smooth muscle cells that express all three caveolin proteins (278). The loss of caveolin-1, but not -2 or -3, resulted in the absence of identifiable caveolae in the bladder, indicating that it is caveolin-1 that is necessary for
caveolar biogenesis in smooth muscle cells. By 12 mo of
age, male Cav-1 null mice exhibited significant bladder
hypertrophy and several pathophysiological abnormalities, including increased baseline and spontaneous pressures of micturation and decreased agonist-induced bladder contraction. Furthermore, these mice exhibited significant fluid accumulation in the prostate, seminal
vesicles, and kidneys, which eventually results in marked
dilation and even necrosis of these organs. In addition,
Cav-1 null prostates from 12-mo-old mice show a hypercellular and thickened fibromuscular stroma (Fig. 16)
(278). In contrast to the study mentioned above, these
authors did not observe any urinary bladder calculi, perhaps due to the differing genetic backgrounds of the mice
used in each study (17, 278).
9. Ocular disease
The role of caveolin-1 in ocular disease has been
proposed based in part on the observation that caveolin-1
is a specific component of rod cell outer segments disk
membranes (10, 44). This pattern of caveolin-1 expression
suggests a possible role in retinal degenerative diseases,
such as retinitis pigmentosa.
Retinitis pigmentosa (RP) refers to a group of genetically heterogeneous progressive inherited retinopathies,
characterized clinically by gradual visual field narrowing
and classical pigmented lesions on fundoscopic exam. RP
is generally the result of a single gene defect (monogentic); however, in at least one case, inheritance of defects
in two unrelated genes (peripherin/rds and rom-1) appears necessary to cause RP (41, 93, 106, 141, 180, 205).
While a multitude of genes have now been implicated in
the pathogenesis of RP, the role of caveolin-1 is only now
being elucidated (93).
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to be due to hyperactivation of the Ras-p42/44 MAP kinase
cascade and upregulation of Cyclin D1 in Cav-1 null vascular smooth muscle cells.
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COHEN, HNASKO, SCHUBERT, AND LISANTI
The rod cell is a highly complex, specialized cell type
composed of an inner and outer segment connected by a
thin cilium. The inner segment is responsible for most of
the cells metabolic needs, while the outer segment, composed of stacked membrane-bound disks, is responsible
for light sensation. The aforementioned peripherin/rds
and rom-1 proteins form oligomeric transmembrane complexes that localize to rod outer segment disks (141). In a
recent study, rom-1 was found to be resistant to extraction with Triton X-100 at 4°C and shown to associate with
lipid rafts and caveolin-1 (10). Because of this association,
it is possible that defects in the Cav-1 gene may lead to a
similar RP phenotype via mislocalization of rom-1. To
address this possibility, we examined the eyes of 1-yr-old
Cav-1 null mice for any histopathological changes consistent with RP. However, there were no significant differences between the retinal architecture of Cav-1 null mice
and wild-type mice, in hematoxylin and eosin-stained sections (Fig. 17). Similarly, fundoscopic examination and
fluorescein angiography revealed no retinal pigmentation
or arteriolar narrowing consistent with RP (Fig. 17). Although these findings suggest that loss of caveolin-1 does
not contribute to the pathogenesis of RP, it is possible
that like rom-1 and peripherin/rds, mutations in Cav-1
constitute a yet unidentified cause of digenic RP.
10. Reductions in life span
On the basis of the myriad of phenotypes described
above, it was not surprising to find that loss of caveolin-1
conferred a significant survival disadvantage (182). Our
group followed a large cohort of male and female wildtype, Cav-1 heterozygous, and Cav-1 null mice over a
Physiol Rev • VOL
period of 2 years. There was an ⬃50% reduction in life
span, most evident between 27 and 65 wk of age, in Cav-1
null mice with no change in heterozygote animals (Fig.
18). Interestingly, Cav-1 male and female mice experienced similar declines in life span, thus abolishing the
normal sexual dimorphism observed in the murine species. Histopathological examination of several organ systems indicated that the most likely cause of increased
mortality in Cav-1 null animals was a combination of
pulmonary fibrosis and cardiac hypertrophy. We found
that 1-yr-old Cav-1 null animals showed progressive thickening of the left ventricular and intraventricular septal
walls, as assessed by cardiac gated MRI and TEE (182). In
addition, the lungs of Cav-1 null animals were severely
fibrotic and hyperplastic, leading to increased right ventricular strain and most likely cardiac failure. We speculate that these mice die from an acute arrythmia secondary to hypertrophic cardiomyopathy.
B. Caveolin-2
Caveolin-2 null mice were generated by targeted disruption of exons 1 and 2 of the murine Cav-2 gene (199). Initial
characterization of these mice demonstrated unequivocally
that caveolin-2 is not necessary for caveolae formation or
the proper membrane localization of caveolin-1. In addition,
analysis of several phenotypes described in the Cav-1 null
mouse (i.e., vascular dysfunction and lipid imbalance) revealed that caveolin-2 plays no observable role in the pathogenesis of these abnormalities. However, Cav-2 null mice do
display marked lung pathology, similar to that described in
Cav-1 null mice (40, 198, 199). Because caveolin-2 expres-
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FIG. 16. Histopathological examination of
prostate specimens from 12-mo-old wild-type and
Cav-1 null male mice. Microscopic evaluation of
hematoxylin and eosin-stained prostate sections
reveals severe thickening of the smooth muscle
layer in Cav-1 null mice compared with wild-type
mice (arrowheads) at 12 mo of age. However,
there appears to be no histological change in the
prostate epithelial lining (arrows). Medium: reduced from ⫻16; High: reduced from ⫻31.5.
[Adapted from Woodman et al. (278).]
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
sion is drastically reduced in Cav-1 null mice, the identification of this phenotype in Cav-2 null animals directly implicates a selective loss of caveolin-2 as the primary cause of
these abnormalities.
1. Interstitial lung disease
A thorough histopathological examination of Cav-2 null
lung specimens revealed significant abnormalities with
marked alveolar hypercellularity (⬃2-fold increase in nuclei)
and septal thickening, compared with wild-type alveoli
(199). Further analysis revealed that the lung hypercellularity appeared to be due to an increase in the number of
vascular endothelial growth factor-receptor (VEGF-R) posiPhysiol Rev • VOL
tively stained endothelial cells, corresponding to an approximately three- to fivefold increase in endothelial cell number.
Also contributing to the noticeable septal thickening observed in these mice was as increase in extracellular matrix
deposition as revealed by reticulin staining of basement
membrane components (199). It should be noted that the
exact composition of this matrix is variable, as Drab et al.
(40) found significant fibrosis (as identified by trichrome
staining) in Cav-1 null lungs during their description of this
phenotype. Regardless, one measurable physiological consequence of these pulmonary abnormalities was severe exercise intolerance, as manifested by the early onset of exhaustion in Cav-1 null mice during a swimming test.
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FIG. 17. Histological and fundoscopic evaluation of wild-type and Cav-1
null mouse eyes. Top: paraffin-embedded
hematoxylin and eosin (H&E)-stained
sections of 12-mo-old wild-type and
Cav-1 null mouse retinas reveal no difference between the two genotypes and no
pathological changes, consistent with an
absence of retinal degeneration in Cav-1
null mice. Original magnification, ⫻20.
Middle: visualization of the fundus and
vasculature does not reveal any noticeable differences between wild-type and
Cav-1 null mice at 12 mo of age. Bottom:
similarly, fluorescein angiography of
mouse retinal vessels does not show any
changes in Cav-1 null mice, compared
with wild-type mice. Note the web of interlaced capillaries observed by this
method.
COHEN, HNASKO, SCHUBERT, AND LISANTI
These pathological abnormalities are reminiscent
of the interstitial lung diseases (ILDs) of humans,
which, although due to a multitude of disorders, are all
similarly characterized by progressive, irreversible fibrosis and severely compromised gas exchange (150).
Most ILDs are caused by the inhalation of known
agents, such as silica or coal dust pneumoconiosis, or
occur secondary to systemic disorders, such as collagen vascular disease or sarcoidosis (23, 61, 248). However, a subset of ILDs remain idiopathic, or of unknown
origin, and even a few of these have been found to
cluster in families, suggestive of a genetic defect (283).
The clinical course of idiopathic pulmonary fibrosis
(IPF) is relatively variable; however, most patients
progress to end-stage severe pulmonary fibrosis and
death despite current therapy (54). Thus the Cav-2 null
mouse may serve as a useful model organism to enhance further study of this disease process.
C. Caveolin-3
1. Specialized function: a muscle-specific isoform
Following the initial identification of caveolae in epithelial cells, several ultrastructural studies quickly identified abundant caveolae in striated muscle tissue, where
they were originally thought to play a role in the formation of the T-tubule system (interconnected transverse
membrane pockets that penetrate into the muscle fibers)
(11, 102). With the discovery of the muscle-specific caveolin family member caveolin-3, the role of this protein in
muscle function could finally be evaluated (256, 272).
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Caveolin-3 is most closely related to caveolin-1, sharing
65% identity and 85% similarity. However, unlike caveolin-1,
the expression of caveolin-3 is restricted to muscular tissue
where it appears to be the exclusive caveolin member
present (except in smooth muscle where all three caveolin
family members are present) (256, 278). Similarly to caveolin-1, the sole expression of caveolin-3 has been shown to be
sufficient to drive the formation of morphologically identifiable caveolae in cells (125). During the differentiation of
skeletal myoblasts in culture, the levels of caveolin-3 mRNA
and protein increase dramatically (242). Furthermore, antisense-mediated downregulation of caveolin-3 is sufficient to
inhibit myotube fusion in vitro (69), suggesting that caveolin-3 expression is intimately linked to proper myocyte development. Caveolin-3 is transiently associated with the Ttubule system during early development, whereas in mature
myocytes it is localized to caveolae of the muscle cell
plasma membrane (sarcolemma) (189, 242).
Numerous studies have shown that the functional role
of caveolin-3 in muscle cells is similar to that of caveolin-1 in
other cell types. For instance, with regard to signal transduction, an analogous cohort of signaling molecules are
known to localize to caveolin-3 generated caveolae, including NOS isoforms, various protein kinase C isoforms, ␣- and
␤-adrenergic receptors, and Src-family tyrosine kinases (55,
207, 208, 266). In addition to their role in sequestering signaling molecules, it also appears that muscle cell caveolae
and caveolin-3 are important modulators of dystrophin-glycoprotein complex function, abnormalities of which are associated with a variety of human muscle diseases (242).
2. Pathogenesis of muscular dystrophy
The term muscular dystrophy (MD) refers to a broad
range of phenotypically similar inherited myopathies
characterized by progressive muscle degeneration and
replacement with fibrous connective tissue. The prognosis of the disease is determined by the type of MD, as is
the progression and severity of muscle weakening. The
most common and severe form of MD is Duchenne
(DMD), an X-linked (Xp21) recessive disease afflicting 1
in 3,500 young males, resulting in morbidity by early
adulthood from respiratory failure (45). DMD is caused by
a variety of mutations in the open reading frame of the
dystrophin gene, resulting in a deficiency of the protein
product and loss of function of the dystrophin-glycoprotein complex (DGC) (96, 97). The DGC is composed of
several protein components, including the dystroglycans
and the sarcoglycans, and spans the muscle sarcolemma,
linking the cortical cytoskeleton with the extracellular
matrix (96, 97, 227).
Dystrophin, as well as several members of the DGC
including ␣-sarcoglycan and ␤-dystroglycan, cofractionate with caveolin-3 in cultured mouse C2C12 myocytes
(242). In addition, coimmunoprecipitation experiments
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FIG. 18. Survival curves for wild-type, heterozygous, and Cav-1 null
mice. Following a cohort of 180 mice for 2 years revealed that loss of
caveolin-1 confers a significant survival disadvantage (an ⬃50% reduction in life span) for both male and female Cav-1 null (KO) mice,
compared with wild-type (WT) mice. This difference was most noticeable between 27 and 65 wk of age. Note that complete absence of
caveolin-1 is necessary for this phenotype, as heterozygous (Het) mice
did not show any changes in longevity. [Adapted from Park et al. (182).]
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ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
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in the intracellular retention of both wild-type and mutant
caveolin-3 proteins at the level of the Golgi and leads to their
ubiquitination and proteasomal degradation (71, 72). More
recently, a novel missense mutation (A45T) in the NH2terminal domain of caveolin-3 has been identified in a patient with LGMD-1C (92). In contrast to the mutations discussed above, which occur within a highly conserved portion of caveolin-3, the A45T mutation does not. However,
this mutation is also thought to result in the production of a
dominant negative caveolin-3 protein, leading to similar retention and degradation of wild-type caveolin-3. Of interest,
loss of caveolin-3 expression in skeletal muscle leads to a
decrease in the expression of nNOS as well as ␣-dystroglycan, while ␤-dystroglycan levels remain unaffected (92).
Additional studies have shown that dysferlin, a skeletal muscle membrane protein whose deficiency causes
distal and proximal recessively inherited forms of Miyoshi
myopathy (MM) and limb-girdle muscular dystrophy type
2B (LGMD-2B), is mislocalized in skeletal muscle biopsies
taken from LGMD-1C patients. Dysferlin contains caveolin-3 binding motifs and coimmunoprecipitates with
caveolin-3 from normal skeletal muscle, suggesting that
its interaction with caveolin-3 may serve an important
function that remains undetermined (148).
Patients with LGMD-1C present with moderate proximal muscle weakness, with muscle cramps, calf-hypertrophy, and elevated serum creatine kinase (CK) levels, which
are typical of muscle pathology (154). Elevated levels of
serum CK (hyperCKemia) without muscle weakness have
been linked to sporadic CAV-3 gene mutations that result in
reduced caveolin-3 protein levels in skeletal muscle. Therefore, idiopathic hyperCKemia may be indicative of a partial
caveolin-3 deficiency in skeletal muscle (19, 153).
Rippling muscle disease (RMD) is a relatively benign
myopathy, first described in 1975, characterized by
stretch-induced muscle contractions which spread to
neighboring muscle fibers and give the appearance of
ripples moving over the muscle (261, 284). Generally
showing an autosomal dominant pattern of inheritance,
RMD was originally thought to be caused by alterations in
sarcoplasmic reticulum calcium homeostasis (261). However, using linkage analysis and positional cloning to identify the gene responsible for the pathogenesis of RMD in
a large cohort of German families, Betz et al. (5) discovered that four previously identified missense mutations
(R26Q, A45T, A45V, and P104L) in the CAV-3 gene on
chromosome 3p25 were responsible for this phenotype.
Specifically, the R26Q has been identified in patients with
asymptomatic hyperCKemia, while the other mutations
are associated with LGMD-1C (19).
Mutations in the CAV-3 gene provide an explanation
for the allelism identified in the dystrophic (LGMD-1C)
and nondystrophic (hyperCKemia and RMD) muscle pathologies (Table 5). Recently, Sotgia and co-workers (244,
246) addressed this issue directly, examining the behavior
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have shown that dystrophin forms a stable complex with
caveolin-3 and that dystrophin and caveolin-3 colocalize
by immunofluorescence microscopy in myocytes. The
WW-like domain within caveolin-3 directly interacts with
the COOH terminus of ␤-dystroglycan, a region containing
a PPXY motif (245). Because dystrophin also contains a
WW domain, it is thought that caveolin-3 can competitively inhibit dystrophin binding and can consequently
alter the recruitment of dystrophin to the sarcolemma. In
patients suffering from DMD, muscle biopsies show that
there is both an upregulation of caveolin-3 expression as
well as an increase in the number and size of caveolae at
the sarcolemma (11, 201). This is consistent with the
dystrophin-deficient mdx mouse, which has a similar phenotypic increase in the number of caveolae with elevated
levels of the caveolin-3 protein in skeletal muscle (262).
While these studies provided preliminary suggestive
evidence that changes in caveolin-3 expression might be
linked to the pathogenesis of DMD, it remained unknown
whether this was indeed the case. To address this issue
directly, Galbiati et al. (68) used a transgenic approach to
broadly overexpress caveolin-3 in mice. Pathophysiological
examination of these mice revealed that they exhibit a DMDlike phenotype as early as 3 wk of age, characterized by 1)
a dramatic increase in the number of skeletal muscle caveolae; 2) numerous hypertrophic, immature, and necrotic muscle fibers; 3) a preponderance of connective tissue infiltrates
in skeletal muscle; 4) a near-compete loss of skeletal muscle
dystrophin; and 5) a dramatic (⬃4-fold) reduction in the
expression of skeletal muscle ␤-dystroglycan. These
changes were accompanied by elevations in serum creatine
kinase levels, indicative of and consistent with the observed
myonecrosis (68). Although this study provided strong support for the role of strictly regulated caveolin-3 expression in
the pathogenesis of MD, it was not until the identification of
two mutations in the human CAV-3 gene resulting in an
autosomal-dominant form of limb-girdle MD (LGMD-1C),
that the specific role of caveolin-3 in muscular pathogenesis
emerged (152, 155).
Limb-girdle MD (LGMD) is a group of hereditary myopathies, including autosomal dominant and recessive forms
that are clinically and genetically heterogeneous. Several
autosomal dominant forms of LGMD have been recognized,
including 1) LGMD-1A, linked to chromosome 5q; 2) LGMD1B, associated with cardiac defects and linked to chromosome 1q11–21; and 3) LGMD-1C, linked to a missense mutation (P104L) in the membrane-spanning region and a 9-bp
in-frame deletion in the CAV-3 gene that removes residues
63– 65 (⌬TFT) in the scaffolding domain. These two mutations result in similar histopathological abnormalities, including a severe (⬃95%) reduction of caveolin-3 expression
in muscle tissue and a concomitant loss of sarcolemmal
caveolae (154, 155). The LGMD-1C caveolin-3 mutant protein acts in a dominant-negative manner by forming unstable
aggregates with the wild-type caveolin-3 protein. This results
COHEN, HNASKO, SCHUBERT, AND LISANTI
5. Disease-related mutations in the human
caveolin-3 (CAV-3) gene
TABLE
MUT CAV-3
LGMD-1C
RMD
hyperCKemia
P104L
⌬TFT63–65
G55S
C71W
A45T
A45V
R26Q
D27E
L86P
A92T
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
1369
(246). Thus the R26Q mutant does not behave in a dominant negative fashion.
Studies of the P104L mutant in a transgenic mouse
model have confirmed that this mutant results in a severe
myopathic phenotype, loss of caveolin-3 expression, and
identifiable sarcolemmal caveolae (251). In addition,
these mice show significant increases in skeletal NOS
activity, suggesting that nitric oxide may play a role in
muscle fiber degeneration.
3. Caveolin-3 null mice
of three mutants (P104L, ⌬TFT, and R26Q) in cultured
cells (see Fig. 19). In these studies, it was shown that both
the P104L and ⌬TFT mutants result in the formation of
unstable aggregates between wild-type and mutant caveolin-3 proteins that are targeted for proteasomal degradation. This leads to an ⬃95% reduction in caveolin-3 protein levels. On the other hand, overexpression of the R26Q
mutant did not reduce wild-type caveolin-3 levels and did
not effect the cellular distribution of the wild-type protein
FIG. 19. In L6 skeletal muscle myoblasts, pathogenic mutants of caveolin-3
(P104L, ⌬TFT, and R26Q) are all retained
in a perinuclear/Golgi-like compartment.
Caveolin-3 wild-type (WT) or mutant
cDNAs (R26Q, P104L, or ⌬TFT mutant)
were used to transiently transfect L6
cells. At 36 h posttransfection, the cells
were formaldehyde-fixed and immunostained with antibodies directed against
caveolin-3. Unlike caveolin-3 WT, which
localizes to the plasma membrane as expected, the R26Q, P104L, and ⌬TFT mutants were all retained intracellularly,
showing a perinuclear distribution pattern (see arrows). It is important to note
that L6 myoblasts do not express endogenous caveolin-3 (not shown). N, nucleus. [Adapted from Sotgia et al. (246).]
Physiol Rev • VOL
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MUT CAV-3, mutations in human caveolin-3 gene; LGMD, limb
girdle muscular dystrophy; RMD, rippling muscle disease; hyperCKemia,
elevated levels of serum creatine kinase.
The generation of Cav-3 null mice provided a means
by which to assess the functional role of genetic ablation
of this gene in a model organism. Characterization of
these mice by two independent groups showed that they
lack muscle cell caveolae yet maintain normal levels of
caveolin-1 and -2 expression as well as normal caveolae in
nonmuscle tissue (67, 86). Both groups found that Cav-3
null mice exhibit mild myopathic changes, similar to
those seen in patients with LGMD-1C. Muscle degeneration in Cav-3 null mice was recognized in the soleus
muscle as early as 8 wk of age, while degeneration was
noted in the diaphragm between 8 and 30 wk (67, 86).
However, no growth or movement differences were identifiable in Cav-3 null mice, even at the extremes of age. In
addition, heterozygous Cav-3 (⫹/⫺) mice did not demon-
1370
ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
4. Cardiomyopathy
Further characterization of Cav-3 null mice revealed
that they also develop a cardiomyopathic phenotype similar to that described above in Cav-1 null animals. Using
cardiac gated MRI and transthoracic echocardiography,
Woodman et al. (279) found that, at 4 mo of age, Cav-3
null mice showed significant cardiac hypertrophy and
reduced fractional shortening (279). Histopathological examination of cardiac sections revealed perivascular fibrosis, myocyte hypertrophy, and cellular infiltration. Mechanistically, these changes were characterized by an exclusion of the dystrophin-glycoprotein complex from cardiac
myocyte lipid rafts and hyperactivation of Ras-p42/44
MAP kinase cascade in Cav-3 null cardiac tissue. These
changes are consistent with the known role of p42/44
MAP kinase activation in cardiac myocyte hypertrophy
and the role of caveolin-3 as a negative regulator of the
Ras-p42/44 MAP kinase cascade, similar to that of caveolin-1 (28, 279).
Interestingly, in screening patients with hypertrophic
and dilated cardiomyopathies, Hayashi et al. (89) found
that a caveolin-3 mutation (T63S) was associated with
siblings with hypertrophic cardiomyopathy. This mutation occurs in the same position as the ⌬TFT deletion
associated with LGMD-1C (71). However, when expressed in cell culture, the T63S mutant does not reduce
the expression of wild-type caveolin-3 to the same extent
as the ⌬TFT mutant, suggesting that subtle changes in
caveolin-3 expression may result in a myriad of clinically
relevant phenotypes.
D. Caveolin-1/3
The generation of truly caveolae-deficient animals
was accomplished by interbreeding Cav-1 and Cav-3 null
mice, to produce caveolin-1/3 double knockout mice
(Cav-1/3 dKO). Surprisingly, although caveolae have been
implicated in a plethora of cellular functions, Cav-1/3 dKO
Physiol Rev • VOL
mice are viable and fertile, despite a complete absence of
morphologically identifiable caveolae in muscle and nonmuscle cells (184). Additionally, these mice are deficient
in the expression of all three caveolin family members, as
caveolin-2 is unstable and degraded in the absence of
caveolin-1. General phenotypic evaluation of these mice
revealed that a combined loss of caveolin protein expression did not produce any alterations in the phenotypes
previously identified in single knockout animals, with the
exception of the cardiac defects.
These mice exhibit a combined phenotype of the
individual caveolin null mice and present with a severe
cardiomyopathy. At 2 mo of age, these mice show more
pronounced increases in several parameters, including
left ventricular wall thickness and septal thickness, than
either the Cav-1 and Cav-3 null animals individually (184).
Histopathologically, cardiac tissue appears hypertrophic,
with areas of interstitial inflammation, perivascular fibrosis, and myocyte necrosis. Thus a combined loss of caveolin-1 and -3 has profound effects on cardiac structure and
function, a tissue that normally expresses an abundance
of both caveolin proteins, but in different cell types (cardiac fibroblasts vs. cardiac myocytes).
VI. CONCLUSIONS AND FUTURE DIRECTIONS
Curious plasma membrane-associated “little caves”
were first recognized over 50 years ago by electron microscopists and dubbed “caveolae.” Since this time, the
importance of these abundant organelles has proven both
thought provoking and elusive. Caveolae have remained
the focus of numerous research efforts to probe their
biochemical properties and to explore their functional
roles in various cellular processes.
With the cloning of the structural subunits of caveolae in the 1990s, the caveolin gene family emerged, finally
allowing researchers a biochemical tool through which
they could dissect caveolar function. A rapid succession
of publications implicated caveolae and caveolins in a
variety of important cellular processes, including vesicular trafficking, cholesterol homeostasis, and signal transduction. The identification of caveolins as oligomeric multivalent scaffolding proteins, responsible for the subcellular
regulation of numerous signaling molecules, led to the proposal of the “caveolae signaling hypothesis” and implicated
caveolins and caveolae in numerous disease processes. Ultrastructural, genetic, and molecular biological analysis of
caveolae and caveolins in cell culture systems provided
convincing evidence that these components were indeed
involved in the pathogenesis of diseases, as disparate as
muscular dystrophy, cancer, and diabetes. Thus it was with
great enthusiasm that the development of caveolin-deficient
mice was received, as these whole animal models provide
the first opportunity to examine the role of caveolins and
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strate any noticeable muscle pathology, indicating that
this phenotype was inherited in a recessive manner (86).
These findings stand in contrast to those observed clinically, where LGMD-1C has been shown to be inherited in
a dominant-negative manner (155).
Biochemical and ultrastructural analysis of skeletal
muscle samples from Cav-3 null mice showed that the
dystrophin-glycoprotein complex no longer associated
with lipid rafts and that the T-tubule system was markedly
disorganized, containing dilated and abnormally oriented
tubules (67). Thus, unlike the severely myopathic transgenic P104L mutant mouse discussed above, a complete
ablation of caveolin-3 produces a much milder phenotype,
suggesting that an expressed dominant negative caveolin-3
mutant effects more than just caveolin-3 protein levels (251).
COHEN, HNASKO, SCHUBERT, AND LISANTI
Physiol Rev • VOL
debated topic; however, findings in Cav-1 null mice
strongly indicate that insulin signaling is directly relayed
via caveolae. Furthermore, the now dubious role of adipose tissue in whole body insulin resistance is supported
by findings in Cav-1 null mice showing a ⬃90% reduction
in insulin receptor levels in adipocytes, without severe
whole body insulin resistance, similar to findings reported
in an adipose-tissue specific insulin receptor knockout
(FIRKO) mouse (9, 30). It is through such studies that the
true contribution of each insulin-responsive tissue to the
diabetic phenotype can be assessed.
In addition, analysis of the various Cav-null mice has
greatly expanded the current repertoire of diseases
thought to be influenced by these proteins. Indeed, caveolin-1 has now been implicated in urinary tract function, a
potential pathogenic component of both urinary calculi
formation and LUTD, diseases that cause immense morbidity in the human population (17, 278). Similarly, caveolin-2 may be involved in the pathogenesis of idiopathic
pulmonary fibrosis (IPF), and thus the Cav-2 null mouse
may serve as an important model for this disease (199).
Cav-3 null mice have served as a “proof of principle”
showing that caveolin-3 is indeed important for muscular
dystrophy, while also enhancing our understanding of the
mechanisms by which mutations in CAV-3 may lead to
muscle-related diseases (67, 86).
Undoubtedly, the next step in the process of understanding the role of the various caveolin family members
in human disease is to apply what we have learned from
the murine models to the human population. This has
already occurred to some extent, as identification of the
hypertrophic cardiac phenotype in Cav-3 null mice was
rapidly followed by the identification of a mutation in
CAV-3 causing hypertrophic cardiomyopathy in humans
(89, 175, 279). In terms of caveolin-1, mutations in this
gene have been found in a variety of human cancers,
while no reports have been published identifying caveolin-1 mutations in other human diseases, such as diabetes
and cardiomyopathy. Therefore, it will be necessary to
examine other relevant human populations, to identify
genetic mutations in caveolin family members, and thus
potentially provide a molecular basis for the many human
diseases that still remain idiopathic in nature.
Additionally, while the generation of the various
caveolin-deficient mice has proven extremely useful in
furthering our understanding of the role of these proteins
in a variety of diseases, these model systems are inherently complex, in part, because the gene of interest is
eliminated in the entire organism. This fact may partially
obscure the nature of a given phenotype and the particular contribution of the cell type(s) involved. For instance,
in studies regarding the role of caveolin-1 in the pathogenesis of breast cancer, it is difficult to distinguish the
contribution of mammary epithelial cells from that of
mammary adipocytes, both cell types that normally ex-
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caveolae in an intact living organism. To date, the Cav-null
mice have proven powerful tools in our understanding of
caveolae/caveolins and have both provided fundamental
support for previous hypotheses as well as given cause to
reassess prior suppositions.
Despite the ablation of an entire cellular organelle following the deletion of either caveolin-1 or caveolin-3, it is
rather surprising that all of the Cav-null mice are viable and
fertile, including the complete “caveolae-less mouse” (Cav1/3 dKO). These findings suggest that, like so many other
disease-related genes, caveolins are not essential for life and
that, indeed, mutations in the genes encoding the caveolins
may be relevant to the pathogenesis of human disease.
It was similarly surprising to find that, although experiments in cell culture have demonstrated an intricate
role for caveolae and caveolins in cholesterol trafficking,
no abnormalities in plasma cholesterol levels were observed in Cav-1 null mice (197). However, when Cav-1 null
mice were interbred with ApoE null mice, creating ApoE/
Cav-1 dKO mice, Frank et al. (62) found noticeably higher
plasma cholesterol levels in these mice than in the single
null ApoE mice. These findings illustrate certain discrepancies observed between the properties of a protein in
vitro compared with that observed in the context of a
whole animal model. For instance, techniques involving
ablation of a gene in vivo are confounded by inherent
variables such as unknown compensatory mechanisms
that exist in the setting of an entire organism but may not
exist in cell culture models. In the case of cholesterol
homeostasis, it may be that cholesterol and fatty acid
transport proteins are upregulated in response to caveolin-1 ablation, thus accounting for the limited changes
observed in Cav-1 null mice in this regard. Furthermore,
these findings exemplify the unpredictable nature of physiological ablation of a gene and the subtle phenotypic
variations that can only be deciphered when the model is
challenged with a particular stressor.
Indeed, while a plethora of cell culture data indicate
a predominant role for caveolin-1 in a variety of cancers,
initial evaluation of Cav-1 null mice showed no increased
incidence of spontaneous tumor formation. Subsequent
challenges of these mice with further carcinogenic “stressors” (i.e., crossing the mice with MMTV-PyMT mice or by
applying the carcinogen DMBA) increased their tumor
incidence and showed that disruption of caveolin-1, in the
context of additional tumor-promoting perturbations, facilitates the progression of cancer (18, 274). Thus it appears that loss of caveolin-1, like many other cancerrelated genes, works in concert with coincident genetic
mutations to enhance cell survival and accelerate tumor
growth (a phenomenon known as cooperativity).
Analysis of Cav-1 null mice has also helped to further
our understanding of insulin signaling and the pathogenesis of type II diabetes. Whether functional insulin signaling is truly dependent on intact caveolae has been a hotly
1371
1372
TABLE
ROLE OF CAVEOLAE AND CAVEOLINS IN HEALTH AND DISEASE
6.
Modifiers of raft function (MORFs)
Protein
References
Caveolin (⫺1, ⫺2, ⫺3)
Flotillin (⫺1, ⫺2)
MAL/BENE
VIP36
LAT/PAG
Stomatin
116
6, 269
38
57
98
210, 240
We are gratefully indebted to Dr. Paul Latkany (Assistant
Professor, New York Medical College, Codirector of Uveitis,
New York Eye and Ear Hospital, New York, NY) for help in
evaluating the eyes of these mice.
This work was supported by grants from the National
Institutes of Health and the Susan G. Komen Breast Cancer
Foundation (to M. P. Lisanti). A. W. Cohen was supported by
National Institutes of Health Medical Scientist Training Grant
T32-GM-07288. M. P. Lisanti is the recipient of a Hirschl/WeilCaulier Career Scientist Award.
Address for reprint requests and other correspondence: M. P.
Lisanti, Dept. of Molecular Pharmacology and the Albert Einstein
Cancer Center, Albert Einstein College of Medicine, 1300 Morris
Park Ave., Bronx, NY 10461 (E-mail: [email protected]).
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