Physiol Rev
83: 673– 686, 2003; 10.1152/physrev.00033.2002.
Functions of Pulmonary Epithelial Integrins:
From Development to Disease
DEAN SHEPPARD
Lung Biology Center, Department of Medicine, University of California, San Francisco, California
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I. Introduction to Integrins
A. Integrins as adhesion receptors
B. Integrins as signaling receptors
C. Integrins as components of mechanotransducers
II. Pattern of Integrin Expression on Airway Epithelial Cells
III. Role(s) of Integrins in Repair of Wounded Epithelia
A. Integrins can serve as both positive and negative regulators of cell proliferation
B. Regulation of epithelial cell survival
IV. Clues From the Extracellular Matrix in Establishing and Responding to Epithelial Cell Polarity
V. Integrins as Receptors for Respiratory Pathogens
A. Viruses
B. Bacteria
VI. Role(s) of Integrins in Epithelial Neoplasia
VII. Specialized Roles of Specific Integrins in Lung and Airway Epithelium
A. ␣9␤1
B. ␣v␤6
C. ␣v␤8
VIII. Conclusions/Future Directions
Sheppard, Dean. Functions of Pulmonary Epithelial Integrins: From Development to Disease. Physiol Rev 83:
673– 686, 2003; 10.1152/physrev.00033.2002.—Signals from integrins are now known to play critical roles in virtually
every aspect of the behavior of epithelial cells, including survival, proliferation, maintenance of polarity, secretory
differentiation, and malignant transformation. The cells that line the conducting airways and alveoli of the lung, like
most surface epithelia, simultaneously express multiple members of the integrin family, including several with
broadly overlapping ligand binding specificities. Although multiple integrins on airway epithelial cells may support
adhesion to the same ligands, the functional roles of each integrin that has been examined in detail are quite distinct.
Findings from mice expressing null mutations of some of these integrins have identified roles for epithelial cells and
epithelial integrins in lung development and in the regulation of lung inflammation, macrophage protease expression,
pulmonary fibrosis, and the pulmonary edema that follows acute lung injury. Epithelial integrins are thus attractive
targets for intervention in a number of common lung disorders.
I. INTRODUCTION TO INTEGRINS
Integrins are a large family of heterodimeric transmembrane glycoproteins that were initially identified as
cation-dependent receptors for components of the extracellular matrix (50, 91, 92, 97) and as antigens that increased after prolonged activation of lymphocytes (41–
43). The first members of the family were identified biochemically in the mid 1980s by affinity chromatography
passing lysates of surface-labeled cells over Sepharose
beads cross-linked to the adhesive ligands fibronectin
(92) or vitronectin (91) and named the fibronectin recepwww.prv.org
tor and the vitronectin receptor, respectively. Demonstration that cells could utilize these receptors to specifically
attach to each of these substrates led to initial hopes that
a similar approach might identify a series of individual
integrins that would each allow cells to specifically attach
to distinct biochemical components of the extracellular
matrix. Over the subsequent decade, a combination of
biochemical approaches and molecular cloning strategies
succeeded in identifying a modest-sized protein family
that does allow cells to attach to a wide variety of components of the extracellular matrix. Each integrin is composed of a single ␣-subunit and a single ␤-subunit, and
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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DEAN SHEPPARD
from studies of the effects of inactivation of individual
integrin subunit genes in mice (52, 101). Most of the
integrin subunits have now been inactivated in mice, and
the phenotypes of the resultant animals have been distinct
and sometimes quite surprising. Lessons from integrin
knock-out mice have been extensively reviewed elsewhere (52, 101), but specific results directly relevant to
the roles of integrins in pulmonary epithelial cells will be
alluded to throughout this review.
Recently, the first crystal structure for the extracellular domains of an intact integrin has been solved (129,
130). The structure demonstrates the presence of a cation-binding site in an exposed face of the ␤-subunit that
coordinates all but one of the free sites on bound cation,
leaving a free coordination site to interact with a negatively charged residue in integrin ligands. This finding
helps to explain the presence of a negatively charged
amino acid within the short peptide sequences that have
been identified as critical for integrin binding to ligands
(e.g., the aspartic acid residue in the arginine-glycineaspartic acid sequence recognized by a substantial subset
of integrins). The close apposition of this site to the
adjacent exposed face of the ␣-subunit helps to explain
how the combined effects of both subunits can influence
ligand binding specificity. A subset of integrin ␣-subunits
also contains an additional inserted domain (I domain)
that extends from the ␣-subunit face and can also directly
interact with ligands. The crystal structure of a subset of
isolated I domains has also been solved and contains a
similar cation-coordinating motif (69).
A. Integrins as Adhesion Receptors
FIG. 1. The integrin family. Solid lines indicate ␣,␤ pairings that
have been identified to date on mammalian cells. Since, despite near
completion of the human and murine genomes, no new integrin subunit
has been identified in the past 2 years, it is likely that this subunit list is
near complete. Additional heterodimeric pairings may remain to be
identified.
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The first members of the integrin family were identified through efforts to find the receptors that mediate
adhesion to specific components of the extracellular matrix (50, 97). The initial conception of adhesion receptors,
and therefore of integrins, was as a form of cellular glue
that contributes to the integrity of multicellular tissues
and organisms. This core function of integrins does seem
to be an important one, since it is shared across evolution
from primitive organisms. Indeed, the two integrins
present in C. elegans appear to mediate adhesion to two
classes of extracellular matrix proteins (laminins and proteins that share the tripeptide recognition motif arginineglycine-aspartic acid, Ref. 53) and homologs of these C.
elegans integrins are present in most higher organisims.
Furthermore, the phenotypes of mutations in C. elegans
integrins involve a defect in muscle contraction that could
be explained by a defect in adhesion. Because of this
history, one of the first experiments done after identification of each of the members of the family shown in Figure
1 has been cell adhesion assays, usually beginning with
assays of adhesion to culture plates coated with specific
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once the nucleotide sequence of a few integrins was
solved, it became apparent that ␣-subunits and ␤-subunits
each constitute a distinct gene family. Both families have
been highly conserved during evolution, with obvious
orthologs in Caenorhabditis elegans and Drosophila (53).
Thus some core functions of these receptors play critical
roles in processes that are shared by all multicellular
organisms.
The identification of regions of very high sequence
conservation among integrin ␣-subunits and ␤-subunits
made this family one of the earliest targets for gene
discovery by homology-based polymerase chain reaction
amplification (27, 93, 102). Intense application of this
approach and traditional methods of purification, amino
acid sequencing, and library screening led to the identification of 18 human ␣-subunits and 8 ␤-subunits that can
form a total of 24 integrin heterodimers (see Fig. 1). The
success of these approaches is supported by the absence
of any reports identifying additional integrin subunits
from the nearly completed human or murine genome
sequences.
As noted above, the high sequence conservation
among ␣-subunits and ␤-subunits across a broad range of
species strongly suggests the existence of core integrin
functions that have been retained through millions of
years of evolution (53). However, the number of integrin
subunits and heterodimers has dramatically expanded in
higher organisms, with only 2 integrins in C. elegans and
at least 24 in humans. It is thus likely that this family of
receptors has been enlisted to carry out a range of specialized functions in complex organisms such as mammals. This hypothesis has received additional support
PULMONARY EPITHELIAL INTEGRINS
matrix components. This approach, combined with affinity chromatography (92) or other cell-free methods to
demonstrate direct binding of integins to putative ligands,
has been quite successful in identifying members of this
family that bind to nearly every major protein component
of the extracellular matrix.
B. Integrins as Signaling Receptors
can direct divergent cellular responses to a single ligand
and that specific integrins perform different functions in
different cells. Epithelial cells, in particular, perform a
number of in vivo functions that are tightly regulated by
integrins (100, 123). These include wound repair, establishment of polarity, differentiation into specialized secretory cells, and modulation of local inflammation. In this
review, I will draw heavily on studies of integrins in the
airway epithelium, skin, and mammary gland to review
some of the emerging information about the functions of
integrins on epithelial cells in general and on airway
epithelial cells in particular.
It is now clear that integrins do not themselves contain any catalytic activity and are thus unable to independently initiate signaling cascades. Rather, the short cytoplasmic domains of integrin ␣- and ␤-subunits serve as
scaffolds for the assembly of multiprotein signaling complexes (see Fig. 2). In this regard, they function in similar
fashion to T-cell and B-cell receptors for antigen. The
integrins themselves serve as the extracellular detectors
in these complexes. The recent solution of the crystal
structure of the integrin ␣v␤3 (129, 130) demonstrates a
potential basis for signal initiation, with a massive change
in conformation suggested when the integrin is occupied
by ligand. Although crystals have thus far been obtained
only with truncated integrins composed of ␣- and ␤-subunit extracellular domains, modeling suggests that this
conformational change would result in a substantial shift
in the orientation of the cytoplasmic domains. Such a
change is likely to be the basis for signals initiated (or
repressed) by integrin ligation.
The list of other proteins contained in integrin signaling complexes is large and rapidly growing (35, 76).
One key protein in these complexes is the focal adhesion
kinase (FAK) (65). FAK is a 125-kDa protein that binds to
integrin ␤-subunit cytoplasmic domains and to multiple
other components of focal adhesions and is highly con-
FIG. 2. Simplified model of integrin signaling complexes. The model depicts the types of components that
are organized by integrins at points of close apposition
to integrin ligands. These include kinases, such as src
family members, dual kinase/adaptor proteins, such as
the focal adhesion kinase (FAK), scaffolding proteins,
such as paxillin, and links to the actin cytoskeleton, such
as ␣-actinin and vinculin.
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In vitro, integrins have been shown to mediate cell
adhesion to a wide variety of extracellular matrix proteins, to cell surface counter-receptors, including members of the immunoglobulin and cadherin families (15),
and to members of several other protein families, including growth factors (78) and proteases (8). This diverse
repertoire of ligands made it apparent a number of years
ago that integrins were not likely to be limited to mediating cell adhesion. Over the past decade, thousands of
studies performed in cultured cells and whole animals
have made it clear that integrins play central roles in
modulating virtually every aspect of cell behavior, including migration, establishment of polarity, growth, survival,
and differentiation (18, 35, 51). Furthermore, integrin ligation can have profound effects on expression of a number of other genes (22, 98), including those encoding
metalloproteinases (127), milk proteins (110), and cytokines (75). Alveolar epithelial cells increase their expression of the gap junction protein connexin 43 when they
are plated on fibronectin (3), an effect that is presumably
due to integrin ligation.
Members of the integrin family are expressed in virtually every cell of most multicellular organisms. In adult
mammals, most cells constitutively express multiple integrins (21, 74, 85, 107) and simultaneously express more
than one receptor for the same ligand. In addition, the
same integrins are often expressed on cells with markedly
divergent functions. It is thus clear that different integrins
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C. Integrins as Components
of Mechanotransducers
In addition to organizing signaling complexes, integrin cytoplasmic domains bind directly to adaptor proteins that themselves bind directly to cellular actin. In this
fashion, integrins are ideally positioned to connect
changes in cell shape or mechanical stresses applied to
cells to initiation of biochemical signals that modify cell
behavior (2, 121). It is now clear that many (most?) cells
can dramatically change their behavior in response to
mechanical deformation. In virtually every case, these
responses can be attenuated or abolished by blocking one
or more integrin. However, because integrins are required
to maintain firm substrate adhesion, and because substrate adhesion itself clearly modulates the direct mechanical effects of deforming extracellular forces, it is not
simple to determine whether these effects of integrin
blockade reflect direct roles for integrins in mechanotransduction or secondary roles in allowing other receptors or channels to be subjected to deformation (33).
II. PATTERN OF INTEGRIN EXPRESSION
ON AIRWAY EPITHELIAL CELLS
Although specialized epithelia express unique integrin repertoires, there are broad similarities in the pattern
of integrin expression in most surface epithelia. Several
groups have characterized the expression of integrins on
the epithelial cells that line the conducting airways (see
Fig. 3) and alveolar spaces of the lung. Integrin expression
is dynamically regulated during lung development (19,
20). At least seven different integrins (␣2␤1, ␣3␤1, ␣6␤4,
␣9␤1, ␣v␤5, ␣v␤6, and ␣v␤8) are expressed on airway
epithelial cells of healthy adults (11, 21, 26, 74, 86, 87,
FIG. 3. Cartoon demonstrations of the changes
in the level and distribution of expression of integrins in the airway epithelium in response to injury. Normal, uninjured epithelium is shown on
the left, and a theoretical site of denudation is to
the far right. Levels of expression are depicted by
the size of the text characters corresponding to
individual integrins. Patterns of integrin expression in healthy and injured alveolar epithelial cells
have not been as thoroughly evaluated.
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centrated at these sites. FAK also has binding sites for the
SH2 domain of the ras-associated protein Grb2, the SH2
domains of src, phosphatidylinositol-3-kinase and phospholipase C-␥, and for a number of adaptor proteins that
can further expand the potential downstream targets of
integrin signaling (18). These include, for example, the
proteins crk-associated substrate (CAS) and paxillin,
which are themselves adaptors allowing binding of several other signaling intermediates. In this fashion, the
potential range of signaling pathways that can be modulated by integrin ligations is greatly expanded. Phosphorylation of FAK and activation of its kinase activity are
induced by interaction of multiple integrins with their
ligands. FAK phosphorylation and activity can also be
regulated by several growth factors (64), suggesting one
mechanism by which signals from growth factor receptors and integrins might be integrated (see below).
Additional transmembrane proteins are also components of integrin signaling complexes. These include a
large family of proteins (tetraspanins) that each contains
four membrane-spanning domains (40). These proteins
are emerging as organizers of transmembrane signaling
complexes, localizing a subset of integrins and other receptors to the same membrane sites. Tetraspanins bind to
integrins through interactions of their extracellular domains (133) but can also participate in initiation of integrin-dependent signals [e.g., protein kinase C activation
(140) or phosphorylation of lipid signaling intermediates
(132)] through distinct sites in their cytoplasmic domains.
Another transmembrane protein that participates in integrin signaling is the so-called integrin-associated protein
(IAP) (9, 72). IAP also interacts with a subset of integrins
(especially ␣2␤1 and ␣v␤3) through one of its extracellular domains (71) but can initiate distinct signals, including
activation of G proteins (29), through cytoplasmic sequences.
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PULMONARY EPITHELIAL INTEGRINS
TABLE
Surface epithelia all have the capacity to repair areas
of denudation. This repair process involves at least three
functional changes in the epithelial cells themselves:
spreading, migration, and proliferation. Each of these processes requires the participation of integrins. The effects
of wounding on the local expression of integrins and their
ligands have been most extensively studied in squamous
epithelia, such as the skin. Cutaneous wounds contain a
provisional matrix that is rich in the integrin ligands fibronectin, osteopontin, and tenascin. In response to epithelial injury, there are dramatic changes in the spatial
distribution and level of expression of epithelial integrins.
Expression of ␣2␤1, ␣3␤1, ␣5␤1, ␣6␤4, ␣v␤5, and ␣v␤6 is
Known Ligand(s)
Distribution
␣2␤1
␣3␤1
Collagen I (IV), tenascin C, echovirus
Laminins 5, 10, 11
␣6␤4
␣9␤1
Laminins 5, 10, 11
Tenascin C, osteopontin, VCAM-1, L1-CAM, vWF, factor
XIII, tissue transglutaminase, fibronectin EIIIA,
angiostatin, ADAMS 1,2,3,9, 15 (at least)
Fibronectin
Vitronectin, adenovirus, ostepontin
LAP of TGF-␤1, -3, fibronectin, tenascin C, osteopontin,
vitronectin, foot and mouth disease virus
LAP of TGF-␤1, -3, vitronectin
␣v␤8
III. ROLE(S) OF INTEGRINS IN REPAIR
OF WOUNDED EPITHELIA
1. Integrins on pulmonary epithelial cells
Integrin
␣5␤1
␣v␤5
␣v␤6
uncertain in light of other reports that have not been able
to substantiate significant roles for either integrin in homotypic interactions (116, 125, 126) and the absence of
any described defects in epithelial cell-cell interactions in
mice lacking either ␣3 (66) or ␣2 (45).
The other integrins that are expressed on basal airway epithelial cells, ␣5␤1, ␣9␤1 (28, 70, 103, 104, 114,
137), ␣v␤5, ␣v␤6 (10, 46, 78, 136), and ␣v␤8 (77, 81),
recognize a wide array of ligands that are not components
of healthy epithelial basement membranes. Many of the
ligands recognized by these integrins [e.g., fibronectin,
tenascin C, and osteopontin (10, 104, 136, 137)] are among
the most highly induced proteins at sites of epithelial
injury (61, 124, 139). Vitronectin, the best characterized
ligand for ␣v␤5, is principally a plasma protein and is thus
also likely to be enriched in the airways after injury or
other increases in vascular permeability. These receptors
are thus good candidates to serve as sensors that would
allow epithelial cells to rapidly detect and respond to the
changes in the extracellular matrix that accompany lung
and airway inflammation and injury.
Diffusely expressed; more intensely on basal cells
Diffusely expressed with highest level expression on
basal surface of basal cells
Restricted to basal surface of basal cells
Diffusely expressed, especially on basal cells
Diffusely expressed, but only after injury
Diffusely expressed, principally expressed on basal cells
Diffusely expressed on all cells (dramatically induced
by injury)
Diffusely expressed on basal cells
VCAM, vascular cell adhesion molecule; VWF, von Willebrand factor; LAP, latency-associated peptide; TGF, transforming growth factor.
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124). Although the original “fibronectin receptor” ␣5␤1 is
generally not seen on healthy adult airway epithelium in
vivo, this integrin is rapidly induced by injury in most
epithelia, including in the airways (87). The integrins expressed, and some of their known ligands, are listed in
Table 1. Of these, ␣3␤1 and ␣6␤4 are the only receptors
for matrix proteins known to be present in normal epithelial basement membranes (laminins 5, 10, and 11) (13,
25). However, even with respect to these integrins, the
spatial patterns of constitutive expression are distinct.
␣6␤4 is completely restricted to the basal surface of basal
cells, where it serves as a central component of hemidesmosomes (108). In this role, ␣6␤4 is critical for maintenance of epithelial integrity, since mice homozygous for
null mutations of either the ␣6- or the ␤4-subunit die soon
after birth with severe blistering of the skin (34, 119). The
identification of severe blistering in a human infant homozygous for a mutation of the ␤4-subunit provided further confirmation that ␣6␤4 plays the same role in humans (80). ␣3␤1 is concentrated at the basal surface, but
is also expressed at lower levels around the lateral and
apical surfaces of cells throughout the epithelium (unpublished observations). Mice lacking the ␣3-subunit have
defects in branching morphogenesis in the lung and kidney (66). In addition, these animals have dramatic defects
in the structural organization of epithelial basement membranes (23). This finding led to the identification of a
direct role for ␣3␤1 in organizing the basement membrane
into an ordered structure. Studies with isolated cells from
␣3 knock-out mice also demonstrated an important role
for ␣3␤1 in epithelial cell migration (44).
␣2␤1 may also interact with collagen IV present in
the basement membrane, but its preferred ligands are
other collagen isoforms, especially collagen 1 (62). The
diffuse surface expression of this integrin suggests other
functions, and possibly other biologically important ligands. It has been suggested that ␣2␤1 and ␣3␤1 play
roles in homotypic cell-cell interaction in epithelia (14,
106, 113). However, the significance of these findings is
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DEAN SHEPPARD
A. Integrins Can Serve as Both Positive and
Negative Regulators of Cell Proliferation
Integrins also play a critical role in regulating cell
proliferation (38, 39). Most adherent cells are incapable of
proliferating without signals from the extracellular matrix, signals that are transmitted by integrins. Integrins are
frequently enriched in membrane microdomains that are
also enriched for other cell surface receptors (e.g., growth
factor receptors) that contribute to cell proliferation.
These sites include structures called focal adhesions, regions of close apposition to the underlying matrix, that
are organized around links between integrins and the
ends of actin filaments and include a large number of
adaptor proteins, signaling kinases, and other components of intracellular signaling pathways (18, 35, 76, 89).
Although signals initiated by integrins have been shown
to enhance cell proliferation under in vitro conditions
without addition of exogenous, soluble growth factors, it
is likely that these results are explained in part by autologous production of growth factors by the cultured cells.
Ligation of integrins can activate several kinases known
to be activated by growth factor receptors, including src,
ras (99), and mitogen-activated protein kinases (17). A
specific subset of integrins (including ␣1␤1, ␣5␤1, and
␣v␤3) can induce or enhance cell proliferation through
interaction of the integrin ␣-subunit cytoplasmic domain
with caveolin-1, a membrane protein that plays a role in
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organizing membrane microdomains. In this pathway,
caveolin-1 recruits the src family kinases, yes or fyn,
which recruit the adaptor protein shc that in turn leads to
recruitment and activation of the well-characterized Ras
pathway (122).
Integrins can regulate both increases and decreases in cell proliferation. Several studies have suggested that overexpression of the fibronectin receptor
␣5␤1 results in inhibition of proliferation (36, 112, 120).
One mechanism by which such inhibition occurs has
been demonstrated in studies utilizing the colon carcinoma cell line HT-29, a cell line that normally does not
express this integrin. Heterologous expression of ␣5␤1
in these cells diminished their proliferative capacity, an
effect that appeared to be mediated by induction of the
growth arrest specific gene gas-1, a gene known to be
involved in the induction of cellular quiescence (120).
Interestingly, this effect was a consequence of expression of unligated integrin, since plating of transfected
cells on dishes coated with the ␣5␤1 ligand fibronectin
reversed gas-1 induction and growth inhibition. If a
similar pathway is operative in normal epithelial cells,
the combined effects of the growth-promoting role of
ligated integrin and the growth inhibitory role of unligated integrin would provide an elegant mechanism by
which cells in normal adult epithelia (which would not
be in contact with fibronectin) are kept out of the cell
cycle, but cells as sites of injury (where fibronectin in
greatly enriched) can be stimulated to proliferate.
The biological significance of integrin-mediated epithelial cell proliferation has been most extensively
examined in the skin (123). Basal keratinocytes in the
skin can be divided into three populations based on
analysis of proliferative capacity: stem cells, transitamplifying cells, and committed cells. Stem cells have a
high capacity for continued proliferation; transit-amplifying cells have the capacity to undergo a few rounds of
proliferation, but usually divide to form daughter cells
that permanently withdraw from the cell cycle and
undergo terminal differentiation; and committed cells
are already permanently withdrawn from the cell cycle.
Stem cells can be identified, and sorted in vitro, based
on higher levels of expression of ␤1-integrins (59, 60).
Furthermore, in committed cells, integrin function is
inhibited, and integrins are lost from the cell surface as
keratinocytes move upward away from the basement
membrane. The in vivo significance of these changes in
integrin expression and function is apparent from the
observation that expression of integrin ␣-subunit partners of ␤1 in the suprabasal cells of the epidermis of
transgenic mice produces a hyperproliferative phenotype reminiscent of the hyperproliferative skin disease
psoriasis (12).
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dramatically upregulated along the injured surface (6, 31,
68). Tightly regulated spatial and temporal patterns of
expression of each of these integrins have suggested that
each of them might play a unique role in orchestrating the
normal healing of injured epithelium. However, there
must also be substantial redundancy in this process, since
inactivation of single integrins [e.g., ␣v␤5 (47) or ␣v␤6] or
even two integrins simultaneously (e.g., ␣v␤5 and ␣v␤6;
unpublished observations) does not lead to significant
impairment in the rate or quality of cutaneous wound
healing.
The most careful study of the effects of airway epithelial wounding on integrin expression was performed
utilizing human bronchial grafts placed under the skin of
SCID mice (87). In this system, the pattern of integrin
expression seen in the absence of injury was quite similar
to the pattern seen in normal human airways. After injury,
the most prominent changes were upregulation of expression of ␣5␤1, ␣v␤5, and ␣v␤6 along the wounded edge. As
in cutaneous wounds, ␣2␤1 and ␣6-containing integrins
were diffusely expressed on cells above the basal layer.
The relevance of these findings to in vivo injury in humans
was confirmed by the observation that integrin expression in airways from patients with cystic fibrosis was
similar to that seen in the injured xenografts (87).
PULMONARY EPITHELIAL INTEGRINS
B. Regulation of Epithelial Cell Survival
IV. CLUES FROM THE EXTRACELLULAR
MATRIX IN ESTABLISHING AND
RESPONDING TO EPITHELIAL CELL
POLARITY
In vivo, surface epithelial cells, including those lining
the conducting airways and alveoli of the lung, are polarized and establish specialized structures along their basal,
lateral, and apical surfaces. The establishment of appropriate epithelial polarity requires input from integrins (84,
95, 105). In most epithelia, normal polarity is principally
dependent on interactions of integrins with the basement
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membrane constituent laminin (83, 105). Furthermore,
many of the normal differentiated functions of epithelial
cells cannot be induced in nonpolarized cultures (110,
118). Considerable insight into the mechanisms underlying establishment of polarity has come from studies of
mammary gland epithelial cells. In tissue culture, these
cells can be induced to form polarized glandlike structures with a central lumen by overlaying cultures with
epithelial derived basement membrane proteins, or with
purified laminin (79). Only under these circumstances will
mammary epithelial cells fully differentiate in response to
lactogenic hormones. These responses to laminin are mediated by input from both ␣3␤1 and ␣6␤4 integrins and
require input from nonintegrin receptors as well (79).
Renal epithelial cells [for example, Mardin-Darby canine kidney (MDCK) cells] can also be induced to form
polarized structures containing an apical epithelium facing a lumen if they are plated in a three-dimensional
culture environment. In this case, MDCK cells produce
their own laminin and organize it into a basement membrane along the basal surface utilizing the integrin ␣3␤1.
Recent studies utilizing inducible forms of the small GTPase
Rac1 have demonstrated a critical role for Rac1-induced
reorganization of the actin cytoskeleton in establishing
epithelial polarity (83). Although airway epithelial cells
have not been directly studied in as much detail, a similar
role for polarity in secretory cell differentialtion has been
demonstrated in serous cells derived from airway submucosal glands, which clearly require input from a ␤1-integrin and laminin to express their differentiated secretory
cell phenotype (118).
V. INTEGRINS AS RECEPTORS
FOR RESPIRATORY PATHOGENS
A. Viruses
Viruses and other microorganisms that infect mammals frequently utilize mammalian cell surface receptors
to attach to and enter host cells. Integrins are among the
important targets used for this purpose (56). At least three
of the integrins expressed on airway epithelial cells play
important roles in infection of these cells by viruses. ␣2␤1
serves as an Echovirus receptor, mediated by direct binding of proteins in the virus coat to the ligand-binding
insertional domain of the ␣2 subunit (4). ␣v␤5 is one of
several adenovirus receptors (128). In cultured cells,
␣v␤5 has been shown to mediate productive adenovirus
infection through its dual roles in facilitating viral internalization and subsequent escape from endosomes (128).
Areas of normal airway epithelium, where ␣v␤5 is generally not present on the luminal surface, are more resistant
to adenoviral infection and transduction with adenovirally encoded transgenes than are injured areas in which
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Nontransformed epithelial cells cannot survive in the
absence of anchorage to the extracellular matrix and die
by apoptosis soon after detachment, a process that has
been termed anoikis (30). This process, like the withdrawal of growth and survival factors from other primary
cells, is mediated, at least in part, by activation of a
cascade of caspase proteases that lead to rapid and efficient cell death. Epithelial cells are thus primed to activate a classical caspase-mediated execution program and
require input from ligated integrins to negatively regulate
this program. Anoikis likely plays the important role of
preventing detached epithelial cells in hollow organs like
the lung or gastrointestinal tract from reattaching at inappropriate sites. However, anoikis does not appear to be
a universal feature of all epithelia. For example, rather
than die in this absence of input from integrins, keratinocytes terminally differentiate and begin the process of
keratinization (123). In the mammary gland, where involution is a normal phenomenon that follows termination
of breast-feeding, apoptosis in the involuting gland is
associated with degradation of the stromal matrix by
metalloproteinases, a process that also presumably results in unligated integrins. Indeed, in this system, apoptosis can be induced by either antibodies to ␤1-integrins
or by overexpression of the matrix-degrading protease
stromelysin-1 in the absence of obvious cell detachment
(5). A similar process has been described for endothelial
cells (73), which could also cause serious problems if they
were capable of surviving detachment and reattachment.
In endothelial cells, one pathway for inducing rapid cell
death in response to unligated integrin involves direct
association of the cytoplasmic domain of the integrin
␤3-subunit and the initiator caspase caspase 8 (111). A
more general pathway appears to involve activation of a
caspase 8-mediated execution program that is downstream of activation of protein kinase A by unligated
integrins. In endothelial cells, individual unligated integrins can induce this apoptosis pathway, even in cells that
remain adherent with other integrins ligated (63).
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DEAN SHEPPARD
B. Bacteria
Integrins can also mediate adhesion and internalization of bacteria into epithelial cells. The best-described
interaction of bacteria with epithelial integrins involves
infection with the gastrointestinal pathogen Yersenia
pseudotuberculosis (54, 55). Yersenia expresses a protein
called invasin on its cell wall that serves as a specific
ligand for ␤1-integrins. Binding of invasin to integrins on
M cells in the gut epithelium is an absolute requirement
for uptake of the bacteria into these cells and plays a
central role in Listeria pathogenesis (54). The best-characterized role for interactions between integrins and bacteria in the respiratory epithelium involves the preferential attachment of the respiratory pathogen Pseudomonas
aeruginosa to sites of epithelial injury. In that case, attachment is not direct, but rather utilizes the extracellular
matrix protein fibronectin as a bridge (96). Fibronectin
binds to an as yet unidentified Pseudomonas surface protein and simultaneously fibronectin binds to ␣5␤1 on
epithelial cells. Because little ␣5␤1 is expressed on respiratory epithelial cells in the absence of injury, and little
integrin at all is expressed on the uninjured luminal surface, epithelial injury greatly enhances Pseudomonas attachment.
VII. SPECIALIZED ROLES OF SPECIFIC
INTEGRINS IN LUNG AND AIRWAY
EPITHELIUM
VI. ROLE(S) OF INTEGRINS IN EPITHELIAL
NEOPLASIA
Initial efforts to understand the roles that integrins
might play in the development of epithelial tumors involved descriptive immunohistochemistry. It soon became apparent, however, that the changes in integrin
protein expression in epithelial tumors are complex and
heterogeneous. As noted above, many primary epithelial
cells undergo apoptosis in response to loss of integrin
ligation, but this response is lost in most epithelial tumors, so carcinomas do not require input from integrins
Physiol Rev • VOL
for survival. Similarly, loss of anchorage dependence (i.e.,
integrin dependence) for growth is a common feature of
carcinomas. In their role in anchoring epithelial cells to
the normal basement membrane, integrins could also impede tumor cell migration and subsequent invasion and
metastasis. Because tumors can survive and grow without
integrins and may be impeded by integrins in invasion and
metastasis, it is not surprising that a general decrease in
integrin expression is seen in many invasive carcinomas.
However, because integrins can also enhance growth factor-mediated proliferation, provide traction for cell migration through the extracellular matrix, and localize matrixdegrading proteases to the leading edge of migrating cells
(8), it is also not surprising that many epithelial tumors
utilize integrins to enhance their growth and/or invasion.
In at least one case, the same integrin (␣6␤4) that restricts
movement of normal epithelial cells through anchorage of
hemidesmosomes is utilized by malignant epithelial cells
to support migration and invasion (16, 32, 94). In that
case, a key step is the relocalization of ␣6␤4 from
hemidesmosomes to the leading edge. In this case, tumor
cells are apparently taking advantage of a normal homeostatic mechanism through which ␣6␤4 is similarly relocalized to support migration of epithelial cells at the
leading edge of wounds.
The integrin ␣v␤6 also appears to modify malignant
transformation and enhance the growth and invasion of
epithelial tumors (131). In this case, the same integrin
affects carcinogenesis growth and invasion through distinct mechanisms. In the early stages of malignant transformation, the ability of this integrin to locally activate
transforming growth factor (TGF)-␤ (see below) would
be expected to inhibit carcinogenesis. However, independently of TGF-␤ activation, the cytoplasmic domain of the
␤6 subunit specifically supports tumor cell proliferation,
both in vivo and in vitro (1), and also induces expression
of the metalloprotease MMP-9 (82, 117), which enhances
tumor cell invasion. Thus this integrin plays distinct roles
at different steps in the process of malignant transformation and tumor growth and invasion.
A. ␣9␤1
The integrin ␣9-subunit was initially identified by
homology polymerase chain reaction amplification from
primary cultures of guinea pig airway epithelial cells (27).
This subunit is present in a single integrin, ␣9␤1, that is
diffusely expressed on the surface of basal airway epithelial cells (86). ␣9␤1 was first identified as a receptor for
the provisional matrix protein tenascin C, where it binds
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this integrin is expressed on the luminal surface of remaining basal cells (37). This finding led to the suggestion
that access to ␣v␤5 might be a critical determinant of
susceptibility to adenoviral infection and adenoviral gene
delivery. However, cells from the airways of ␤5 knock-out
mice are as susceptible to adenoviral infection as cells
from wild-type mice, demonstrating that the roles of this
integrin can be effectively performed by other cell surface
receptors. At least three integrins, ␣v␤3, ␣v␤6 (58), and
␣v␤1 (57), have been shown to be capable of supporting
infection by the foot and mouth disease virus that has
recently been the cause of an economically costly epidemic in the United Kingdom. Of these, ␣v␤6 is the only
one known to be expressed on the mucosal epithelial cells
that are the primary site of infection by this virus.
PULMONARY EPITHELIAL INTEGRINS
B. ␣v␤6
The integrin ␣v␤6 was initially identified as a receptor for the extracellular matrix proteins fibronectin (10)
and tenascin C (90, 136). This integrin is unusual in that its
Physiol Rev • VOL
expression is highly restricted to a subset of epithelial
cells, including the epithelium lining the conducting airways and alveoli (6, 7, 124). Studies with in situ hybridization and the first developed monoclonal antibodies that
specifically recognize this integrin suggested that the integrin was not expressed at these sites in healthy adults.
However, the development of better antibodies has made
it clear that ␣v␤6 is constitutively expressed at low levels
in uninjured epithelia. However, ␣v␤6 expression is markedly upregulated on epithelial cells in multiple epithelial
organs, including the lung, in response to injury and acute
and chronic inflammation (6, 124). One of the first functions identified for this integrin was enhancement of the
proliferation of carcinoma cells in three-dimensional cultures and in vivo in nude mice (1). Expression of ␣v␤6
also induces expression of the matrix-degrading protease
MMP-9 (82, 117). These effects are dependent on an 11amino acid carboxy-terminal sequence within the ␤6 cytoplasmic domain that is not present in other integrins (1,
24, 82). Because ␣v␤6 is commonly expressed in carcinomas, especially on cells adjacent to or invading the underlying stroma, ␣v␤6 might play an important role in the
growth, invasion, and/or spread of carcinomas derived
from lung epithelial cells.
Introduction of a null mutation of the ␤6 subunit in
mice identified additional functions for this integrin that
are likely to be even more important for normal lung
homeostasis and responses to lung injury. ␤6-Subunit
knock-out mice develop exaggerated inflammatory responses to injury in the lungs and skin (48) but are
protected from one of the usual consequences of lung
inflammation, pulmonary fibrosis (78). This phenotype
suggested a possible interaction between this integrin and
the growth factor TGF-␤1, since TGF-␤ is a known central
regulator of tissue fibrosis, but TGF-␤1 knock-out mice
also develop exaggerated inflammation (67). These observations led to identification of a direct interaction between the ␣v␤6 integrin and a region of TGF-␤1 called the
latency-associated peptide (LAP) (78). LAP, which is
formed by cleavage of the amino terminus of the TGF-␤1
gene product, normally forms a homodimer which noncovalently associates with a homodimer of the mature,
active TGF-␤ (the carboxy-terminal fragment of this same
cleavage reaction). In this form, LAP prevents mature
TGF-␤ from binding its receptors and inducing TGF-␤
effects. ␣v␤6 can bind LAP in latent TGF-␤1 and TGF-␤3
complexes and alter the confomation of the complexes,
effectively activating TGF-␤1. Importantly, binding of the
integrin is not sufficient to activate latent complexes, and
this process itself can be activated through a signaling
pathway in the integrin-expressing epithelial cell. Furthermore, the conformational change in the latent complex
does not appear to release free active TGF-␤, providing an
elegant mechanism to tightly regulate the activity of this
potent cytokine both in space and in time (Fig. 4A).
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to a novel sequence adjacent to an exposed loop that
contains the arginine-glycine-aspartic acid site that serves
as a ligand for at least five other integrins (134). It is now
clear that ␣9␤1 is a rather promiscuous integrin that can
bind to a large group of rather diverse ligands, including
the provisional matrix protein osteopontin (104, 135), an
alternatively spliced region of cellular fibronectin (70),
the coagulation protein factor XIII (114), the extracellular
enzyme tissue transglutaminase (114), the immunoglobulin family members vascular adhesion molecule-1 (115)
and L1-CAM (103), and several members of the ADAMs
family of transmembrane metalloproteinases (28). Because mice homozygous for a null mutation of the ␣9subunit die soon after birth from chylothorax (an apparent consequence of defective lymphatic development), it
has not been possible yet to utilize these mice to directly
examine the roles of ␣9␤1 and each of these ligands in the
adult lung or airways (49). Nonetheless, some clues are
provided by descriptive studies of the pattern of ␣9␤1
expression and by studies of its function in vitro on
cultured cells.
The observation that ␣9␤1 is generally expressed on
cells with the capacity to proliferate (e.g., the limbic cells
of the cornea and the basal cells of the conducting airways and skin), but that ␣9␤1 expression is rapidly lost in
vitro in primary epithelial cultures, suggests that interaction of ␣9␤1 with one or more of its ligands might be
important in maintaining differentiated cells with regenerative capacity in epithelial organs (109). Further clues
come from studies demonstrating critical roles for ␣9␤1
in mediating rapid cell migration of both neutrophils (115)
and nonleukocytic cells (138). Studies utilizing heterologously expressed deletion mutants of the ␣9 cytoplasmic
domain and chimeras of ␣9 and other integrins have
identified a short region of the ␣9 cytoplasmic domain
that is directly responsible for the ability of this integrin to
enhance cell migration (138). Perhaps this function plays
a role in the rapid migration of precursor cells across the
diverse ligands present at sites of epithelial denudation.
Recent observations have demonstrated that ␣9␤1 is the
preferred receptor for most members of the ADAMs family, a widely expressed family of transmembrane proteases that also contain a so-called disintegrin domain (a
binding site for integrins) and a domain that resembles
viral fusion peptides. It is tempting to hypothesize that
interactions of ADAMs family members with ␣9␤1 could
play a role in modulating either their proteolytic or membrane fusion functions (28).
681
682
DEAN SHEPPARD
emphysema. This response is completely dependent on
induction of MMP12, since mice homozygous for null
mutations of MMP12 and the ␤6-subunit are protected
from the development of emphysema (76a).
C. ␣v␤8
Further studies in ␤6 knock-out mice demonstrated a
role for the ␣v␤6 integrin in the development of pulmonary edema in two models of acute lung injury (88). Mice
lacking this integrin were protected from the pulmonary
edema that occurred in wild-type controls following instillation of intratracheal bleomycin or endotoxin. Blockade of TGF-␤ resulted in similar protection in wild-type
mice, identifying a role for ␣v␤6-mediated activation of
TGF-␤ in the development of pulmonary edema following
acute lung injury, at least in mice.
Analysis of global gene expression in the lungs of
wild-type and ␤6 knock-out mice identified dramatic induction of the macrophage-restricted metalloproteinase
MMP12 in the lungs of the ␤6 knock-out (61). This integrin
thus also appears to play an unexpected role in a negative
feedback system by which injured epithelial cells can turn
off protease expression in macrophages that are likely to
be activated in the setting of alveolar injury. Loss of this
signal has biologically important consequences, since ␤6
knock-out mice eventually develop slowly progressive
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FIG. 4. Models of transforming growth factor (TGF)-␤ activation by
the integrins ␣v␤6 (A) and ␣v␤8 (B). A: integrin ␣v␤6 binds to the RGD
site in TGF-␤1 or -␤3 latency-associated peptides (LAP). Binding is not
sufficient to induce activation of the latent complex. Activation requires
rearrangement of the actin cytoskeleton and allows the active site on
TGF-␤ to bind and activate receptors on adjacent cells. B: integrin ␣v␤8
also binds to the RGD site in TGF-␤1 or -␤3 LAP, but in this case binding
presents the latent complex to the transmembrane protease MT1MMP,
which cleaves LAP and releases active TGF-␤, which is free to diffuse
from the cell surface.
Human airway epithelial cells also express the integrin ␣v␤8. Until recently, the only known ligand for this
integrin was the plasma protein vitronectin (81), and the
function(s) of ␣v␤8 was obscure. In contrast to most
integrin ␤-subunits, which contain several functionally
important conserved sequence motifs in their short cytoplasmic domains, the ␤8 cytoplasmic domain is completely unique. Recent studies have demonstrated that
␣v␤8 is also functionally distinct from most other integrins in that its principal effect on cell proliferation is
inhibitory (11). ␣v␤8-mediated inhibition of cell proliferation is, not surprisingly, directly dependent on sequences
within its divergent ␤-subunit cytoplasmic domain. Interestingly, though, ␣v␤8, like ␣v␤6, can bind to the LAP of
TGF-␤1 and -␤3 and activate latent complexes (77). However, the mechanism of TGF-␤ activation by these two
integrins is completely different. ␣v␤6-mediated TGF-␤
activation depends on interaction of sequences in the ␤6
cytoplasmic domain with the actin cytoskeleton, leads to
a local change in conformation of latent complexes without releasing free diffusible active TGF-␤, and appears not
to involve the activity of any known proteases. In contrast, ␣v␤8-mediated TGF-␤ activation does not require
the ␤8 cytoplasmic domain and does lead to release of
free TGF-␤ (Fig. 1B). Furthermore, ␣v␤8-mediated activation is inhibited by the metalloprotease inhibitor GM6001
and depends on the presence of the transmembrane metalloprotease MT1MMP (77). Thus it appears likely that
␣v␤8 binds latent complexes and presents them to
MT1MMP, which can directly cleave LAP, leading to the
release of active TGF-␤. The biological significance of the
simultaneous existence of two distinct pathways for integrin-mediated TGF-␤ activation in airway epithelial cells
has not been directly examined. However, possible clues
come from the reported patterns of expression of these
integrins. ␣v␤8 is highly expressed in resting airway epithelial cells, but expression is decreased when these cells
are placed in culture, after injury, and in most epithelial
tumors, all conditions that are associated with epithelial
cell proliferation. In contrast, ␣v␤6 expression is dramatically increased in cultured cells, injured cells, and many
carcinomas. Thus one plausible hypothesis is that ␣v␤8mediated TGF-␤ activation produces low local concentrations of this cytokine that maintain growth arrest in resting, confluent epithelial cells, an effect that is enhanced
by the growth inhibitory effects of the divergent ␤8 cytoplasmic domain. In contrast, in the setting of injury, ␣v␤6
PULMONARY EPITHELIAL INTEGRINS
serves to spatially concentrate TGF-␤ at sites where it is
needed for repair, while the growth-promoting ␤6 subunit
cytoplasmic domain can counteract the normal growth
inhibition induced by any active TGF-␤ that is presented
to adjacent epithelial cells.
6.
7.
VIII. CONCLUSIONS/FUTURE DIRECTIONS
Address for reprint requests and other correspondence: D.
Sheppard, Univ. of California, San Francisco, Box 0854, San
Francisco, CA 94143-0854 (E-mail: [email protected]).
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