Am J Physiol Gastrointest Liver Physiol
279: G961–G974, 2000.
Retention of mutant ␣1-antitrypsin Z in endoplasmic
reticulum is associated with an autophagic response
JEFFREY H. TECKMAN1 AND DAVID H. PERLMUTTER1,2
Departments of 1Pediatrics and 2Cell Biology and Physiology, Washington University School
of Medicine, Division of Gastroenterology and Nutrition, St. Louis Children’s Hospital,
St. Louis, Missouri 63110
Received 5 April 2000; accepted in final form 22 June 2000
described specific morphological alterations in subcellular structure in response
to the accumulation of misfolded or unassembled proteins in the endoplasmic reticulum (ER). Raposo et al.
(26) examined the accumulation of unassembled class I
major histocompatibility complex (MHC) molecules in
the ER of thymic epithelial cells, which overexpress
heavy chains on a genetic background that is deficient
for the peptide transporter TAP1. In these cells, class I
MHC heavy-chain molecules accumulated in an expanded post-ER/pre-Golgi network, or ER-Golgi intermediate compartment, that consists of tubulated and
fenestrated smooth membranes. Johnston et al. (18)
examined the accumulation of misfolded cystic fibrosis
transmembrane conductance regulator (CFTR) ⌬F508
and presenilin-1 (PS1) molecules in human embryonic kidney 293 cells. Undegraded CFTR and PS1
molecules, modified by ubiquitination, were found in
pericentriolar cagelike structures surrounded by the
intermediate filament protein vimentin. These “aggresomes” were induced by high levels of expression of
misfolded CFTR, PS1 molecules, or unassembled T cell
receptor ␣-subunits or by chemical inhibition of proteasomal degradation in the presence of lower levels of
expression of the misfolded membrane proteins. Aggresomes have also recently been described in cells in
which there has been cytoplasmic accumulation of certain viral proteins (2).
It has been known for some years that the ER
possesses complex machinery, called the quality control apparatus, by which it can recognize, retain, and
degrade misfolded proteins (11, 29). This includes
proteins that are unable to fold properly, unable to
undergo posttranslational modifications such as glycosylation or formation of intra- or intermolecular disulfide bonds, or unable to assemble into hetero- or homooligomers because of naturally occurring mutations
associated with disease states/deficiency disorders or
experimental conditions. Recent studies have shown
that the ER degradative mechanism(s) involves retrotranslocation, or dislocation, of misfolded proteins
across the ER membrane into the cytoplasm and that
dislocation, for the most part, is coupled to covalent
modification by polyubiquitin chains for proteolysis by
the proteasome (11). Recent studies have also shown
that the ER possesses signaling pathways, such as the
unfolded protein response, that permit it to respond to
the presence of retained misfolded proteins by altering
expression of its chaperones and components of its
membrane structure (29).
In the classic form of ␣1-antitrypsin (␣1AT) deficiency (homozygous PIZZ ␣1AT deficiency), the mutant
␣1ATZ molecule is retained in the ER of liver cells
rather than secreted into the blood and extracellular
Address for reprint requests and other correspondence: J. Teckman, Dept. of Pediatrics, Washington Univ. School of Medicine at St.
Louis Children’s Hospital, 1 Children’s Place, St. Louis, MO 63110
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
autophagy; quality control; liver disease
SEVERAL RECENT STUDIES HAVE
http://www.ajpgi.org
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Teckman, Jeffrey H., and David H. Perlmutter.
Retention of mutant ␣1-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am J Physiol
Gastrointest Liver Physiol 279: G961–G974, 2000.—
Although there is evidence for specific subcellular morphological alterations in response to accumulation of misfolded
proteins in the endoplasmic reticulum (ER), it is not clear
whether these morphological changes are stereotypical or if
they depend on the specific misfolded protein retained. This
issue may be particularly important for mutant secretory
protein ␣1-antitrypsin (␣1AT) Z because retention of this
mutant protein in the ER can cause severe target organ
injury, the chronic hepatitis/hepatocellular carcinoma associated with ␣1AT deficiency. Here we examined the morphological changes that occur in human fibroblasts engineered
for expression and ER retention of mutant ␣1ATZ and in
human liver from three ␣1AT-deficient patients. In addition
to marked expansion and dilatation of ER, there was an
intense autophagic response. Mutant ␣1ATZ molecules were
detected in autophagosomes by immune electron microscopy,
and intracellular degradation of ␣1ATZ was partially reduced
by chemical inhibitors of autophagy. In contrast to mutant
CFTR⌬F508, expression of mutant ␣1ATZ in heterologous
cells did not result in the formation of aggresomes. These
results show that ER retention of mutant ␣1ATZ is associated with a marked autophagic response and raise the possibility that autophagy represents a mechanism by which
liver of ␣1AT-deficient patients attempts to protect itself from
injury and carcinogenesis.
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
MATERIALS AND METHODS
Materials. Antibodies used against ␣1AT included rabbit
anti-human ␣1AT from DAKO (Santa Barbara, CA), goat
anti-human ␣1AT from Cappel (Durham, NC), and monoclonal mouse anti-human ␣1AT from Zymed (San Francisco,
CA). Antibodies against human calnexin included rabbit
anti-human calnexin (DP23 and DP33) generated in our
laboratory (33), SPA-865 from StressGen (Victoria, BC, Canada), and mouse anti-human calnexin from Chemicon (Temecala, CA). Lyso-tracker Red (LTR) and ER-tracker Blue
(ETB) were purchased from Molecular Probes (Eugene, OR).
Anti-dinitrophenyl antibody, Cy3-conjugated anti-vimentin
antibody, monodansylcadaverine (MDC), 3-methyladenine
(3MA), and wortmannin were purchased from Sigma (St.
Louis, MO). LY-294002 was purchased from Biomol Research
Laboratories (Plymouth Meeting, PA). Liver tissue from
three patients with ␣1AT deficiency and one normal liver
transplant donor was used for electron microscopy (EM). One
of the deficient livers and the normal liver were processed for
EM at the time of harvest. One of the deficient livers had
been fixed in glutaraldehyde and stored until processed for
EM for this study. The third deficient liver was reprocessed
for EM from paraffin-embedded tissue. Liver tissue from four
PiZ mice (7) and four wild-type mice of the same genetic
background was freshly prepared for EM as described in EM.
Cell lines and labeling. Human fibroblast cell lines engineered for stable expression of mutant ␣1ATZ by transduction of amphotropic recombinant retroviral particles have
been previously described (33). The same cell lines transduced with wild-type ␣1ATM or with vector alone or not
transduced at all were used as additional controls. Cell lines
from three PIZZ ␣1AT-deficient patients with liver disease
(susceptible hosts) and two PIZZ individuals without liver
disease (protected hosts) were used. For this study, the same
human fibroblast cell lines as well as Chinese hamster ovary
(CHO) cells were transduced with amphotropic recombinant
retroviral particles bearing mutant CFTR⌬F508 cDNA
(kindly provided by Dr. Richard Gregory, Framingham, MA).
Two hepatoma cell lines were used. The murine hepatoma
cell line Hepa1–6 engineered for stable constitutive expression of human ␣1ATZ (Hepa1–6N2Z9) has previously been
described (6). This study also used a rat hepatoma, H11
(kindly provided by K. Fournier, Seattle, WA), which has the
properties of a well-differentiated hepatocyte but does not
express endogenous ␣1AT because hepatocyte nuclear factor-1␣ and -4 gene expression have been extinguished (27).
The H11 cell line was engineered for stable constitutive
expression of human ␣1ATZ exactly as previously described
for human fibroblast cell lines (33). Pulse radiolabeling experiments showed that human ␣1AT gene expression had
been conferred and that there was intracellular retention of
␣1ATZ in this cell line, H11N2Z1 (data not shown).
This study also used a HeLa cell line engineered for inducible expression of human ␣1ATZ, HTO/Z. The tet-off system
of Bujard was used (14). Pulse radiolabeling showed that
human ␣1ATZ gene expression was absent in the presence of
doxycycline at 1 ng/ml, that it could be induced in a timedependent and concentration-dependent manner by removal
of doxycycline from the cell culture fluid, and that, once
induced, there was intracellular retention of ␣1ATZ (unpublished observations).
Cell lines were subjected to pulse-chase radiolabeling, and
samples were analyzed by immunoprecipitation and SDSPAGE analysis of immunoprecipitates as previously described (33). Results were quantified by scanning of PhosphorImager plates (Storm System; Molecular Dynamics,
Sunnyvale, CA) exposed to the radiolabeled gels. Values are
reported as means ⫾ SD.
For immunofluorescent staining of vimentin fibers, cells
were fixed, permeabilized, and stained with Cy3-conjugated
antivimentin antibody exactly as described by Johnston et al.
(18). In experiments with proteasome inhibitors, the cells
were incubated at 37°C for 18 h in N-acetyl-leu-leu-norleucinol (ALLN) (10 ␮g/ml for CHO cells, 50 ␮g/ml for fibroblasts)
in normal growth media before fixation. All fluorescent micrographs and photomicrographs were obtained with standard techniques using a Zeiss Axioskop microscope.
Cell-free translation and translocation. The pGEM-4Z vector (Promega) containing either ␣1ATM cDNA or ␣1ATZ
cDNA was linearized beyond the 3⬘ end of the cDNA using
Hind III. SP6 RNA polymerase was used for in vitro transcription in the presence of m7G(5⬘)ppp(5⬘)G (Pharmacia,
Uppsala, Sweden) to generate 7mG-capped mRNAs following
the protocol provided by Promega and previously described
(24). ␣1ATM and ␣1ATZ polypeptides were synthesized in the
reticulocyte lysate cell-free system according to the protocol
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fluid, where it ordinarily functions as an inhibitor of
neutrophil elastase. Carrell and Lomas (8) have shown
that the mutation that characterizes the ␣1ATZ molecule results in aberrant polymerization in the ER by a
loop-sheet insertion mechanism. Retention of the misfolded mutant ␣1AT protein in the ER is thought to
cause severe liver injury and hepatocellular carcinoma
in a subgroup of deficient individuals (31). In fact, this
deficiency constitutes the most common genetic cause
of liver disease in children. Studies in genetically engineered cell lines from patients with ␣1AT deficiency
have shown that there is a correlation between susceptibility to liver disease and delayed ER degradation of
␣1ATZ (33). Deficient individuals who are “protected”
from liver disease have more efficient ER degradation
of ␣1ATZ and a lesser net burden of mutant ␣1AT
molecules retained in the ER. Recent studies have
shown that ␣1ATZ is similar to other mutant proteins
that are retained in the ER in that its degradation in
the ER appears to involve the proteasome (5, 20, 24).
Studies in intact genetically engineered cell lines and
in the cell-free microsomal translocation system indicate that degradation involves at least several steps,
including, first, stable binding to the transmembrane
ER chaperone calnexin and then polyubiquitination of
calnexin and proteolysis of the ␣1ATZ-polyubiquitinated calnexin complex by the 26S proteasome (24).
Fractionation of reticulocyte lysate used in the cell-free
microsomal translocation system and reconstitution
with purified ubiquitin proteins has shown that the
ubiquitin-conjugating enzyme E2-F1 plays a role in the
ubiquitin-dependent proteasomal mechanism for degradation of ␣1ATZ (30). Moreover, studies in the cellfree system indicate that ubiquitin-independent proteasomal and nonproteasomal mechanisms may also
contribute to intracellular ␣1ATZ degradation.
In this study, we examined the effect of ER retention
of ␣1ATZ in human fibroblast cell lines to determine
whether there are specific morphological changes that
will ultimately provide more information about its hepatotoxic and oncogenic properties.
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
oles, we examined EM photomicrographs of 25 whole, intact
cells containing a nucleus. Quantitative grids of the appropriate size were superimposed on the photomicrographs, and
the grid area occupied by nascent (AVi) and degradative
(AVd) autophagic vacuoles was compared with the grid area
occupied by the cytoplasm.
RESULTS
Structural changes in human fibroblasts engineered
for expression and ER retention of ␣1ATZ. First, we
used transmission EM to determine whether there
were specific morphological changes in human fibroblasts engineered for stable expression of mutant
␣1ATZ compared with wild-type ␣1ATM. Previous
studies of these cell lines have shown that the wildtype molecule is rapidly and completely secreted,
whereas the mutant is retained and ultimately degraded in the ER, recapitulating the known defect in
␣1AT deficiency (24, 33). In the cell line expressing
wild-type ␣1ATM (Fig. 1), the ultrastructure was identical to that of the untransfected human fibroblasts,
with thin, closely apposed rough ER (rER) cisternae in
the perinuclear region interspersed among normalappearing mitochondria. There were no alterations in
any other organelles (data not shown). In the cell line
expressing mutant ␣1ATZ (Fig. 1) there was markedly
dilated rER cisternae filled with granular material.
Furthermore, the architecture of the rER cisternae was
disrupted and widened by networks of multiple intervening electron-dense multilamellar vacuoles.
The network of electron-dense multilamellar vacuoles that surround and separate ER cisternae in cells
expressing ␣1ATZ is shown in greater detail in Fig. 2.
In the cell line expressing mutant ␣1ATZ, viewed under low magnification (Fig. 2A), a striking network of
electron-dense structures is visible in the perinuclear
region. Higher magnification of the cell line expressing
mutant ␣1ATZ (Fig. 2B) revealed that the electrondense structures are multilamellar vacuoles that intervene and widen the spaces between rER cisternae.
Many of the vacuoles are bound by a double, smooth
membrane and surround debris and fragmented membranous structures. In many fields, the smooth surrounding double membranes are contiguous with, or
Fig. 1. Morphology of the endoplasmic reticulum
(ER) in human fibroblast cell lines by electron microscopy (EM). Cell lines expressing either wild-type
␣1-antitrypsin (␣1AT) M (left) or mutant ␣1ATZ
(right) were fixed, embedded in plastic, subjected to
thin sectioning, and examined by transmission EM.
N, nucleus; rER, rough ER; M, mitochondria. Bar ⫽
1 ␮m.
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provided by Promega. The cell-free reaction mixture (50 ␮l)
contained 35 ␮l of micrococcal nuclease-treated rabbit reticulocyte lysate and was supplemented with the following final
concentrations of additional components: 20 ␮M of 19-aminoacid mixture minus methionine, 0.8 U/␮l of RNase inhibitor
RNasin, 0.8 ␮Ci/␮l of [35S]methionine, 4 A260/ml of canine
pancreatic microsomal vesicles, and 20 ␮g/ml of the appropriate mRNAs. The canine pancreatic microsomal vesicles
were prepared by a previously described protocol (25) and
kindly provided by Dr. R. Gilmore (Worcester, MA). The
cell-free translation and translocation assay was performed
for 1 h at 30°C. After the translation reaction, the microsomal
vesicles that contained either ␣1ATM or ␣1ATZ polypeptide
were isolated by centrifugation at 15,000 g for 15 min at 4°C.
The pelleted microsomal vesicles were resuspended in fresh
proteolysis-primed lysate contained in a final volume of 50 ␮l:
40 mM Tris 䡠 HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol,
0.5 mM ATP, 10 mM phosphocreatine, and 15 ␮g creatine
phosphokinase (350 U/mg at 25°C; Boehringer Mannheim,
Indianapolis, IN) and fresh reticulocyte lysate, followed by
incubation at 37°C. After 20 min, the reaction mixture was
fixed for EM as described.
EM. For plastic-embedded EM, cells were grown to 85%
confluence in 10-cm dishes and then trypsinized, pelleted,
and washed. Cells were then fixed in 1% glutaraldehyde-0.1
M Na-cacodylate and embedded in polybed for ultrathin
section transmission EM by standard techniques (26). For
immune EM, a previously established protocol with fixation
in paraformaldehyde glutaraldehyde and embedding in 10%
gelatin for ultrathin sectioning was used (15). Labeling with
the primary antibody was carried out for 2 h, and labeling
with the appropriate species-specific secondary anti-IgG/Au
gold conjugate (Jackson ImmunoResearch, West Grove, PA)
was carried out for 1 h. Sections were stained with uranyl
acetate and embedded in methyl cellulose. Specimens were
viewed and photographed using a Zeiss 902 electron microscope. Where indicated, cells were incubated with 50 ␮M
N-{3-[(2,4-dinitrophenylamino)propyl]}-N-(3-aminopropyl)methylamine d-hydrochloride (DAMP) at 37°C for 30 min in
growth media to label intracellular acidic compartments before fixation for EM (1, 9, 10). For each EM experiment, at
least two samples of the cell lines were prepared, immunostained, and examined separately. All double-label immune
EM experiments were performed at least twice with each
primary antibody. To ensure specificity, separate experiments were also done with several different preparations of
each primary antibody, including preparations made in rabbits, goats, and mice. For quantitation of autophagic vacu-
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
budding from, the nearby rER membranes. This appearance is the hallmark of AVi (9, 10, 23). Other
neighboring vacuoles enclose even more electrondense, lamellar accumulations of membranes, characteristic of the maturation of AVi into AVd. These AVi
and AVd also have unique asymmetric and elongated
shapes and form complex nests adjacent to and in
between rER cisternae (Fig. 2B). The rER cisternae
themselves are markedly dilated and filled with granular material. Dilation of rER cisternae, disruption of
the rER architecture, and intense accumulation of networks of multiple intervening electron-dense multilamellar vacuoles were seen in cell lines from three PIZZ
␣1AT-deficient patients with liver disease (susceptible
hosts) and from two PIZZ ␣1AT-deficient patients without liver disease (protected hosts; data not shown).
This marked alteration of the ER and the intense
accumulation of autophagic vacuoles was specific for
␣1ATZ, as shown by the ultrastructural characteristics
of the cell line expressing ␣1ATM (Fig. 2, C–E). At low
magnification (Fig. 2C) there is no evidence for perinuclear accumulation of autophagosomes. At higher magnification, there are thin, normal-appearing rER cisternae and normal mitochondria near the nucleus (Fig.
2D). Only a few spherical AVi and AVd can be found in
the cytoplasm (Fig. 2E). These AVi and AVd are not
elongated, do not form nests, and are typical for cultured cells in general. Quantitative morphometry
showed that AVi and AVd occupied 4 ⫾ 2.5% of the
cytoplasm in cells expressing wild-type ␣1ATM compared with 17.5 ⫾ 4.5% in cells expressing mutant
␣1ATZ. There was no difference in percent cytoplasm
occupied by autophagic vacuoles in the nontransduced
parent fibroblast cell line or the fibroblast cell line
transduced with the expression vector alone compared
with the cell line expressing ␣1ATM (data not shown).
Characterization of the electron-dense vacuoles in
human fibroblast cell lines engineered for expression
and ER retention of mutant ␣1ATZ. To provide further
evidence that the electron-dense vacuoles were in fact
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Fig. 2. Electron-dense vacuoles in human fibroblast cell lines by EM. Cell lines expressing either mutant ␣1ATZ
(A and B) or wild-type ␣1ATM (C–E) were fixed, embedded in plastic, subjected to thin sectioning, and examined
by transmission EM. The ultrastructural characteristics of the cell line expressing wild-type ␣1ATM were identical
to this cell line untransfected (data not shown). G, Golgi; AVi, nascent autophagosome; AVd, degradative
autophagosome. Bar ⫽ 1 ␮m.
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
G965
Fig. 3. Intravital staining of human fibroblast cell lines
with monodansylcadaverine (MDC) and ER-tracker
Blue (ETB). The previously described cell lines expressing wild-type ␣1ATM (A and C) or mutant ␣1ATZ (B and
D) were incubated with the MDC (A and B) or ETB (C
and D) vital dyes, and representative living cells were
photographed under fluorescent microscopy. Bar ⫽
10 ␮m.
ETB, a fluorescent vital dye that selectively stains ER
membranes in living cells (32) (Fig. 3, C and D). ETB
stains structures that are closer to the nucleus but
overlapping with the structures stained by MDC.
There is a marked increase in ETB staining of the cells
expressing mutant ␣1ATZ (Fig. 3D) than in cells expressing ␣1ATM (Fig. 3C), corresponding to the expansion and dilation of the ER membrane observed at the
ultrastructural level.
Second, we examined whether the structures stained
by MDC were also stained by the fluorescent vital dye
LTR, which accumulates and labels acidic intracellular
compartments in living cells (Fig. 4). Acidification is
known to occur early in the formation of autophagic
Fig. 4. Intravital staining with Lyso-tracker Red (LTR) and ETB. Cells expressing wild-type ␣1ATM (A–C) or
mutant ␣1ATZ (D–F) were incubated with the LTR and ETB vital dyes, and representative living cells were
photographed under fluorescent microscopy. A and D: LTR labeling; B and E: ETB staining; C and F: double
labeling for LTR and ETB. Bar ⫽ 10 ␮m.
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autophagic, three approaches were used. First, we subjected fibroblasts expressing wild-type ␣1ATM or mutant ␣1ATZ to intravital staining with MDC, a fluorescent reagent known to specifically label autophagic
vacuoles in vivo (3). The resulting fluorescent photomicrographs demonstrate intense staining of vacuolar
structures in the center of the cytoplasm of the cells
expressing the mutant protein (Fig. 3B). The staining
is significantly increased in intensity compared with
cells expressing the wild-type protein (Fig. 3A). Moreover, the staining corresponds in location with the
electron-dense vacuoles observed by EM in close proximity to, but distinct from, the ER. This is shown more
clearly by comparing the MDC staining with that of
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
tant ␣1ATZ had the known acidic properties of autophagic vesicles and that they could be specifically labeled
by immune EM for DAMP.
Next, we used immune EM to examine the possibility that the autophagic vesicles contained mutant
␣1ATZ. The results show immunogold labeling of dilated ER membranes (Fig. 5C) and tangled nests of AVi
and AVd (Fig. 5D). Beads were only rarely present in
the nucleus, mitochondria, or other structures (data
not shown). The same cells were then double labeled
for DAMP (small, 12 nm beads) and ␣1AT (large, 18 nm
beads) to determine whether ␣1ATZ is colocalized to
autophagosomes. The result is illustrated by a highmagnification view within a cluster of autophagosomes
that have engulfed multilamellar debris (Fig. 5E).
These structures are labeled by both DAMP and ␣1AT.
As a control (Fig. 5F), examination of cells transduced
with the expression vector alone reveals only rare,
round DAMP-positive AVi and acidified simple vacuoles but no complex nests of autophagosomes and no
␣1AT labeling. These results demonstrate that the appearance of nests of DAMP-labeled autophagosomes
containing ␣1AT is specific for cells that express and
retain the mutant ␣1ATZ.
Next we examined the possibility that calnexin was
also present in autophagosomes by double labeling
with antibodies to calnexin (small, 12 nm beads) and
␣1AT (large, 18 nm beads). Previous studies have sug-
Fig. 5. Immune EM of human fibroblast cell lines for DAMP, ␣1AT, and calnexin. Cells expressing ␣1ATZ were
incubated with DAMP and then with anti-dinitrophenyl antibodies and immunogold beads (A and B). The same
cells were incubated with anti-␣1AT antibodies and immunogold beads (C and D). Cells expressing mutant ␣1ATZ
(E) and cells transduced with the expression vector alone (F) were then double labeled with DAMP (small, 12 nm
immunogold beads; small arrows) and ␣1AT (large, 18 nm immunogold beads; large arrows). Cells expressing
mutant ␣1ATZ (G and H) were also double labeled with antibody to calnexin (small, 12 nm beads; small arrows)
and ␣1AT (large, 18 nm beads; large arrows). Bars ⫽ 200 nm.
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vacuoles (1, 9, 10). The results show that intense staining of vacuolar structures in the cytoplasm of cells
expressing mutant ␣1ATZ (Fig. 4D) is significantly
increased over that in cells expressing ␣1ATM (Fig.
4A). This staining overlaps in location with that of rER
membranes by ETB (Fig. 4, B and E), and dual-labeled
images (Fig. 4, C and F) show that these acidic vacuoles are closely apposed to the ER.
Third, we examined the possibility that the electrondense vacuoles were acidified by immune EM for
DAMP (Fig. 5, A and B). DAMP accumulates in acidic
vesicles and can be immunolabeled with anti-dinitrophenyl antibodies and therefore can identify AVi and
AVd, which acidify early in their biogenesis (1). In Fig.
5, A and B, there is intense accumulation of gold beads
in double membrane-bound AVi containing debris.
Elongated nests of multiple, DAMP-labeled AVd also
containing electron-dense debris and lamellar arrangements of membranes are also identified (Fig. 5B). Because the vacuoles form a complex nest around the ER
it sometimes looks like the beads labeling DAMP are
outside the vacuoles, but in every case, when examined
in another field of view, these beads were in fact in
adjacent vacuoles. There was an occasional gold bead
found free in the cytoplasm and in other nonacidic
organelles, probably representing background staining
(data not shown). These results indicate that the electron-dense vacuoles found in cells expressing the mu-
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
G967
gested that calnexin plays a critical role in the ER
degradation of ␣1ATZ and, moreover, that it is the
␣1ATZ-calnexin complex that is attacked by the ER
degradation pathway (24, 30). The result shows colocalization of calnexin and ␣1ATZ in ER membranes
(Fig. 5G) and in AVi and AVd (Fig. 5H). Many individual AVi and AVd had both small and large beads,
indicating colocalization of calnexin and ␣1ATZ.
To exclude the possibility that the autophagic response to ER retention of ␣1ATZ is peculiar to fibroblasts, we used EM to examine model lines derived
from several other cell types. First, we examined the
mouse hepatoma cell line engineered for expression of
␣1ATZ Hepa1–6N2Z9 (Fig. 6). In cells from the parent
untransfected Hepa1–6 cells, the nucleus is surrounded by normal cytoplasm and a few electron-dense
structures representing simple lysosomes and an occasional multilamellar autophagic vacuole (Fig. 6A). In
contrast, Fig. 6B shows a similar region from the
Hepa1–6N2Z9 cell line, engineered for expression of
␣1ATZ; there are many electron-dense, multilamellar
autophagic vacuoles. The typical multilamellar structure and close proximity to rER is particularly evident
under higher magnification of one of the vacuoles in
Fig. 6C. There is also an increased number of autophagic vacuoles observed in the rat hepatoma cell line
engineered for expression of ␣1ATZ, H11N2Z1 (data
not shown).
Second, we examined by EM a HeLa cell line engineered for inducible expression of ␣1ATZ (Fig. 7). When
␣1ATZ expression is suppressed in the presence of
doxycycline (Fig. 7A), a few electron-dense structures
are visible near the nucleus. In the absence of doxycycline for a period of time associated with induction of
␣1ATZ expression and ER retention in Fig. 7B, there is
a significantly increased focal, perinuclear accumula-
Fig. 7. Autophagic vacuoles in electron micrographs of HeLa cells engineered for inducible expression of ␣1ATZ. A:
representative low-magnification photomicrograph of the HTO/Z cell line with expression of ␣1ATZ suppressed in
the presence of doxycycline. B: representative low-magnification photomicrograph of the HTO/Z cell line with
␣1ATZ expression induced in the absence of doxycycline. C: high-magnification view of the nest of multilamellar,
electron-dense autophagic vacuoles in the cytoplasm to the right of the nucleus in HTO/Z cells in the absence of
doxycycline. Bar ⫽ 1 ␮m.
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Fig. 6. Autophagic vacuoles in electron micrographs of mouse hepatoma cell line Hepa1–6 engineered for
expression and ER retention of ␣1ATZ. A: low-magnification photomicrograph of a representative cell from the
parent, untransfected Hepa1–6 cell line. B: low-magnification photomicrograph of a representative cell from the
Hepa1–6N2Z9 cell line engineered for expression and ER retention of ␣1ATZ. C: high-magnification view of 1 of the
many electron-dense, multilamellar autophagic vacuoles found in abundance in the Hepa1–6N2Z9 cell line. Bar ⫽
1 ␮m.
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
Fig. 8. Effect of 3-methyladenine (3MA) on the degradation of mutant ␣1ATZ. A: cells expressing mutant ␣1ATZ were subjected to
pulse-chase radiolabeling in the absence and presence of 2.5 mM
3MA. The cell lysates were then analyzed by immunoprecipitation
followed by SDS-PAGE/fluorography. The migration of the intracellular 52-kDa ␣1ATZ polypeptide is indicated by arrows at left. B:
phosphorimaging analysis of 3 separate experiments is shown. In
each of these experiments, there was a similar amount of radiolabeled ␣1ATZ present at time 0 in control and experimental conditions. Results are reported at each time point as means ⫾ SD.
appears between 2 and 6 h of the chase period. Only a
trace amount of ␣1ATZ is secreted into the extracellular fluid (data not shown). This result is consistent
with our previous studies showing retention and degradation of ␣1ATZ in the ER as a 52-kDa intermediate
with high-mannose-type oligosaccharide side chains
(24, 33). In the presence of 3MA, the ␣1ATZ is also
initially synthesized as a 52-kDa precursor polypeptide. However, the rate of disappearance is reduced.
There is a greater amount of ␣1ATZ remaining at 3 h
and at each subsequent time point of the chase period.
There is no increase or decrease in the trace amount of
␣1AT secreted into the extracellular fluid (data not
shown). Three identical experiments were used for
quantification of the kinetics of disappearance by phosphorimaging analysis, as shown in Fig. 8B. The results
show a decrease in rate of degradation beginning at 3 h
and particularly apparent at later time points. Experiments with wortmannin and LY-294002, two other
chemical inhibitors of autophagy (4), had similar results, with a decrease in rate of degradation of ␣1ATZ
especially apparent after 3 h of the chase period (data
not shown). Together with the observation that ␣1ATZ
can be detected in autophagic vacuoles by immune EM,
these data provide evidence that autophagy plays a
role in ER degradation of misfolded ␣1ATZ molecule.
Morphological evidence for autophagic vacuoles in
the liver of PiZ transgenic mice. Misfolded ␣1ATZ molecules are retained in the ER of liver cells in the PiZ
mouse model transgenic for the human ␣1ATZ gene (7).
Here we examined the livers of four PiZ mice and four
wild-type mice of the same genetic background by EM
for the presence within hepatocytes of multilamellar,
electron-dense structures indicative of autophagic
vacuoles (Fig. 9). The results showed that in some
hepatocytes there were nearly normal areas of cytoplasm with normal-appearing rER and other organelles (Fig. 9A). However, in most hepatocytes there
were areas of dilated rER membranes filled with granular deposits as previously described (data not shown),
as well as focal nests of electron-dense, multilamellar
vacuoles (Fig. 9B). When these vacuoles were examined under higher magnification (Fig. 9C), they were
clearly multilamellar structures containing electrondense debris that were closely apposed to rER membranes. Interestingly, nests of these vacuoles were
most commonly found within hepatocytes containing
dilated rER membranes. Examination of the livers
from the wild-type mice revealed occasional multilamellar vacuoles within the cytoplasm of hepatocytes,
but they were much fewer in number than seen in the
PiZ mice and were not localized around the rER (data
not shown).
Morphological changes in liver tissue from patients
with ␣1AT deficiency. It is well known from many
clinical studies that the ER in liver cells of ␣1ATdeficient patients is dilated by granular ␣1AT deposits.
Early clinical studies also noted areas of ribosome-free
ER membrane and surrounding vacuoles (12, 16, 34),
but it is not entirely clear from these studies whether
these vacuoles were truly autophagic, whether the
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tion of electron-dense, multilamellar vacuoles to the
right of the nucleus. This intense, focal area of autophagic activity is shown under higher magnification in
Fig. 7C. Examination by fluorescence microscopy revealed that this perinuclear nest of vacuoles stained
positively for acidity by LTR and stained positively by
the autophagic marker MDC (data not shown). Together, these data provide evidence that vacuoles with
the structural characteristics of autophagosomes are
associated with expression of ␣1ATZ in several cell
types and include cells of hepatocyte lineage and that
autophagosomes are specifically induced by expression
of ␣1ATZ in the inducible cell line.
Effects of chemical inhibitors of autophagy on the fate
of mutant ␣1ATZ. To investigate the possible role of
autophagy in the degradation of ␣1ATZ, we examined
the effect of 3MA, a chemical inhibitor of autophagy
(28), on the fate of ␣1ATZ in pulse-chase radiolabeling
experiments (Fig. 8A). The results show that, in the
absence of 3MA, ␣1ATZ is synthesized as a 52-kDa
polypeptide at time 0 and is retained for ⬃1 h of the
chase period. This polypeptide then progressively dis-
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
G969
autophagic response was prominent, and whether it
was only present in liver cells with dilated ER. Here we
examined by transmission EM liver tissue from three
patients with liver disease caused by ␣1AT deficiency
(Fig. 10) to determine whether an autophagic response
is also present in vivo. The results showed normalappearing rER, mitochondria, Golgi, nucleus, and
other structures in some cells (Fig. 10A). However, in
most cells there were areas of markedly dilated rER
membranes filled with granular, proteinaceous material (Fig. 10B). In many of these cells, multilamellar
autophagic vacuoles budding from, and still contiguous
with, ER membranes were observed (Fig. 10C). Numerous, fully formed, double membrane-bound AVi
containing debris could be seen that had freshly budded from nearby ER membranes (Fig. 10D). In other
areas, nests of multilamellar, electron-dense structures with the appearance of AVd could be easily identified (Fig. 10E). These autophagic structures are remarkably similar to those seen in fibroblast cell lines
that express ␣1ATZ. Moreover, autophagic vacuoles
were almost always seen in the cells that had dilated
ER cisternae. This type of intense autophagic response
was seen in the livers of all 3 ␣1AT-deficient patients
but not in the liver from the normal individual (data
not shown). The autophagic response in the ␣1ATdeficient liver could not be attributed to the method by
which the liver tissue was stored or processed because
each of these specimens was stored and/or processed
differently. Moreover, one ␣1AT-deficient liver and one
normal liver were processed immediately after harvest
by an identical protocol. Thus the results indicate that
autophagosomes are also induced by ␣1ATZ in vivo.
Morphological changes in microsomes that specifically degrade mutant ␣1ATZ in a cell-free system. Our
previous studies have shown that ␣1ATZ is specifically
degraded in a cell-free microsomal translocation sys-
tem and that the biochemical characteristics of its
degradation in the cell-free system recapitulate those
that occur in intact cells (24). Here we examined the
possibility that degradation of ␣1ATZ in this cell-free
system was associated with morphological changes in
the microsomal vesicles (Fig. 11). The vesicles were
first subjected to cell-free translation/translocation reaction for 1 h at 30°C. These conditions were associated
with translocation of wild-type ␣1ATM and mutant
␣1ATZ in similar amounts (data not shown) (24, 30).
Moreover, similar amounts of wild-type ␣1ATM and
mutant ␣1ATZ were protected from protease digestion
under these conditions (24). The vesicles were then
pelleted, resuspended in proteolysis-primed reticulocyte lysate, and incubated at 37°C for 20 min. By this
time, mutant ␣1ATZ, but not wild-type ␣1ATM, had
begun to undergo degradation (data not shown) (24,
30). The results show that native microsomal vesicles
(Fig. 11A) and microsomal vesicles that had translocated wild-type ␣1ATM (Fig. 11B) contained intact
round vesicular structures studded with ribosomes and
surrounded by proteinaceous reticulocyte lysate. Microsomes that had translocated ␣1ATZ were only rarely
round and spherical. In almost every field, there was
budding and elongation of these microsomes (Fig. 11,
C–F). Some of the microsomal membrane was devoid of
ribosomes, and there was formation of many nests of
ribosome-free membrane ghosts (Fig. 11E). The budding, elongation, loss of ribosomes, and nesting of vesicles were rarely, if ever, seen in the controls. These
data indicate that there are indeed morphological
changes in microsomal vesicles associated specifically
with the translocation and degradation of ␣1ATZ in
vitro.
Expression and ER retention of ␣1ATZ does not induce aggresome formation. A recent study by Johnston
et al. (18) has shown that when mutant CFTR⌬F508 or
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Fig. 9. Autophagic vacuoles in electron micrographs of liver from PiZ mice. The livers from 4 PiZ transgenic mice
were examined by EM, and representative fields were photographed. A: hepatocyte from 1 region that contains
rough ER that is not dilated, mitochondria, and a few electron-dense autophagic vacuoles. B: nest of multilamellar,
electron-dense autophagic vacuoles in the cytoplasm of a neighboring hepatocyte. C: high-magnification view of the
multilamellar autophagic vacuoles. Bar ⫽ 1 ␮m.
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
PS1 A246E molecules accumulate in the ER of transfected CHO cells there is formation of non-membranebound cagelike structures adjacent to the pericentriolar region of the nucleus, called aggresomes (18).
Aggresome formation was induced by expression of the
mutant proteins at high levels or by expression of the
mutant proteins at lower levels but in the presence of
inhibitors of their degradation by the proteasome. Aggresome formation also occurred at a low rate in cells
that did not express a mutant protein but were treated
with chemical proteasomal inhibitors. Here we examined the possibility that expression of ␣1ATZ was also
associated with aggresome formation.
Human fibroblast cell lines transduced to express
moderately high levels of wild-type ␣1ATM, mutant
␣1ATZ, or mutant CFTR⌬F508 were incubated in the
absence or presence of proteasomal inhibitor ALLN
and then fixed and stained with fluorescent antibodies
to vimentin (Fig. 12). In the absence of proteasome
blockade by ALLN, all of the cells maintained a spindle
or fan-shaped typical fibroblast morphology, with networks of vimentin fibers visible throughout the periphery of the cells (Fig. 12, A–C). In the presence of ALLN,
there was no collapse of peripheral vimentin fibers in
the majority (⬃65%) of cells expressing wild-type
␣1ATM or mutant ␣1ATZ (Fig. 12, D and E). The
remaining 35% of the cells had variable levels of vimentin fiber collapse, with a few cells demonstrating a
compact aggresome. There was no difference between
cells expressing mutant ␣1ATZ, wild-type ␣1ATM, and
the nontransduced parent fibroblast cell lines (data not
shown). However, in the fibroblast cell lines expressing
CFTR⌬F508 and incubated with ALLN (Fig. 12F), the
majority of the cells (70%) demonstrated complete collapse of vimentin fibers from the periphery of the cell
into a basketlike, perinuclear structure indicative of
aggresome formation.
A similar analysis was also performed in CHO cells
transduced to express moderately high levels of ␣1ATZ
or CFTR⌬F508. In the absence of ALLN, ⬍1% of CHO
cells expressing ␣1ATZ demonstrated the vimentin fiber collapse typical of aggresomes (Fig. 13). This was
similar to the rate of aggresome formation observed in
nontransduced parent CHO cells and in CHO cells
transduced with wild-type ␣1ATM (data not shown).
In the absence of ALLN, ⬃5% of cells expressing
CFTR⌬F508 spontaneously formed an aggresome with
a perinuclear, basketlike structure of collapsed vimen-
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Fig. 10. Autophagic vacuoles in electron micrographs of human liver tissue. A liver biopsy specimen from an
␣1AT-deficient patient was examined by EM. In C, an AVi is budding off and contiguous with ribosome-free rER
membranes just to the right of the rER label. Bar ⫽ 100 nm.
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
G971
Fig. 12. Identification of aggresomes
by fluorescent staining of vimentin fibers in human fibroblasts. Human fibroblasts transduced to express wildtype ␣1ATM, mutant ␣1ATZ, or cystic
fibrosis transmembrane conductance
regulator mutant ⌬F508 were fixed
and immunostained with fluorescent
antibodies to vimentin (A–C, respectively). The same cell lines were then
incubated in 50 ␮g/ml ALLN for 18 h
before immunostaining (D–F).
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Fig. 11. EM of microsomal vesicles that
have translocated wild-type ␣1ATM or mutant ␣1ATZ in a cell-free system. Canine
pancreatic microsomal vesicles were used in
cell-free microsomal translocation assays
programmed with wild-type ␣1ATM or mutant ␣1ATZ mRNA and then subjected to a
chase in proteolysis-primed reticulocyte lysate. Aliquots of microsomes were removed
during the degradation reaction and examined by EM. A: native canine pancreatic
rER microsomal membrane vesicles. B: microsomes that had translocated wild-type
␣1ATM. C–F: microsomes that had translocated and were degrading mutant ␣1ATZ.
Bar ⫽ 300 nm.
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AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
Fig. 13. Identification of aggresomes by fluorescent
staining of vimentin fibers in Chinese hamster ovary
(CHO) cells. CHO cells transduced to express mutant
␣1ATZ (left) or CFTR⌬F508 (right) in the absence of
ALLN (top) or following a 18 h incubation with 10 ␮g/ml
ALLN (bottom) were immunostained for vimentin.
Arrows show aggresomes in CHO cells expressing
CFTR⌬F508 and incubated with ALLN. Bar ⫽ 10 ␮m.
DISCUSSION
These results indicate that retention of mutant
␣1ATZ in the ER is associated with a marked autophagic response. There is mention of electron-dense vacuoles within the expanded ER-Golgi intermediate compartment of thymic epithelial cells that accumulate
unassembled MHC class I molecules (26) and of double-membrane vesicular structures in the immediate
vicinity of aggresomes in cells that accumulate
CFTR⌬F508, suggesting an autophagic response in
these cases (18). However, it is not clear at this time
whether the autophagic response in those cases is as
intense or as generalized as the one seen here in cells
in which there is ER retention of ␣1ATZ. There was a
marked difference in the degree of autophagy seen here
in human fibroblasts transduced with the CFTR⌬F508
gene compared with those transduced with the ␣1ATZ
gene.
Even though ␣1ATZ and CFTR⌬F508 are both mutant proteins that are degraded in the ER, there are
differences in the properties of the two proteins that
could explain the differences in cellular responses.
CFTR⌬F508 is a membrane protein with multiple
membrane-spanning domains expressed at lower concentrations than ␣1ATZ. The proteasome is involved in
degradation of both proteins, but there is evidence that
other mechanisms play a contributory role or, at least,
that the proteasomal mechanism cannot fully account
for degradation of either protein (17, 30). Because
CFTR⌬F508 is degraded more rapidly than ␣1ATZ, it is
likely that there are differences in how CFTR⌬F508
and ␣1ATZ reach the proteasomal machinery in the
cytoplasm or differences in the nonproteasomal mechanisms for degradation. It is also possible that differences in the absolute concentration of mutant protein,
the duration of time it has accumulated at a certain
concentration, and the rate or mechanism by which it
is extruded into the cytoplasm account for the differences in morphology and response. Moreover, it is
possible that intrinsic properties of the mutant protein
determine whether the autophagic or aggresomal responses are invoked; i.e., aggregated ␣1ATZ molecules
are so toxic when free in the cytoplasm that cells only
survive when capable of engulfing them within a membranous subcompartment. This is a particularly important issue for ␣1AT deficiency because a subgroup of
individuals affected by this deficiency develop severe
liver injury that is thought to be caused by the hepatotoxic effects of the retained ␣1ATZ molecule. There
are now many other naturally occurring human deficiency syndromes in which abnormal proteins are retained in the ER (22). In some of these cases, misfolded
secretory proteins such as mutant fibrinogen, coagulation, or complement proteins are retained in the ER of
liver cells without apparent hepatotoxic effects, implying that there is something intrinsically different
about the retention of ␣1ATZ, or the response to it, that
is associated with hepatotoxicity and carcinogenesis.
Although the results of this study provide evidence
that autophagy itself contributes to the ER degradation pathway for ␣1ATZ, it is still difficult to ascertain
the relative significance of its contribution. Previous
studies have shown that there is a ubiquitin-dependent
proteasomal mechanism that targets the ␣1ATZ-calnexin complex for degradation as well as a ubiquitin-
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tin fibers (Fig. 13). In the presence of ALLN, ⬃50% of
the CHO cells expressing mutant ␣1ATZ demonstrated
the collapse of vimentin fibers into aggresomes (Fig.
10), which is identical to the rate of aggresome formation for nontransduced parent CHO cells (data not
shown). However, in the presence of ALLN, nearly
100% of the CFTR⌬F508-expressing CHO cells demonstrated the collapse of cytoplasmic vimentin fibers into
tightly packed, perinuclear aggresomes (Fig. 13). Examination of these cell lines by EM and fluorescent
microscopy after intravital staining with MDC showed
a marked autophagic response only in the cell line
expressing mutant ␣1ATZ (data not shown). These
results indicate that the accumulation of ␣1ATZ does
not induce the formation of aggresomes and suggest
that the morphological changes that characterize the
response of cells to the accumulation of mutant proteins in the ER are, at least in part, substrate specific.
AUTOPHAGY IN ␣1-ANTITRYPSIN DEFICIENCY
the efficiency of translocation of ␣1ATZ is identical to
that of wild-type ␣1ATM, which undergoes two distinct
endoproteolytic cleavages but is not degraded over
many hours in isolated microsomal vesicles in vitro.
Electron microscopic analysis of vesicles from these
microsomal translocation reactions shows that translocation and degradation of ␣1ATZ is specifically accompanied by a marked expansion and distortion of the
vesicles that in some ways resembles the alterations of
ER seen in intact cells and in human liver. There were
even areas of ribosome-free microsomal membranes
and invaginations that clearly resemble the initial
stages of autophagosome formation in vivo. These results suggest that at least some of the profound morphological alterations that accompany accumulation of
the mutant secretory protein ␣1ATZ in the ER are
intrinsic to the ER or the combination of ER membrane
vesicles and proteolysis-primed reticulocyte lysate.
The authors are indebted to Mary Pichler for preparing this
manuscript.
These studies were supported in part by National Institutes of
Health grants HL-37784, DK-52526, P01-DK-56783, P30-DK-52574,
and DK-02379 (J. H. Teckman).
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