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 1 Pediatrics and 2 Cell Biology and
Physiology, Washington University School of Medicine, Division of
Gastroenterology and Nutrition, St. Louis Children's Hospital, St.
Louis, Missouri 63110
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ABSTRACT |
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.
autophagy; quality control; liver disease
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INTRODUCTION |
SEVERAL RECENT STUDIES
HAVE 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
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
cell-free 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.
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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 time-dependent
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 SDS-PAGE 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 µg/ml for CHO cells, 50 µg/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 provided by Promega. The
cell-free reaction mixture (50 µl) contained 35 µl of micrococcal
nuclease-treated rabbit reticulocyte lysate and was supplemented with
the following final concentrations of additional components: 20 µM of
19-amino-acid mixture minus methionine, 0.8 U/µl of RNase inhibitor
RNasin, 0.8 µCi/µl of [35S]methionine, 4 A260/ml of canine pancreatic microsomal vesicles, and 20 µg/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 µl: 40 mM Tris · HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 0.5 mM ATP, 10 mM
phosphocreatine, and 15 µg 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 µM
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 vacuoles, 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.
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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 wild-type 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 normal-appearing 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.

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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 µm.
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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 electron-dense 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
budding from, the nearby rER membranes. This appearance is the hallmark
of AVi (9, 10, 23). Other neighboring vacuoles enclose
even more electron-dense, 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).

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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 µm.
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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 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 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.

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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 µm.
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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 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.

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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 µm.
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Third, we examined the possibility that the electron-dense 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 mutant
1ATZ had the known acidic
properties of autophagic vesicles and that they could be specifically
labeled by immune EM for DAMP.

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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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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 high-magnification 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 suggested 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).

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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 µm.
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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
accumulation 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.

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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 µm.
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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 disappears 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.

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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.
|
|
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 electron-dense 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).

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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 µm.
|
|
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
1AT-deficient 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 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
normal-appearing 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
1AT-deficient 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.

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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.
|
|
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 system
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.

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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.
|
|
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 PS1 A246E molecules accumulate in the ER of
transfected CHO cells there is formation of non-membrane-bound 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.

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Fig. 12.
Identification of aggresomes by fluorescent staining of
vimentin fibers in human fibroblasts. Human fibroblasts transduced to
express wild-type 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 µg/ml ALLN for 18 h before immunostaining
(D-F).
|
|
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 vimentin 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.

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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 µg/ml ALLN (bottom) were
immunostained for vimentin. Arrows show aggresomes in CHO cells
expressing CFTR F508 and incubated with ALLN. Bar = 10 µm.
|
|
 |
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-independent proteasomal mechanism and nonproteasomal
mechanism, or mechanisms, for degradation of
1ATZ
retained in the ER (24, 30). The results reported here
show that
1ATZ and calnexin molecules are present in
autophagic vacuoles. Alone, these data do not prove that
1ATZ is degraded in autophagosomes and certainly do not
address the relative significance of autophagy in degradation of
1ATZ. In fact, because there is substantial evidence
that autophagosomes form at least in part from ER (9, 10),
it is possible that a certain number of
1ATZ molecules
that are retained in the ER and calnexin molecules that are integral to
the ER are nonspecifically carried into the autophagosomes. The results
of the current study do show that degradation of
1ATZ is
partially abrogated by 3MA, wortmannin, and LY-294002. However, the
effect of these chemical inhibitors of autophagy was significantly
lower in magnitude than that of the proteasomal inhibitors lactacystin
and MG132 previously reported (24, 30). Neither
lactacystin nor MG132 completely inhibit degradation of
1ATZ, and the presumed nonproteasomal component of ER
1ATZ degradation is particularly apparent at later time points (24, 30). The inhibitory effects of 3MA,
wortmannin, and LY-294002 in this study were, in fact, observed at
later time points, raising the possibility that proteasomal and
autophagic pathways constitute independent mechanisms for ER
degradation of
1ATZ, perhaps acting at different stages
and/or on different pools of retained
1ATZ molecules.
Nevertheless, because it is difficult to know the relative inhibitory
efficacy in our current model cell culture systems of 3MA, wortmannin,
or LY-294002 on autophagy compared with that of lactacystin on
proteasomal activity, it is not yet possible to determine whether
autophagy plays a relatively minor role in the overall degradation of
1ATZ retained in the ER. Sophisticated genetic
techniques for abrogating autophagy may be required to more
definitively address this issue in the future.
In addition to playing a protective role by contributing to the
degradation of mutant
1ATZ molecules, the autophagic
response may represent a mechanism for preventing carcinogenesis.
Recent studies have suggested that autophagic activity/proteins are
decreased in tumors and that reconstitution of autophagic activity
inhibits tumorigenesis in vivo (19, 21). In this regard,
it is of some interest that autophagic vacuoles were most commonly seen
in liver cells with dilated ER in the
1AT-deficient
patient. Moreover, in one genetically engineered mouse model of
1AT deficiency, hepatocarcinogenesis appeared to evolve
in nodular aggregates of hepatocytes that were negative for
1AT expression by immunohistochemical staining
(13).
A striking alteration in morphology was also induced by translocation
and degradation of the
1ATZ polypeptide in isolated microsomal vesicles in vitro. Our previous studies have
shown that
1ATZ is rapidly and specifically degraded
after it is translocated into microsomal vesicles in vitro and that the
biochemical characteristics of its degradation in vitro are very
similar to those that occur in intact cells (24).
Moreover, 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.
 |
ACKNOWLEDGEMENTS |
The authors are indebted to Mary Pichler for preparing this manuscript.
 |
FOOTNOTES |
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).
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: teckman{at}kids.wustl.edu).
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.
Received 5 April 2000; accepted in final form 22 June 2000.
 |
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