The Pancreatitis-induced Vacuole Membrane Protein 1 Triggers Autophagy in Mammalian Cells

Article (PDF Available)inJournal of Biological Chemistry 282(51):37124-33 · January 2008with69 Reads
DOI: 10.1074/jbc.M706956200 · Source: PubMed
Abstract
Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.
The Pancreatitis-induced Vacuole Membrane Protein 1
Triggers Autophagy in Mammalian Cells
*
Received for publication, August 20, 2007, and in revised form, October 4, 2007 Published, JBC Papers in Press, October 16, 2007, DOI 10.1074/jbc.M706956200
Alejandro Ropolo
‡1
, Daniel Grasso
‡2
, Romina Pardo
‡1
, Maria L. Sacchetti
, Cendrine Archange
§
, Andrea Lo Re
‡3
,
Mylene Seux
§
, Jonathan Nowak
§
, Claudio D. Gonzalez
, Juan L. Iovanna
§4
, and Maria I. Vaccaro
‡5
From the
Department of Physiology, School of Medicine, University of Buenos Aires, 2155 Paraguay p7,
Buenos Aires C1121ABG, Argentina,
CEMIC University Hospital, 4102 Av. E. Galvan, Buenos Aires C1431FWO, Argentina,
and
§
INSERM Unite 624, 163 Avenue de Luminy, Case 915, Marseille 13288, France
Autophagy is a degradation process of cytoplasmic cellular
constituents, which serves as a survival mechanism in starving
cells, and it is characterized by sequestration of bulk cytoplasm
and organelles in double-membrane vesicles called autophago-
somes. Autophagy has been linked to a variety of pathological
processes such as neurodegenerative diseases and tumorigene-
sis, which highlights its biological and medical importance. We
have previously characterized the vacuole membrane protein 1
(VMP1) gene, which is highly activated in acute pancreatitis, a
disease associated with morphological changes resembling
autophagy. Here we show that VMP1 expression triggers auto-
phagy in mammalian cells. VMP1 expression induces the forma-
tion of ultrastructural features of autophagy and recruitment of
the microtubule-associated protein 1 light-chain 3 (LC3), which
is inhibited after treatment with the autophagy inhibitor 3-me-
thiladenine. VMP1 is induced by starvation and rapamycin
treatments. Its expression is necessary for autophagy, because
VMP1 small interfering RNA inhibits autophagosome forma-
tion under both autophagic stimuli. VMP1 is a transmembrane
protein that co-localizes with LC3, a marker of the autophago-
somes. It interacts with Beclin 1, a mammalian autophagy initi-
ator, through the VMP1-Atg domain, which is essential for
autophagosome formation. VMP1 endogenous expression co-
localizes with LC3 in pancreas tissue undergoing pancreatitis-
induced autophagy. Finally, VMP1 stable expression targeted to
pancreas acinar cell in transgenic mice induces autophagosome
formation. Our results identify VMP1 as a novel autophagy-re-
lated membrane protein involved in the initial steps of the mam-
malian cell autophagic process.
Autophagy is an evolutionarily preserved degradation proc-
ess of cytoplasmic cellular constituents, which serves as a sur-
vival mechanism in starving cells (1–3). This catabolic process
is involved in the turnover of long lived proteins and other
cellular macromolecules, and it might play a protective role
in development, aging, cell death, and defense against intra-
cellular pathogens (4 8). By morphological studies, autoph-
agy has been linked to a variety of pathological processes
such as neurodegenerative diseases and tumorigenesis,
which highlights its biological and medical importance (4, 9,
10). Early reports of autophagy in human disease include the
ultrastructural autophagic features described in pancreas
from human pancreatitis (11, 12).
Autophagy is characterized by sequestration of bulk cyto-
plasm and organelles in double-membrane vesicles called auto-
phagosomes, which eventually acquire lysosomal-like features
(13, 14). Autophagy is mediated by a set of evolutionarily con-
served gene products (termed the Atg proteins) originally dis-
covered in yeast (15). In mammalian cells, Beclin 1 (3, 16 –18)
promotes autophagosome formation when it functions as part
of a complex with the Class III phosphatidylinositol 3-kinase
(PI3K)
6
mediating the localization of other autophagic proteins
to the autophagosomal membrane (19). However, despite the
advances in understanding autophagy, autophagosome forma-
tion in mammalian cells is a complex process, and neither the
molecular mechanisms nor all the implicated genes involved in
its formation are fully elucidated (20, 21).
The pancreatitis-associated protein named vacuole mem-
brane protein 1 (VMP1) is a transmembrane protein with no
known homologues in yeast. We identified the VMP1 tran-
script because it is highly activated in acinar cells early during
experimental acute pancreatitis and its transient expression
induced the formation of numerous cytoplasmic vacuoles in
mammalian cells (22). VMP1 mRNA in situ expression corre-
lated with the formation of vacuoles in acinar cells during
experimental acute pancreatitis and in the course of chronic
pancreatitis in WBN/Kob rats (23, 24). In this study we tested
the hypothesis that VMP1 expression is involved in the autoph-
agic process. We show that VMP1 is a novel autophagy-related
membrane protein that triggers autophagosome formation in
mammalian cells. VMP1 is induced by autophagy stimuli; it is
required for autophagosome development and its expression
* This work was supported in part by grants from the Agencia Nacional de
Promocio´ n Cientı´fica y Tecnolo´ gica, Consejo Nacional de Investigaciones
Cientı´ficas y Te´cnicas, University of Buenos Aires, INSERM, and Ligue Con-
tre le Cancer. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas Fellows.
2
A University of Buenos Aires Fellow.
3
An Agencia Nacional de Promocio´ n Cientı´fica y Tecnolo´ gica Fellow.
4
To whom correspondence may be addressed: Tel.: 33-491-828-803; Fax:
33-491-266-219; E-mail: iovanna@marseille.inserm.fr.
5
To whom correspondence may be addressed: Tel.: 5411-5950-9500 (ext.
2127); Fax: 5411-4433-4539; E-mail: mvaccaro@fmed.uba.ar.
6
The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; VMP1, vac-
uole membrane protein 1; LC3, light-chain 3; siRNA, small interfering RNA;
3-MA, 3-methyladenine; GST, glutathione S-transferase; GFP, green fluo-
rescent protein; mTOR, mammalian target of rapamycin; Atg, autophagy-
related domain; RFP, red fluorescent protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 51, pp. 37124 –37133, December 21, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
37124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 51 DECEMBER 21, 2007
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IF: 5.581
triggers autophagy, even under nutrient-replete conditions.
VMP1 interacts with Beclin 1 through its hydrophilic C-termi-
nal region, which we named Atg domain because it is essential
for autophagosome formation. We also show that VMP1 co-
localizes with the microtubule-associated protein 1 light chain
3 (LC3) in vacuole membranes in pancreas tissue undergoing
pancreatitis-induced autophagy. Finally, we found that VMP1
expression promotes the formation of LC3-positive vacuoles
when specifically targeted to the pancreas acinar cells in trans-
genic mice.
EXPERIMENTAL PROCEDURES
Mammalian Cell Lines, Transfections, and Treatments—Hu-
man HeLa, 293T and MCF7, and mouse NIH3T3 (ATCC) cell
lines were used. Low-Beclin 1 levels in MCF7 cells were verified
by Western blot. Cells were transfected using FuGENE-6 rea-
gent (Roche Applied Science). Plasmids were pcDNA4-V5-His
(Invitrogen) and pEGFP-N1 (Clontech), containing full-length
rat VMP1 cDNA (NM_138839) (22) or VMP1
Atg
subcloned
within the HindIII and BamHI restriction sites. Full-length
human Beclin 1 cDNA (NM_003766) was subcloned into
pECFP-C1 (Clontech) within the EcoRI and SalI or into
pcDNA3 (Invitrogen) within EcoRI and ApaI restriction sites.
pRFP-C1 containing full-length rat LC3 was kindly provided by
Dr. Maria I. Colombo (Universidad Nacional de Cuyo, Consejo
Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET),
Argentina). Cells were also transfected using Oligofectamine
(Invitrogen)-mediated transfer with siRNA pre-designed for
human VMP1 mRNA using 5-GGCAGAAUAUUGUCCU-
GUGtt-3, as sense, and 5-CACAGGACAAUAUUCUCUG-
CCtt-3, as antisense (Ambion ID32935) and 24 h later, cells
were subjected to autophagy stimuli for an additional 6-h
period. For autophagy inhibition, cells were treated with 10 m
M
3-methyladenine (3-MA) (Sigma) 2 h before and during co-
transfection with pcDNA4-VMP1 and pRFP-LC3. The efficacy
of 3-MA treatment was verified on starved cells (data not
shown). For lysosomal hydrolase inhibition, cells were treated
with E64d (10
g/ml) for 4 h before processed. Autophagy was
induced by amino acid/serum-deprived medium using Earle’s
balanced salt solution (Invitrogen) or 55
M rapamycin (Calbio-
chem) treatments. Fluorescence was observed using a fluores-
cence microscope Nikon Eclypse 200 (Plan100), an inverted
fluorescence microscopy Olympus GX71, a confocal micro-
scope Nikon Eclipse C1 (Plan40/0.95 and Plan60/1.40), or
an inverted confocal microscope Olympus FV1000 (PLAPO
N/1.42).
Transmission Electron Microscopy—Cells were fixed without
being brought into suspension and processed for transmission
electron microscopy by standard procedures. Grids were exam-
ined under a Carl Zeiss C-10 electron microscope (Laboratorio
Nacional de Investigacio´n y Servicios en Microscopı´a Elec-
tro´nica, University of Buenos Aires).
Percentage of RFP-LC3 Cells with Punctate Staining—The
number of cells with punctate staining per 100 fluorescent RFP-
LC3 transfected cells was determined in three independent
experiments. To quantify, the number of fluorescent cells with
punctate staining was counted in six random fields represent-
ing 100 fluorescent cells and expressed as the mean S.D. of
combined results. We consider an RFP-LC3 cell to have punc-
tate staining when all the red fluorescence is present as punc-
tate and no diffused protein remains.
Recombinant GST-VMP1 Peptides and His
6
-Beclin 1 Protein
VMP1 hydrophilic peptides were amplified from pcDNA4-VMP1
by PCR and subcloned into pGEX-5X-2 (Amersham Biosciences)
to obtain GST-VMP1 peptides. Peptides were expressed in
Escherichia coli BL21 and purified by glutathione affinity
chromatography (Amersham Biosciences). Beclin 1 cDNA
was obtained from HeLa cell total RNA extracts and cloned
into pQE31 (Qiagen). His
6
-Beclin 1 was expressed in E. coli
M15 and purified using nickel-nitrilotriacetic acid-agarose
beads (Qiagen).
Membrane Isolation—Cells were washed and homogenized
with a motor driven Teflon pestle homogenizer in ice-cold SBE
buffer (250 m
M sucrose, 1 m M EGTA, 10 mM Hepes/KOH, pH
7.5) containing protease inhibitors. Homogenates were centri-
fuged twice at 500 g for 15 min. The resulting supernatants
were centrifuged at 100,000 g for 1 h. Membrane pellets were
resuspended in ice-cold SBE buffer and treated with 1% Non-
idet P-40 (Nonidet P-40), 0.2
M Na
2
CO
3
, pH 11.0, or 1.5 M NaCl.
All steps were performed at 4 °C. The membrane pellets were
obtained by centrifugation, and retained proteins were sepa-
rated by SDS-PAGE. Western blot analysis was performed
using as secondary antibody a peroxidase-labeled IgG antibody
provided with the ECL-kit (Amersham Biosciences). Immuno-
blotting was performed using the ECL kit and membranes were
exposed to a Kodak BioMax film.
Pulldown Assays—HeLa cells transfected with the pcDNA4-
VMP1, pcDNA4-VMP1
Atg
, and pcDNA4-empty plasmids
were lysed by sonication and solubilized with 1% Triton X-100,
followed by a 30-min centrifugation at 100,000 g. The
supernatants containing His-tagged proteins were incubated
with nickel-nitrilotriacetic acid-agarose beads (Qiagen) for
2 h at 4 °C, extensively washed with a buffer containing 5 m
M
imidazole, and eluted with a buffer containing 120 m M imid-
azole. Proteins were separated by SDS-PAGE and trans-
ferred to nitrocellulose for immunoblotting. In another
experiment His
6
-Beclin 1 produced in E. coli was incubated
in nickel-nitrilotriacetic acid-agarose beads with the puri-
fied GST-VMP1 peptides.
Co-immunoprecipitation Assays—For the co-immunoprecipi-
tation assays, the supernatant from lysates of pcDNA4-VMP1-
transfected or rapamycin-treated cells were incubated with anti-
sera bound to protein A-Sepharose
TM
Cl-4B beads (Amersham
Biosciences) for2hat4°Candwashed extensively. Bound pro-
teins were eluted with 100 m
M glycine-HCl, pH 2.5, precipi-
tated by 5% trichloroacetic acid, washed with ice-cold acetone,
and resuspended in a SDS sample buffer.
Antibodies—Polyclonal rabbit antiserum to VMP1 against
the peptide MAQSYAKRIQQRLNSEEKTK (residues 386
406) was obtained and used at 1:100 dilution. Polyclonal goat
anti-LC3, goat anti-Beclin 1, rabbit anti-GST, monoclonal
mouse anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA),
monoclonal mouse anti-V5 (Invitrogen) antibodies were used
according manufacturer. Donkey anti-rabbit and anti-goat
Alexa Fluor 488 and 590, and rabbit anti-mouse Alexa Fluor 590
(Molecular Probes) antibodies were used for immunofluores-
VMP1 Triggers Autophagy
DECEMBER 21, 2007 VOLUME 282NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 37125
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cence. Peroxidase-labeled anti-rabbit, anti-mouse, and anti-
goat IgG antibodies were used for Western blot according
Amersham Biosciences.
Caerulein-induced Pancreatitis—Male Wistar rats weighing
200–250 g were used. Animals were housed with free access to
food and water. Experiments were performed according to the
standard ethical and<