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Deficiency of hepatocystin induces autophagy through an mTOR-dependent pathway

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China.
Autophagy (Impact Factor: 11.75). 07/2011; 7(7):748-59. DOI: 10.4161/auto.7.7.15822
Source: PubMed
ABSTRACT
Mutations in the gene encoding hepatocystin/80K-H (PRKCSH) cause autosomal-dominant polycystic liver disease (ADPLD). Hepatocystin functions in the processing of nascent glycoproteins as the noncatalytic beta subunit of glucosidase II (Glu II) and regulates calcium release from endoplasmic reticulum (ER) through the inositol 1,4,5-trisphosphate receptor (IP3R). Little is known, however, on how cells respond to a deficiency of hepatocystin. In this study, we demonstrate that knockdown of hepatocystin induces autophagy, the major intracellular degradation pathway essential for cellular health. Ectopic expression of wild-type hepatocystin, but not pathogenic mutants, rescues the siRNA-induced effect. Our data indicate that the induction of autophagy by hepatocystin deficiency is mediated through mammalian target of rapamycin (mTOR). Despite the resulting severe reduction in Glu II activity, the unfolded protein response (UPR) pathway is not disturbed. Furthermore, the inhibition of IP3R-mediated transient calcium flux is not required for the induction of autophagy. These results provide new insights into the function of hepatocysin and the regulation of autophagy.

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©2011 Landes Bioscience.
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Autophagy 7:7, 1-12; July 2011; © 2011 Landes Bioscience
BASIC RESEARCH PAPER
BASIC RESEARCH PAPER
*Correspondence to: Zhiping Xie and Wei-Guo Zhu; Email: zxie@nankai.edu.cn and zhuweiguo@bjmu.edu.cn
Submitted: 09/14/10; Revised: 04/02/11; Accepted: 04/12/11
DOI:
Introduction
Mutations in the gene encoding hepatocystin (also known as
80K-H) (PR KCS H ) are associated with autosomal-dominant
polycystic liver disease (ADPLD).
1,2
The hallmark of ADPLD is
the formation of multiple cysts scattered in the liver parenchyma.
In late stages, the presence of large cysts may cause increased
intra-abdominal pressure that becomes disabling for certain
patients. Currently, surgical operations to reduce or remove the
cysts are the only option for treatment.
3,4
Hepatocystin normally resides in the lumen of endoplas-
mic reticulum (ER), where it functions as the noncatalytic beta
subunit of glucosidase II (Glu II).
5-9
The heterodimeric Glu II
complex is responsible for the removal of the two innermost glu-
cose residues from N-linked oligosaccharides on newly synthe-
sized glycoproteins.
10,11
These two cleavage events are important
steps during nascent glycoproteins’ entry and exit from calnexin/
calreticulin-assisted folding process.
12
In addition to its role in
glycoprotein folding, hepatocystin has recently been shown to
interact with and regulate the inositol-1,4,5-trisphosphate recep-
tor (IP3R), an intracellular signaling hub on the ER.
13
Mutations in the gene encoding hepatocystin/80K-H (PRKCSH) cause autosomal-dominant polycystic liver disease
(ADPLD). Hepatocystin functions in the processing of nascent glycoproteins as the noncatalytic beta subunit of glucosidase
II (Glu II) and regulates calcium release from endoplasmic reticulum (ER) through the inositol-1,4,5-trisphosphate receptor
(IP3R). Little is known, however, on how cells respond to a deciency of hepatocystin. In this study, we demonstrate
that knockdown of hepatocystin induces autophagy, the major intracellular degradation pathway essential for cellular
health. Ectopic expression of wild-type hepatocystin, but not pathogenic mutants, rescues the siRNA-induced eect.
Our data indicate that the induction of autophagy by hepatocystin deciency is mediated through mammalian target of
rapamycin (mTOR). Despite the resulting severe reduction in Glu II activity, the unfolded protein response (UPR) pathway
is not disturbed. Furthermore, the inhibition of IP3R-mediated transient calcium ux is not required for the induction of
autophagy. These results provide new insights into the function of hepatocysin and the regulation of autophagy.
Deciency of hepatocystin induces autophagy
through an mTOR-dependent pathway
Jing Yang,
1,2,†
Ying Zhao,
1,†
Ke Ma,
1
Fen-Jun Jiang,
3
Wenjuan Liao,
1
Ping Zhang,
1
Jingyi Zhou,
1
Bo Tu,
1
Lina Wang,
1
Harm H. Kampinga,
2
Zhiping Xie
3,
* and Wei-Guo Zhu
1,4,
*
1
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education); Department of Biochemistry and Molecular Biology;
4
School of Oncology;
Peking University Health Science Center; Beijing China;
2
Department of Cell Biology; University Medical Center Groningen; University of Groningen;
Groningen, The Netherlands;
3
School of Medicine; Nankai University; Tianjin, China
These authors contributed equally to this work.
Key words: autophagy, hepatocystin (PRKCSH), glucosidase II, mTOR, polycystic liver
Abbreviations: Atg, autophagy-related genes; GFP, green fluorescent protein; LC3, microtubule-associated protein 1 light chain
3; mTOR, mammalian target of rapamycin; PRKCSH, protein kinase C substrate 80K-H; Rheb, Ras homolog enriched in brain;
UPR, unfolded protein response
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Despite our knowledge of the normal function of hepatocys-
tin, little is known about how cells respond to its deficiency. In
many cases, defective protein glycosylation and folding trigger
the unfolded protein response (UPR), which leads to reduction
of protein translation, upregulation of the chaperones, as well
as retrotranslocation and degradation of misfolded proteins.
14
In addition, recent studies suggest autophagy, the major intra-
cellular degradation pathway, can also be induced in response
to ER stress.
15-19
In this pathway, obsolete or damaged cytosolic
proteins and membrane-bound organelles are sequestered into
specialized degradation vesicles, autophagosomes and degraded
upon autophagosome-lysosome/endosome fusion.
20,21
Although
excessive amounts of autophagy can be harmful, autophagy
within a physiological level is generally cytoprotective; and the
critical roles of autophagy in processes such as tumor suppres-
sion, immune response and prevention of neuron degeneration
are well recognized.
22-26
In particular, studies using mouse mod-
els have shown that autophagy is beneficial for the health of the
liver under normal or pathological conditions.
27, 28
Nevertheless,
direct evidence is lacking as for whether hepatocystin deficiency
affects autophagy.
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2 Autophagy Volume 7 Issue 7
hepatocystin alone or is mediated through the Glu II complex.
As shown in Figure 2A, knockdown of hepatocystin reduced the
activity of Glu II to a comparable level as knockdown of the alpha
catalytic subunit (encoded by GANAB), confirming its function
in the Glu II complex. In cells with the alpha subunit knocked
down, the effects we observed on the formation of GFP-LC3
puncta and on the lysosome-dependent degradation of endoge-
nous LC3 and p62 were comparable to those in cells with hepato-
cystin knocked down (Fig. 2BE). Furthermore, a similar result
was obtained for cells treated with bromoconduritol, a specific
inhibitor of Glu II activity (Fig. 2FI). These results demonstrate
that the deficiency of Glu II induces autophagy.
Pathogenic hepatocystin mutants lack autophagy-suppress-
ing ability. ADPLD-associated pathogenic mutations of hepa-
tocystin are distributed across the entire reading frame of 528
amino acids (GenBank Accession No. NP 002734.2), the major-
ity of which result in truncation of the protein. Accordingly, we
chose three representative truncation mutants (156x, 198x and
414x) and tested their effects on the induction of autophagy.
In Hela cells stably expressing GFP-LC3, the expression of the
full-length hepatocystin completely eliminated the autophagy
response introduced by the knockdown of endogenous protein,
including the formation of GFP-LC3 puncta, the production of
free GFP moiety and the degradation of p62 (Fig. 3A–D). In
contrast, none of the truncation variants retained the autophagy
suppressing ability. These mutants also failed to achieve the sub-
cellular localization pattern of the full-length protein (Fig. 3A).
Moreover, the expression of the two shorter variants alone lead
to increased free GFP and decreased p62 even in the presence of
the endogenous protein (Fig. 3C and D), suggesting that these
pathogenic hepatocystin mutants act dominantly in the induc-
tion of autophagy.
The unfolded protein response remains intact. The removal
of glucose residues by Glu II is critical in the processing and fold-
ing of glycoproteins in the ER.
10-12
Since hepatocystin knockdown
impaired Glu II activity (Fig. 2A), we reasoned that it might
induce autophagy through disturbing the UPR pathway.
18,32,33
To
test this hypothesis, we examined the transcriptional regulation
of UPR pathway proteins. The knockdown of hepatocystin did
not significantly change the mRNA levels of GRP78, CHOP and
calreticulin (Fig. 4A and B). In contrast, inhibition of disulfide
bond formation by dithiothreitol (DTT) caused a drastic increase
in their expression, indicating that the UPR signaling pathway is
intact. The amount of ATF6 remained relatively stable, consistent
Here we set out to study the potential link between hepato-
cystin and autophagy. We present evidence that deficiency of
hepatocystin induces autophagy instead of the UPR, and that the
induction is mediated through mammalian target of rapamycin
(mTOR).
Results
Hepatocystin knockdown impairs the function of Glu II and
induces autophagy. To test whether autophagy is induced by a
deficiency of hepatocystin, we first examined the effect of hepa-
tocystin knockdown in Hela cells stably expressing GFP-LC3.
GFP-LC3 serves as a label for autophagic structures since lipid-
conjugated LC3 is concentrated on autophagosomal mem-
branes.
29
In addition, the GFP moiety is relatively stable in the
autolysosomal lumen after the inner vesicle of an autophagosome
and the LC3 moiety are broken down by lysosomal hydro-
lases; thus the amount of free GFP moiety signifies the level of
autophagosome turnover.
30
As shown in Figure 1A and B, the
knockdown of hepatocystin caused a substantial increase in the
number of GFP-LC3 punta. In contrast, few GFP-LC3 puncta
were observed in nonspecific siRNA treated cells. Furthermore,
the amount of free GFP was significantly higher in hepatocystin
knockdown cells than in control cells (Fig. 1C), indicating that
autophagy is induced. To verify that the phenotype is not an arti-
fact of overexpressed GFP-LC3, we then examined the level of
endogenous proteins in Hela cells. As shown in Figure 1D and
E, the knockdown of hepatocystin enhanced the expression of
LC3, especially the lipid-conjugated form, LC3-II. More impor-
tantly, inhibition of lysosomal proteases by E-64 and pepstatin
A led to a higher boost in the amount of LC3-II, indicating that
the knockdown of hepatocystin causes greater turnover of LC3.
We also examined the amount of p62, an LC3-interacting cargo-
adaptor protein that is degraded by autophagy.
31
Consistent with
an increase of autophagic flux, the amount of p62 was decreased
in hepatocystin knockdown cells and the decrease was repressed
by lysosomal protease inhibitors (Fig. 1D). To rule out the pos-
sibility of any off-target effect of the siRNA oligos, we tested
siRNA oligos targeting different regions of the transcript and
found similar results (Fig. 1F and G). Taken together, these data
indicate that hepatocystin knockdown induces autophagy.
Previous reports have demonstrated that hepatocystin func-
tions as the noncatalytic subunit of Glu II in the ER.
5-9
We
therefore examined whether the effect on autophagy is unique to
Figure 1 (See opposite page). Knockdown of hepatocystin induces autophagy. (A and B) Knockdown of hepatocystin increases GFP-LC3 puncta
formation. HeLa cells stably expressing GFP-LC3 were treated with control (NS) or hepatocystin specic siRNA for 72 h. Cells were then observed by
confocal microscopy for GFP-LC3 puncta. (A) Representative images. Scale bar, 10 μm. (B) Quantication of the number of GFP-LC3 puncta per cell.
Error bar, standard deviation (n = 50, from a total of three experimental repeats). (C) Knockdown of hepatocystin increases the processing GFP-LC3.
HeLa cells stably expressing GFP-LC3 were treated as in (A). Cell lysates were analyzed by immunoblotting using antibody against GFP. The experiment
was repeated three times and a representative image is shown. (D and E) Knockdown of hepatocystin enhances lysosome-dependent degradation of
endogenous LC3 and p62. Hela cells were treated with control (NS) or hepatocystin specic siRNA for 12 h. Cells were then treated with lysosomal pro-
tease inhibitors (10 mg/ml of E64 and Pepstatin-A) or mock treated with DMSO for 48 h. Cell extracts were analyzed by immunobloting as indicated. (D)
Representative images of immunoblotting analysis. (E) Quantication of the amounts of LC3-II relative to actin. The average value in the control cells
without inhibitor treatment was normalized as 1. Error bar, standard deviation from three experimental repeats. (F and G) siRNA oligos targeting dier-
ent regions of the hepatocystin transcript produce the same eect. HeLa cells stably expressing GFP-LC3 were treated as in (A). Oligo 2 targets another
region of the ORF; Oligo 3 targets a noncoding region. (F) Representative images of immunoblotting analysis. (G) Quantication of the amounts of free
GFP relative to actin. The average value in the control cells was normalized as 1. Error bar, standard deviation from three experimental repeats.
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Figure 1. For gure legend, see page 2.
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4 Autophagy Volume 7 Issue 7
Figure 2. Deciency of Glu II complex induces autophagy. (A) Knockdown of hepatocystin reduces Glu II activity. Hela cells were treated with control
(NS), hepatocystin or Glu II alpha subunit specic siRNA for 72 h. Cell lysates were collected and assayed for Glu II activity. The activity in control siRNA
treated cells was normalized as 1. Error bar, standard deviation from three experimental repeats. (B and C) Knockdown of the alpha subunit of Glu II
enhances lysosome-dependent degradation of endogenous LC3 and p62. Hela cells were treated with indicated siRNA oligos and lysosomal protease
inhibitors as in Figure 1D. Immunoblotting results are presented as in Figure 1D and E. (D and E) Knockdown of the alpha subunit of Glu II increases
GFP-LC3 puncta formation. Hela cells expressing GFP-LC3 were treated with indicated siRNA oligos and analyzed by uorescence microscopy as in Fig-
ure 1A and B. (F–I) Inhibition of Glu II activity induces autophagy. Hela cells expressing GFP-LC3 were treated with bromoconduritol, a specic inhibitor
of Glu II or mock treated for 4 h. (F and G) Cell lysates were analyzed by immunoblotting as in Figure 1F and G. (H and I) Fluorescence microscopy
results are presented as in Figure 1A and B. Scale bar, 10 μm.
Page 4
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attenuated starvation-induced autophagy or the inhibition of
mTOR (Fig. 7A and B). These results indicate that even though
both hepatocystin deficiency and nutrient starvation inhibit
mTOR activity, the signaling pathways involved under these two
conditions are not identical.
Discussion
In this study, we show that the deficiency of hepatocystin impairs
the function of Glu II and induces autophagy. The induction
of autophagy is caused not only by the loss of normal endog-
enous proteins, but also by the presence of pathogenic hepatocys-
tin mutants. During this process, the inhibition of mTOR is an
essential step; and the autophagy response can be eliminated if
the activity of mTOR is maintained. The regulation of IP3R by
hepatocystin may contribute to the autophagy-inducing effect,
although it is not necessary. The impairment of Glu II activity
under such condition causes neither the activation nor the dys-
regulation of the UPR pathway. These results shed new light on
the function of hepatocystin and provide a novel dimension on
the regulatory network of autophagy.
The role of Glu II complex in the processing of glycoproteins
in the ER implies that ADPLD might be caused by the folding
defect of certain proteins in the secretory pathway.
3
The UPR
is often the weapon of choice to alleviate the ER stress under
such conditions.
14
Interestingly, this is not the case for cells with
deficiency in hepatocystin. The depletion of hepatocystin and
hence the reduction of Glu II activity does not induce the UPR,
nor does it interfere with the induction of the UPR by a DTT
treatment (Fig. 4). Instead, loss of Glu II activity was associated
with an induction of autophagy. Does autophagy serve to limit
further potential damage caused by the loss of hepatocystin or
contribute to cell damage and eventually to the development
of ADPLD? Currently, several lines of indirect evidence favor a
beneficial role of autophagy under such conditions. Firstly, auto-
phagy is critical for the health of liver under both normal and
pathological conditions. Loss of autophagy in the liver leads to
abnormal enlargement of the organ and accumulation of aber-
rant membrane structures and protein aggregates.
27
In a disease
model with pathogenic aggregate-prone protein in the liver, auto-
phagy is instrumental in its clearance.
28,40
Secondly, autophagy
protects cells from glycosylation-related ER stress. Tunicamycin
is a commonly used antibiotic for ER stress research. By interfer-
ing with the synthesis of oligosaccharide precursors, it acts on the
same pathway where Glu II functions: N-linked protein glycosyl-
ation.
41
For cells challenged with tunicamycin, the induction of
autophagy confers a survival advantage.
15,16
Thirdly, suppression
of mTOR reduces cystogenesis in autosomal-dominant polycystic
kidney disease (ADPKD). ADPLD is closely related to ADPKD,
although the latter is a more severe disease that affects both the
kidney and the liver.
4
Genetic defects in ADPKD lead to acti-
vation of mTOR; and the suppression of mTOR by rapamycin
reduces cyst formation.
42,43
Although these studies did not exam-
ine autophagy directly, it is reasonable to assume that autophagy
was induced by the rapamycin treatment and therefore might
contribute to the observed therapeutic effect.
with its primary mode of regulation being post-translational pro-
cessing, instead of transcriptional variations. Additionally, DTT
treatment did not change the expression of hepatocystin (Fig.
4A); and the DTT-induced UPR was not affected by hepatocys-
tin knockdown. These results suggest that despite the reduction
of Glu II activity, the deficiency of hepatocystin does not disturb
the UPR pathway.
Hepatocystin, but not the catalytic subunit of Glu II,
regulates IP3R-mediated calcium flux. Hepatocystin interacts
directly with the IP3R, an ER-resident calcium channel known
to be involved in the regulation of autophagy.
13,34,35
Consistent
with existing reports, we found that the knockdown of hepato-
cystin significantly attenuated ATP-induced transient calcium
flux (Fig. 5A).
13,36
On the other hand, we found no significant
change in the steady-state concentrations of calcium in the cyto-
sol or in the lumen of ER (Fig. 5B), suggesting that the regula-
tion of autophagy by hepatocystin does not rely on alterations in
resting calcium levels. Interestingly, the knockdown of the cata-
lytic subunit of Glu II did not affect the activity of IP3R (Fig.
5A), even though autophagy was efficiently induced under such
condition (Fig. 2BE). These results indicate that the inhibi-
tion of IP3R-mediated transient calcium flux is not necessary for
the induction of autophagy and that at least another pathway is
involved.
The inhibition of mTOR is critical for the induction of auto-
phagy. To identify the pathway responsible for the induction of
autophagy by hepatocystin deficiency, we next examined the
involvement of mTOR signaling pathway. Although well-known
for its central role in nutrient-deprivation induced autophagy,
recent studies suggest mTOR is also regulated by ER stress.
19,37
As
shown in Figure 6A, nutrient starvation led to a clear reduction
in the phosphorylation level of an mTOR substrate, ribosomal
protein S6 kinase 1 (S6K1).
38
A similar result was observed in
hepatocystin knockdown cells, even though the total amount of
S6K1 remained stable, suggesting that hepatocystin deficiency
inhibits mTOR.
We then investigated whether the inhibition of mTOR is a
necessary step in the induction of autophagy under such con-
dition. For this purpose, we overexpressed a constitutive active
form of Rheb (Rheb Q64L), which positively regulates mTOR.
39
As shown in Figure 6B, the expression of Rheb Q64L restored
the activity of mTOR in hepatocystin knockdown cells.
Concurrently, it also suppressed the degradation of p62 and
the increase in LC3 lipidation, indicating that the induction of
autophagy is abolished. In cells expressing GFP-LC3, the expres-
sion of Rheb Q64L eliminated the increase in the number of
GFP-LC3 puncta and the amount of free GFP (Fig. 6DG). In
contrast, expression of an inactive form of Rheb (Rheb D60K)
affected neither the activity of mTOR nor the level of autophagy
(Fig. 6BG). Taken together, these results indicate that the inhi-
bition of mTOR constitutes the definitive step in the induction
of autophagy by hepatocystin deficiency.
It should be noted, however, that there was no detectable
change in the expression of hepatocystin or the alpha subunit of
Glu II in cells under nutrient starvation (Fig. 7A). Furthermore,
overexpression of neither hepatocystin nor the alpha subunit
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6 Autophagy Volume 7 Issue 7
Figure 3. For gure legend, see page 7.
Page 6
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www.landesbioscience.com Autophagy 7
It should be noted, however, that only a few
clinical studies of polycystic liver have been per-
formed with ADPLD clearly distinguished from
ADPKD.
44,45
At present, it is not known whether
there is a constitutive inhibition of mTOR path-
way in patients affected by P R KCSH mutations.
Furthermore, the effect of mTOR inhibition is not
limited to autophagy induction. A major function of
mTOR substrate S6K1, for instance, is the regula-
tion of general protein translation, which might also
account for the observed therapeutic effect.
46
Future
studies, ideally containing data from both clinical
surveys and animal models, will be needed to pro-
vide a definitive answer for the role of mTOR path-
way and autophagy in the development of ADPLD.
Based on our current data, the inhibition of
mTOR is an essential step in the induction of auto-
phagy by hepatocystin deficiency (Fig. 6). The
regulation of autophagy by TOR is well estab-
lished, with the ULK1/Atg1 kinase complex being
the most prominent downstream effector.
47-50
The
mechanism linking hepatocystin deficiency to the
downregulation of mTOR, however, is unresolved
at present. Alterations in intracellular calcium
level have been shown to regulate the activity of
mTOR.
51,52
Whereas the knockdown of hepatocys-
tin did affect the transient flux of calcium through
IP3R,
13
the knockdown of the catalytic subunit of
Glu II did not (Fig. 5A), even though autophagy was
efficiently induced under such condition (Fig. 2).
Moreover, we did not detect any significant changes
in the resting calcium concentrations in either case
(Fig. 5B). Likewise, although components of the
UPR pathway have also been involved in the regula-
tion of autophagy,
18,32,33
the UPR was not induced by
hepatocystin deficiency (Fig. 4). The identification
of the component that directly senses the deficiency
of hepatocystin would be instrumental in further
elucidating the underlying signaling pathway.
Materials and Methods
Culture media and chemicals. Cells were grown
in DMEM medium (GIBCO, 12800-017) supple-
mented with 10% heat-inactivated fetal bovine
serum (Hyclone, SV30087.02) and 1% penicillin-
streptomycin (Sigma, P4333), in a 37°C incuba-
tor with a humidified, 5% CO
2
atmosphere. The
chemicals used in our experiments were: E-64
Figure 3 (See opposite page). Pathogenic hepatocystin mutants lack autophagy-suppressing ability. (A–D) HeLa cells expressing GFP-LC3 were rst
transfected with an empty vector or vectors expressing indicated variants of hepatocystin. After 24 h, cells were further treated with control (NS)
siRNA oligo or one targeting a noncoding region of the hepatocystin transcript for 72 h. (A and B) For uorescence microscopy analysis, the signal
of GFP-LC3 was imaged directly; the signal of hepatocystin variants was obtained by an immunouorescence procedure using anti-myc primary
antibody and CY3-conjugated secondary antibody. Data are presented as in Figure 1A and B. Scale bar, 10 μm. (C and D) Immunoblotting results are
presented as in Figure 1F and G.
Figure 4. Knockdown of hepatocystin does not induce the UPR. (A and B) Hela cells
were treated with control (NS) or hepatocystin specic siRNA for 72 h. Cells were then
treated with 1 mM DTT or mock treated for 2 h. The relative amounts of mRNA were
analyzed by real-time PCR. The amounts of mRNA in mock treated NS RNAi cells were
normalized as 1. (A) Eciency of siRNA treatment. (B) Eects of hepatocystin silencing
and DTT on the expression of UPR genes. Error bar, standard deviation (n = 3). The
experiment was repeated three times and a representative result is shown.
Page 7
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8 Autophagy Volume 7 Issue 7
CTC GAG CGG CCG CTA GCT GTA CAG GTA AGC
AAA C; 198X Reverse, ATG CTC GAG CGG CCG CTA
CAG CTT CTG GTG CTG CTC T; 156X Reverse, ATG
CTC GAG CGG CCG CTA CTT CTC CTC CCG TGC
CTT C. The amplified fragments were digested with Hind III
and Not I and inserted back to the original pRcCMV-myc-
GluII-beta plasmid treated with the same enzymes.
Cell transfection and establishment of stable cell lines.
Transfection of siRNA oligos and plasmids was performed using
Lipofectamine 2000 (Invitrogen, 11668019) following protocols
from the manufacturer. Cell lines stably expressing G1ER and
TN-XXL were selected in 200 μg/ml G418.
RNAi treatment. siRNA mediated gene knockdown was per-
formed with the following synthetic siRNA duplexes: nonsilenc-
ing, UUC UCC GAA CGU GUC ACG U; Hepatocystin #1,
GCA CCA ACA CUG GCU AUA A, Hepatocystin #2, TAC
GAG CTC ACC ACC AAC G, Hepatocystin #3, GCG CAG
AGA ACC UCA AGA A; Glu II alpha subunit #1, UGA UAU
AAU CCG AGA UGC C, Glu II alpha subunit #2, GCC ATA
(Sigma, E3132), Pepstatin A (Sigma, P5318), Bromoconduritol
(Santa Cruz, sc-201367), DTT (Tiangen, RT103), ATP (Sigma,
A26209), G418 (Sigma, A1720), 4-methylumbelliferyl-α-D-
glucopyranoside (4-MUG) (Sigma, M9766), Paraformaldehyde
(Dingguo, 7B500548).
Cell lines and plasmids. HeLa cell line was purchased from
the American Type Culture Collection. Plasmid pEGFP-LC3
was a gift from Dr. Noboru Mizushima. Hela cell line stably
expressing GFP-LC3 was a gift from Dr. Aviva M. Tolkovsky.
The pRcCMV-myc-GluII-beta and pRcCMV-myc-GluII-alpha
plasmids were gifts from Dr. Kazuo Yamamoto. Plasmid pD1ER
was a gift from Dr. Tullio Pozzan with permission from Dr. Roger
Tsien. Plasmid pTN-XXL was a gift from Dr. Oliver Griesbeck.
The Rheb-D60K-myc and Rheb-Q64L-HA plasmids were gifts
from Dr. Kun-Liang Guan.
Vectors expressing truncated variants of hepatocystin were
constructed from pRcCMV-myc-GluII-beta. The following
primers were used to amplify the truncated fragments: Forward,
AAG CAG AGC TCT CTG GCT AAC; 414X Reverse, ATG
Figure 5. The inhibition of IP3R activity is not required for
autophagy induction by Glu II deciency. (A) Knockdown
of hepatocystin, but not the alpha subunit of Glu II,
reduces IP3R activity. Hela cells stably expressing TN-XXL,
a cytosolic calcium sensor, were treated with indicated
siRNA oligos for 72 h before confocal microscopy ob-
servation. FRET signals were collected every 20 s for a
period of 340 s. 40 s after observation started, cells were
stimulated with 20 μM ATP. (B) Knockdown of hepatocys-
tin does not change resting calcium levels in the cytosol
or the ER lumen. Hela cells stably expressing TN-XXL or
D1ER, an ER-localized calcium sensor, were treated with
control (NS), hepatocystin or IP3R specic siRNA for 72 h
and FRET signals were collected. Error bar, standard devia-
tion (n = 10). The experiment was repeated three times
and a representative result is shown.
Figure 6 (See opposite page). Hepatocystin regulates
autophagy through mTOR. (A) Knockdown of hepatocys-
tin inhibits mTOR. Hela cells were treated with control
(NS) or hepatocystin specic siRNA for 72 h or serum-
starved for 6 h. Cell extracts were analyzed by immunob-
lotting as indicated. The knockdown of hepatocystin led
to reduced phosphorylation of S6K1, an mTOR substrate.
(B–G) Activation of mTOR blocks hepatocystin-knock-
down induced autophagy. (B and C) HeLa cells were rst
transfected with empty vector or vectors expressing con-
stitutively active (Q64L) or inactive (D60K) Rheb mutants.
After 24 h, cells were further treated with control (NS) or
hepatocystin specic siRNA for 48 h. Immunoblotting re-
sults are presented as in Figure 1D and E. (DG) Hela cells
expressing GFP-LC3 were treated as in (B) and analyzed
by uorescence microscopy or immunoblotting. (D and E)
For uorescence microscopy analysis, the signal of GFP-
LC3 was imaged directly; the signal of Rheb variants was
obtained by an immunouorescence procedure using
anti-HA (Q64L) or anti-myc (D60K) primary antibody and
CY3-conjugated secondary antibody. Data are presented
as in Figure 1A and B. Scale bar, 10 μm. (F and G) Immu-
noblotting data are presented as in Figure 1F and G.
Page 8
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Autophagy 9
Figure 6. For gure legend, see page 8.
Page 9
©2011 Landes Bioscience.
Do not distribute.
10 Autophagy Volume 7 Issue 7
AAC G, TCA CCA TTC GGT CAA TCA GAG C; ATF6,
CTG GAT GAA GAT TGG GAT T, TGA CCG AGG AGA
CGA GAC; GAPDH, GAA GGT GAA GGT CGG AGT C,
GAA GAT GGT GAT GGG ATT TC.
Immunoblot analysis. Immunoblot analysis of cellular pro-
teins was performed as described previously using the following
antibodies:
26
Anti-Hepatocystin/PRKCSH (Santa Cruz, sc-10774), anti-
LC3(Cell Signaling Technology, 2775), anti-p62/SQSTM1
(MBL, PM045), anti-phospho-S6K1 thr389 (Cell Signaling
Technology, 9206), anti-S6K1 (Bethyl, A300-510A), anti-mTOR
(Cell Signaling Technology, 2973), anti-myc (MBL, M047-3),
anti-HA (Santa Cruz, sc-805), anti-Actin (Santa Cruz, sc-7210).
Glu II activity assay. The activity of Glucosidase II was mea-
sured as described previously with minor modifications.
53
In
brief, the assay was performed at 37°C in 0.1 ml of reaction buf-
fer containing 2 mM 4-MUG and 50 mM PIPES (pH 7.0). The
reaction was terminated after 3 h by the addition of 1.5 ml of 0.5
M glycine (pH 10.7). Fluorescence of 4-methylumbelliferone was
measured by a Hitachi F-4500 spectrophotometer (excitation:
366 nm, emission: 450 nm).
Confocal microscopy. Cells were plated on coverslips and
treated as indicated. The cells were then fixed with 4% para-
formaldehyde (15 min incubation at room temperature) and
observed by an Olympus FV1000 confocal microscope using
default settings. Images were collected with the center of nucleus
in focus. Immunofluorescence microscopy was performed as
described previously using the following antibodies: anti-myc
(MBL, M047-3), anti-HA (Covance, MMS-101R), Cy3-Linked
Goat-anti-mouse IgG (GE Healthcare, PA43002).
26
Calcium concentration analysis. The concentration of
intracellular calcium was measured as described previously
in reference 54–56. Briefly, Hela cell lines stably expressing
TN-XXL (cytosolic calcium sensor) or D1ER (ER lumenal
calcium sensor) was established. To measure resting calcium
concentrations, FRET signal was collected by confocal micros-
copy from un-stimulated cells. To measure the activity of IP3R,
cells expressing TN-XXL was stimulated with 20 mM ATP and
monitored continuously to record calcium flux. For each experi-
mental group, fluorescence signals from 10 representative cells
were quantified. The software used was Olympus Fluoview.
Acknowledgements
The authors thank Dr. Oliver Griesbeck (Max Planck Institute
of Neurobiology, Germany), Dr. Kun-Liang Guan (University
of California, USA), Dr. Sergio Lavandero (University of Chile),
Dr. Noboru Mizushima (Tokyo Medical and Dental University,
Japan), Dr. Hanne Ostergaard (University of Alberta, Canada),
Dr. Tullio Pozzan (University of Padua, Italy), Dr. Roger Tsien
(University of California, USA), Dr. Aviva M. Tolkovsky
(University of Cambridge), Dr. Kazuo Yamamoto (University of
Tokyo, Japan), Dr. Li Yu (Tsinghua University, China) and Dr.
Dong Zhong (Medical College of Georgia, USA) for technical
advice and gifts of cell lines and plasmids.
This work was supported by National Key Basic Research
Program of China (Grant 2011CB910100 to Y.Z. and Z.X.,
TCG CCT CTA CAA T; IP3R1 #1,GCA CCA GCA GCU
ACA ACU A, IP3R1 #2, CCA GCA GAA UCA AGC UUU G.
Real-time PCR analysis of mRNA. Total RNA was isolated
with TRIzol reagent (Invitrogen, 15596026). Reverse transcrip-
tion was performed from 2 μg of total RNA with oligo(dT)18
primers using QuantScript RT (Tiangen, KR03-03). Primer
sequences for real-time PCR analysis were retrieved from
PrimerBank (http://pga.mgh.harvard.edu/primerbank/). The
sequences are: Hepacystin, TCG CAG AAA CCC AAA CTC
G, CCC GTG CCT TGC TCA TAC; Glu II alpha, AGC CTG
AGG AAA CAC CCA, GCC AGA AGA TGC CCA AGT;
IP3R1, GCG GAG GGA TCG ACA AAT GG, TTT TGG GCA
GAG TAG CGG TTC; GRP78, CAC GCC GTC CTA TGT
CGC, AAA TGT CTT TGT TTG CCC ACC; Calreticulin,
CCC GCC GTC TAC TTC AAG G, GAA CTT GCC GGA
ACT GAG AAC; CHOP, ACC AAG GGA GAA CCA GGA
Figure 7. Overexpression of Glu II subunits does not aect starvation-
induced autophagy. (A and B) Hela cells were transfected with empty
vector or vectors expressing hepatocystin or Glu II alpha subunit. After
48 h, cells were serum-starved or further cultured in complete medium
for 6 h. Immunoblotting results are presented as in Figure 1D and E.
Page 10
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www.landesbioscience.com Autophagy 11
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Page 12
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    • "What kind of role autophagy is playing in these diverse liver pathologies is still largely unknown. Recently, we revealed that in polycystic liver disease (PCLD), autophagy is activated by the deficiency of hepatocystin, of which mutants have been shown to cause PCLD 141. Therefore, in PCLD, autophagy inhibition might be a strategy to delay pathogenesis. "
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