Skip Navigation


Human Molecular Genetics Advance Access originally published online on July 29, 2008
Human Molecular Genetics 2008 17(20):3254-3262; doi:10.1093/hmg/ddn221
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
17/20/3254    most recent
ddn221v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liang, G.
Right arrow Articles by Chen, X.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liang, G.
Right arrow Articles by Chen, X.-Z.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2{alpha}

Genqing Liang{dagger}, JungWoo Yang{dagger}, Zuocheng Wang, Qiang Li, Yan Tang and Xing-Zhen Chen*

Membrane Protein Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

* To whom correspondence should be addressed. Tel: +1 7804922294; Fax: +1 7804928915; Email: xzchen{at}ualberta.ca

Received June 12, 2008; Accepted July 25, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of renal, hepatic and pancreatic cysts and by non-cystic manifestations such as abnormal vasculature and embryo left–right asymmetry development. Polycystin-2 (PC2), in which mutations account for 10–15% of ADPKD, was previously shown to down-regulate cell proliferation, but the underlying mechanism was not elucidated. Here, we demonstrate that PC2, but not pathogenic mutants E837X and R872X, represses cell proliferation through promoting the phosphorylation of eukaryotic translation initiation factor eIF2{alpha} by pancreatic ER-resident eIF2{alpha} kinase (PERK). ER stress is known to enhance eIF2{alpha} phosphorylation through up-regulating PERK kinase activity (assessed by phosphorylated PERK). During ER stress, PC2 knockdown also repressed eIF2{alpha} phosphorylation but did not alter PERK phosphorylation, indicating that PC2 facilitates the eIF2{alpha} phosphorylation by PERK. PC2 was found to be in the same complex as PERK and eIF2{alpha}. Together, we demonstrate that PC2 negatively controls cell growth by promoting PERK-mediated eIF2{alpha} phosphorylation, presumably through physical interaction, which may underlie a pathogenesis mechanism of ADPKD and indicates that PC2 is an important regulator of the translation machinery.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of PKD and occurs in 0.1–0.2% of the adults (1–3). Renal pathogenic polycysts in ADPKD are fluid-filled epithelial-lined cavities arising from glomeruli, nephron tubules and collecting ducts (4,5). ADPKD can also result in cerebral and intracranial aneurysms, cardiovascular abnormalities (e.g. cardiac valve disorders), aberrant embryonic left–right asymmetry development and hypertension (6,7). The majority (~95%) of the ADPKD cases are caused by mutations in the PKD1 or PKD2 gene that encodes polycystin-1 (PC1) or polycystin-2 (PC2) (8,9). PC1 is an integral membrane glycoprotein and acts as a G-protein-coupled receptor. PC2 is a non-selective cation channel and is mainly localized on the ER membrane as a Ca release channel (10,11).

The mechanisms of how ADPKD results in over-proliferation of renal epithelial cells are not clearly understood, and different pathways have been reported to be implicated (12–15). PC1 regulates cell growth of renal tubular epithelial cells through p53 induction and JNK activation (15). PC2 was also reported to suppress cell growth and branching morphogenesis in kidney epithelial cells, but the underlying mechanism remains unknown (13). Bhunia et al. (12) showed that PC1 and PC2 together regulate cell cycle through inducing p21 and activating the JAK-STAT signaling pathway. On the other hand, Li et al. (14) demonstrated that PC2 undergoes PC1-dependent phosphorylation, which enhances its interaction with the helix–loop–helix inhibitor ld2, to regulate cell growth and differentiation.

ER is critical to the synthesis, modification, folding and quality control of both secretory and membrane protein (16). Conditions disrupting the ER homeostasis can cause unfolded protein accumulation that constitutes a fundamental threat to all living cells and triggers unfolded protein response (UPR), which is currently termed ER stress (17). During the ER stress response or UPR, ER-resident molecular chaperones and foldases are induced to enhance the folding capacity of the ER, and translation is attenuated to reduce the biosynthetic load of ER (17,18). Current studies of the UPR mechanism in mammalian cells have identified three branches of the signaling pathway, represented by three types of ER-transmembrane proteins: pancreatic ER eIF2{alpha} kinase (PERK) leading to phosphorylation of eIF2{alpha} that causes translational repression, activating transcription factor 6 (ATF6) which induces the expression of chaperones such as BiP, and inositol requiring 1 (IRE1) which initiates spliceosome-independent splicing of XBP1 mRNA, leading to the activation of ER chaperone genes.

Type I ER membrane protein PERK was recently reported to be involved in controlling cell growth (19,20). PERK contains a luminal ER stress-sensing N-terminal domain and a large C-terminal cytoplasmic kinase domain (18,21). During ER stress, PERK is activated by autophosphorylation via homodimerization. The phosphorylated (or activated) PERK (P-PERK) subsequently binds and phosphorylates its subunit eIF2{alpha} (21,22), which inhibits global protein synthesis and cell-cycle factors such as cyclin D1, and results in the repression of cell proliferation (19,20,23). We demonstrate here that PC2 negatively regulates cell proliferation through enhancing PERK-dependent eIF2{alpha} phosphorylation. Our study suggests that PC2-associated ADPKD pathogenesis is due at least in part to dysregulation of PERK-dependent cell growth.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
PC2 facilitates the eIF2{alpha} phosphorylation by PERK
We recently reported that Herp, a regulator of ER-associated degradation (ERAD) (24–26, 28), interacts with PC2 and regulates its expression (27). Here, we started to test whether PC2 is involved in the regulation of UPR activated during ER stress (17,18). Immunoblotting experiments revealed that PC2 over-expression increases the phosphorylated eIF2{alpha} (P-eIF2{alpha}) level by 227 ± 7% (P < 0.001, N = 3) in human embryonic kidney (HEK) 293T cells (Fig. 1A), with quantitative analysis performed using the program Image J (v1.41c, NIH, http://rsb.info.nih.gov/ij/). Similar results were also shown in human melanoma A7 and Madin–Darby canine kidney (MDCK) cells (Fig. 1B and Supplementary Material, Fig. S1). In the presence of ER stress induced by thapsigargin (Tg) through ER Ca depletion, P-eIF2{alpha} substantially augmented, as expected. The combination of PC2 over-expression and Tg treatment, compared with Tg treatment alone, did not significantly further increase the P-eIF2{alpha} level (103% ± 5%, P = 0.29, N = 3), possibly because of a saturated P-eIF2{alpha} level induced by Tg. Of important note, PC2 over-expression had no effect on immunoglobulin heavy-chain binding protein (BiP, also called GRP78, an ER chaperone protein) (Fig. 1A and B) and splicing of XBP-1 mRNA (Supplementary Material, Fig. S2), both of which are activated during UPR (17,21), indicating that high levels of PC2 do not cause ER stress leading to UPR. This suggests that the effect of PC2 over-expression is not through triggering ER stress. It is known that increased P-eIF2{alpha} attenuates protein synthesis and selectively up-regulates the translation of the activating transcription factor 4 (ATF4) (29). We next tested the effects of PC2 on these downstream signals of P-eIF2{alpha}. Compared with control, GFP-expressing cells, GFP-PC2-expressing cells exhibited profound attenuation of global translation rates, as demonstrated using [35S]methionine/cystine incorporation (Fig. 1C). Consistently, the protein synthesis rate negatively correlated with the level of P-eIF2{alpha} (Fig. 1C). In addition, Western blotting showed that ATF4 expression is increased in cells expressing GFP-PC2, compared with control cells (Fig. 1D). Thus, these effects of PC2 are consistent with the reported downstream signaling of eIF2{alpha} phosphorylation. Also, we found that over-expressed human anion exchanger AE1 (Gift of Dr Joe Casey, University of Alberta) in HEK293T cells has no effect on the P-eIF2 level (Fig. 1E), further supporting the specificity of the PC2-mediated up-regulation of P-eIF2.


Figure 1
View larger version (85K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Effects of over-expressed PC2 on eIF2{alpha} phosphorylation. (A) and (B) Effect of PC2 on eIF2{alpha} phosphorylation. Extracts of HEK293T cells transiently transfected with GFP-PC2 for 40 h (A), and A7 cells with a stable expression of GFP-PC2 (B) were used for immunoblotting with an antibody against P-eIF2{alpha}, eIF2{alpha}, BiP or GFP. Treatment with Tg (0.5 µM for 1 h) was used as a positive control. β-actin was used as a loading control. (C) Effect of PC2 on global protein synthesis. HEK293T cells expressing GFP-PC2 or GFP were labeled with [35S]methionine/cystine for 10 min. Whole-cell extracts were resolved by SDS–PAGE and viewed by autoradiography (upper panel). P-eIF2{alpha} and eIF2{alpha}, in the same cell extracts, were revealed by Western blotting. (D) Effect of PC2 on ATF4 translation. Extracts of HEK293T cells transiently transfected with GFP-PC2 for 40 h were used for immunoblotting with an antibody against ATF4, P-eIF2{alpha} or GFP. (E) Effect of AE1 on eIF2{alpha} phosphorylation. Extracts of HEK293T cells transfected with plasmid pEGFP, pEGFP-PC2, pRBG4 or pRBG4-AE1 were subjected to immunoblotting with a P-eIF2{alpha}, eIF2{alpha}, GFP, AE1 or β-actin antibody. Tg treatment was a positive control.

 
We further tested the effect of PC2 on eIF2{alpha} phosphorylation by small interference RNA (siRNA) knockdown (27,30). PC2 siRNA caused robust reduction in the PC2 and P-eIF2{alpha} levels in HEK293T cells and mouse inner medullary collecting duct (IMCD) cells (Fig. 2A and B). Thus, PC2 is a regulator of eIF2{alpha} phosphorylation. eIF2{alpha} is known to be phosphorylated by four Ser/Thr protein kinases (activated by different stress inducers): GCN2 (activated by nutrition limitation and UV irradiation), PERK (by ER stress), PKR (by double-stranded RNA and viral infection) and HRI (oxidative stress) (31). To identify the kinase(s) that is responsible for the PC2-regulated eIF2{alpha} phosphorylation, we examined effects of different stress inducers on eIF2{alpha} phosphorylation in HEK293T cells with or without PC2 knockdown. Our data showed that the effect of Tg (Fig. 2A), but not that of oxidative inducer arsenite (Fig. 2A), amino acid (aa) depletion or osmotic shock (data not shown), on eIF2{alpha} phosphorylation substantially diminished with the PC2 knockdown. A similar effect of PC2 knockdown was observed in IMCD cells (Fig. 2B). These results indicate that PC2 is crucial for ER stress-induced eIF2{alpha} phosphorylation. As PERK is known to be the major kinase phosphorylating eIF2{alpha} during ER stress (18,21), we tested whether PERK is involved. Indeed, PERK knockdown significantly repressed PC2-induced eIF2{alpha} phosphorylation in HEK293T cells (Fig. 2C). Together, our data showed that PC2 promotes eIF2{alpha} phosphorylation by the ER-resident eIF2{alpha} kinase PERK. In contrast, we found that PERK phosphorylation is not affected by either over-expression or knockdown of PC2 (Fig. 1A and B, and Fig. 2A), which indicates that PC2 is a crucial factor that facilitates eIF2{alpha} phosphorylation by phosphorylated (i.e. activated) PERK.


Figure 2
View larger version (51K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Effects of PC2 and PERK knockdown on eIF2{alpha} phosphorylation. (A) and (B) Alteration of eIF2{alpha} phosphorylation by PC2 knockdown. HEK293T (A) and IMCD (B) cells were transfected with PC2 siRNA or control siRNA (Ctrl) (20 µl of 20 µM in 35 mm dishes) for 40 h with or without arsenite (As, 200 µM for 1 h) or Tg (0.5 µM for 1 h) before immunoblotting. (C) Effect of PERK knockdown on PC2-induced eIF2{alpha} phosphorylation. Extracts of HEK293T cells co-transfected with plasmid GFP-PC2 and PERK siRNA were subjected to immunoblotting with a PERK, P-eIF2{alpha}, eIF2{alpha} or β-actin antibody.

 
Positive correlation between the PC2 and P-eIF2{alpha} levels revealed by immunofluorescence
We performed co-immunofluorescence experiments to further document the regulation of P-eIF2{alpha} by PC2. To enlarge the range of PC2 expression levels in the same images taken by the microscope, 24 h prior to experiments, we mixed HEK293T cells transiently expressing PC2 with those with PC2 siRNA knockdown at the 20:80 ratio. We found that the PC2 expression level significantly correlates with that of P-eIF2{alpha}, with a correlation coefficient of 0.82 based on 290 randomly selected cells (Fig. 3A and B) and that PC2 and P-eIF2{alpha} partially colocalizes in perinuclear regions (Fig. 3A). Mixing native HEK293T cells with those with PC2 knockdown by siRNA at the 20:80 ratio produced similar results (Fig. 3C), with a correlation coefficient of 0.59 based on 107 randomly selected cells. These results support the concept that PC2 promotes the phosphorylation of P-eIF2{alpha} by the ER membrane kinase PERK, leading to the accumulation of P-eIF2{alpha} in perinuclear regions.


Figure 3
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Correlation between the PC2 and P-eIF2{alpha} levels revealed by immunofluorescence. (A) Co-immunofluorescence of PC2 and P-eIF2{alpha} from a representative image. Cells were stained with antibodies against P-eIF2{alpha} and PC2. The arrow indicates a cell with low expression of PC2 and P-eIF2{alpha}. (B) Relationship between normalized cellular levels of PC2 and P-eIF2{alpha} obtained from 290 randomly selected HEK293T cells. Cells were a mix of those expressing Myc-PC2 and those with PC2 siRNA knockdown, at the 20:80 ratio. The straight line was a linear fit using Sigmaplot 10, resulting in a correlation coefficient of 0.82. (C) Co-immunofluorescence of PC2 and P-eIF2{alpha} in a mix of HEK293T cells with PC2 siRNA or control siRNA at the 20:80 ratio. Cells were stained with the P-eIF2{alpha} and PC2 antibodies.

 
PERK is required for PC2-regulated cell proliferation
It was previously shown that PERK represses cell growth by inducing eIF2{alpha} phosphorylation to attenuate protein synthesis and by down-regulating cyclin D1 required for cell-cycle exit from the G1 phase (19,20,23). We thus reasoned that the reported down-regulation of cell growth by PC2 may be through the PERK-eIF2{alpha} signaling pathway. We measured cell growth rate using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) as a function of alterations in the PC2 and PERK levels. We found that an increased (or decreased) PC2 expression significantly down-regulates (or up-regulates) proliferation of MDCK and/or HEK293T cells (Fig. 4), consistent with the reported anti-proliferative effect of PC2 (12–14). The PC2-mediated suppression of cell growth was significantly inhibited in HEK293T cells by PERK knockdown (Fig. 4D), which is in parallel to the inhibition of the PC2-promoted eIF2{alpha} phosphorylation by PERK knockdown (Fig. 2C). In contrast, PERK knockdown slightly, but not significantly, increased the rate of cell growth in our assays. Overall, these results indicate that PERK and eIF2{alpha} play key roles in the negative control of cell proliferation by PC2.


Figure 4
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Effects of PC2 and PERK on cell proliferation. Five thousand cells from MDCK cells stably (A) and HEK293T cells transiently (B) expressing GFP-PC2 or GFP were plated in multiple wells of a 96-well plate and grown for 48 h for cell proliferation assay (see Materials and Methods). (C) Five thousand cells from HEK293T cells with siRNA transfection for 24 h were plated in multiple wells of a 96-well plate and grown for 30 h for cell proliferation assay. (D) Five thousand cells from HEK293T cells expressing GFP-PC2 or GFP with PERK siRNA transfection for 24 h were plated in multiple wells of a 96-well plate and grown for 30 h for cell proliferation assay.

 
PC2 physically interacts with PERK and eIF2{alpha}
We next explored the possible association of PC2 with PERK and eIF2{alpha}. For this end, we immunoprecipitated extracts of IMCD cells using an anti-PC2 antibody (27,30). We found that PERK and eIF2{alpha}, but not the ER membrane protein ATF6{alpha} (negative control) (32), co-precipitate with PC2 (Fig. 5A). Reciprocally, PC2 was immunoprecipitated by PERK in HEK293T cells expressing Myc-tagged PERK (Fig. 5B). Also, PC2 was associated with PERK and eIF2{alpha} in HEK293T cells over-expressing GFP-PC2 (Fig. 5C). These data demonstrate that PC2 is in the same complex as PERK and eIF2{alpha}. Because the eIF2{alpha} phosphorylation by the activated PERK is facilitated by PC2, we wanted to examine whether PC2 also interacts with the activated PERK. For this, ER stress was induced by Tg in HEK293T cells to stimulate the P-PERK level. Our data showed that PC2 indeed interacts with P-PERK (Fig. 5D). Finally, we examined the role of PERK in the PC2-eIF2{alpha} interaction, utilizing PERK+/+ and PERK–/– murine embryonic fibroblast (MEF) cells. We observed that the amount of PC2-bound eIF2{alpha} is sharply decreased in PERK–/– MEF cells (Fig. 5E), suggesting that PERK mediates the PC2-eIF2{alpha} interaction, possibly by forming a PC2-PERK-eIF2{alpha} complex. Together, our data indicate that PC2 promotes the eIF2{alpha} phosphorylation by P-PERK, presumably through physically facilitating the action of P-PERK on eIF2{alpha}. Interestingly, we found that PC2 expression is significantly reduced in PERK-deficient MEF cells (Fig. 5E, input). How PERK reversely regulates PC2 is unclear and may be a subject of future studies.


Figure 5
View larger version (61K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Association of PC2 with PERK and eIF2{alpha}. (A) Extracts of IMCD cells were used for IP with an anti-PC2 antibody. The precipitates and an input (10%) were detected by immunoblotting with a PERK, eIF2{alpha}, ATF6{alpha} or PC2 antibody. (B) HEK293T cells were transiently transfected with a C-terminal Myc-tagged PERK plasmid pcDNA-PERK (21) for 40 h, followed by protein preparation and IP with a Myc antibody. The precipitates and an input (10%) were immunoblotted by PC2 or Myc antibody. (C) and (D) Extracts of HEK293T cells transfected with pEGFP-PC2 or pEGFP (5 µg in 100 mm plates) for 40 h without (C) or with (D) Tg treatment, as described earlier, were subjected to IP with the GFP antibody. The precipitates and an input (30%) were detected by immunoblotting with the PERK, P-PERK, eIF2{alpha} or GFP antibody. (E) Role of PERK in the PC2-eIF2{alpha} interaction. Extracts of PERK+/+ and PERK–/– MEF cells were used for IP with the anti-PC2 antibody. The precipitates and an input (10%) were detected by immunoblotting with the eIF2{alpha} or PC2 antibody.

 
Pathogenic mutations in PC2 abrogate the regulation of eIF2{alpha} phosphorylation and cell proliferation by PC2
We investigated how PC2 mutations affect its effect on cell proliferation and eIF2{alpha} phosphorylation, using HEK293T cells transiently expressing one of the following GFP-tagged PC2 mutants: PC2 (positive control), R872X, E837X, PC2{Delta}C (or S689X, aa 1–688, lacking the C-terminus), PC2{Delta}N (aa 209–968, lacking the N-terminus), PC2{Delta}NC (aa 209–688) and GFP (negative control) (27). We found that, unlike Tg treatment and WT PC2 (positive controls), these truncation mutants were unable to stimulate eIF2{alpha} phosphorylation (Fig. 6A). Using the cell viability assay described earlier, we found that pathogenic mutants R872X and E837X has no effect on the rate of cell growth (Fig. 6B). These data indicate that PC2 exerts its effect on eIF2{alpha} phosphorylation through physical association with PERK. Thus, the inability of PC2 pathogenic mutants in negatively controlling cell growth through stimulating eIF2{alpha} phosphorylation may lead to cyst formation. Further, PC2{Delta}C, PC2{Delta}N, PC2{Delta}NC and E837X lost the association with P-PERK (Fig. 6C), suggesting that the PC2-PERK physical interaction is critical for PC2-mediated cell proliferation and eIF2{alpha} phosphorylation.


Figure 6
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Effects of PC2 mutations on cell proliferation and eIF2{alpha} phosphorylation. (A) Extracts of HEK293T cells transfected with GFP-PC2 or a PC2 mutant were subjected to immunoblotting with a P-eIF2{alpha}, eIF2{alpha}, GFP or β-actin antibody. Tg treatment was a positive control. (B) Five thousand cells from HEK293T cells expressing GFP, GFP-PC2, E837X or R872X were utilized for cell growth assay, as described earlier. (C) Interaction of PC2 mutants with P-PERK. Extracts of HEK293T cells transfected with pEGFP, pEGPF-PC2 or a PC2 mutant for 48 h and treatment by Tg for 1 h were subjected to IP with the GFP antibody and immunoblotting with the P-PERK or GFP antibody. Cell extracts (input, 30%) were utilized for detecting transfection efficiency.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
ER stress increases the kinase activity of PERK to promote eIF2{alpha} phosphorylation, which results in translational repression and activation of downstream signals that down-regulate cell growth (18–23). Renal cystogenesis, caused by loss-of-function mutations in PC1 or PC2, is characterized by over-proliferation, de-differentiation and so on, but how PC2 negatively controls cell growth remains unclear. In this study, we demonstrate that PC2 regulates cell proliferation through the PERK-eIF2{alpha} phosphorylation signaling pathway.

Our data show that induction of eIF2{alpha} phosphorylation and inhibition of cell growth by PC2 are blocked in cells with the treatment of PERK siRNA, indicating that PERK plays a critical role in this event. Interestingly, among the four eIF2{alpha} kinases, only PERK is localized to the ER membrane where the ER Ca release channel PC2 is localized. PC2, PERK and eIF2{alpha} are in the same complex, suggesting that PC2 may play a role in recruiting eIF2{alpha} to form PERK–substrate complexes for eIF2{alpha} phosphorylation. This was confirmed by the observation that repression of PC2 expression dramatically inhibits ER stress-induced eIF2{alpha} phosphorylation, which is promoted mainly by PERK (18,21). Furthermore, PC2 pathogenic mutations lose the capability of inhibiting cell proliferation and of stimulating eIF2{alpha} phosphorylation. On the basis of these data, we propose that the PERK-eIF2{alpha} pathway be part of a novel molecular mechanism underlying the PC2-associated ADPKD.

Controlling cell over-proliferation in PKD is a key principle of therapeutic treatments such as the use of antagonists of vasopressin receptors and mammalian target of rapamycing (mTOR). mTOR antagonist, rapamycin, effectively alleviates cyst development in several animal models of PKD and in human ADPKD patients (33). In fact, PC1 interacts with mTOR to suppress the up-regulation of cell proliferation by mTOR, in which translation initiation factor eIF4E is involved (33). The current study shows that protein synthesis inhibitor P-eIF2{alpha} mediates negative control of cell growth by PC2. Therefore, the negative control of cell growth by PC1 and PC2, either individually or as a complex (12,14) may be through the same thread, i.e. protein synthesis initiation machinery. However, it cannot be excluded that PERK, by interacting with PC2, affects PC1-regulated cell proliferation, in which PC2 phosphorylation is required (12,14). In summary, this study shows that PC2 is an important regulator of the cellular translation machinery and may underlie a pathogenesis mechanism of ADPKD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cell culture, DNA constructs and gene transfection
IMCD, MDCK and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (high glucose; Invitrogen) containing 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2. PERK+/+ and PERK–/– MEF cells were maintained as described previously (34). Plasmids pEGFP-PC2, pEGFP-R872X, pEGFP-E837X, pEGFP-PC2{Delta}C, pEGFP-PC2{Delta}N and pEGFP-PC2{Delta}NC were described previously (27,35). HEK293T cells were grown to ~70% confluency prior to transfection using Lipofectamine 2000 (Invitrogen). MDCK cells stably expressing GFP-PC2 or GFP were selected as previously described (36) and maintained using G418 (300 µg/ml).

Immunoprecipitation and immunoblotting
Protein extraction, immunoblotting and immunoprecipitation (IP) were performed, as described earlier (27). Typically, 20 and 200 µg of total cellular protein were used for immunoblotting and IP, respectively. HEK293T cells were transiently transfected with pEGFP, pEGFP-PC2, pcDNA3.1 (Invitrogen) or pcDNA3.1-PERK (19,21) for IP. At 40 h post-transfection, cells were used for protein extraction and precipitation. To examine the effect of PC2 mutants on eIF2{alpha} phosphorylation, we transfected HEK293T cells with pEGFP-PC2, pEGFP-R872X, pEGFP-E837X, pEGFP-PC2{Delta}C, pEGFP-PC2{Delta}N, pEGFP-PC2{Delta}NC or vector pEGFP. At 40 h post-transfection, cell extraction was prepared for immunoblotting.

Immunofluorescence microscopy and quantitative analysis
Immunofluorescence microscopy was performed as described earlier (27). Briefly, HEK293T cells were transiently transfected with Myc-PC2 or siRNA, mixed at the 20:80 ratio after 24 h of transfection, and then grown on cover slips. At 48 h post-transfection, cells were subject to Tg treatment for 1 h before fixation. P-eIF2{alpha} and PC2 antibodies were used for double staining. Fluorescence images were captured on a motorized Olympus IX81 microscope with a CCD cooling RT SE6 monochrome camera (Diagnostic Instruments). Final composite images were made using Image-Pro Plus 5.0 (Media Cybernetics). The quantification of expression density was performed using Image J. The area of each cell was manually defined for the analysis of both green and red signal densities. A cluster of confluent cells was defined as one cell for density calculation. For each image that contains multiple cells, the average density for red or green color was normalized to 100 after background subtraction. The correlation coefficient was obtained from linear regression using Sigmaplot 10 (Systat Software Inc.).

Antibodies
Rabbit antibodies against eIF2{alpha} and P-eIF2{alpha} (S51), and mouse anti-Myc antibody were purchased from Cell Signaling Technology. Human anti-PERK and anti-P-PERK antibodies were from R&D Systems and Biolegend, respectively. β-actin antibody was from Sigma-Aldrich, Canada. Goat anti-BiP, rabbit anti-ATF6{alpha}/ATF4 and mouse anti-PERK were purchased from Santa Cruz. GFP antibodies were a gift of Dr Luc Berthiaume (University of Alberta; also available at www.eusera.com). Mouse anti-PC2 antibody was described as before (27,30). Secondary antibodies were from Amersham or Promega.

PERK and PC2 knockdown by siRNA
PERK Stealth siRNA (Invitrogen, EIF2AK3-HSS114058, -059 and -060) and control siRNA (Invitrogen, Cat# 46-2002) were used to transfect HEK293T cells using Lipofectamine 2000 reagent following the manufacturer's instructions. PC2 knockdown was described previously (30). The efficiency of the siRNA knockdown was assessed by immunoblotting.

Cell proliferation assays
HEK293T cells were transfected with plasmid pEGFP-PC2 or PC2 siRNA in 35 mm dishes (Fig. 4B and C). At 24 h post-transfection, cells were split and seeded into eight separate wells of a 96-well plate. After incubation for another 30 h, luminescence activity was measured using a cell viability assay kit (Promega, Cat# G7571) and an illuminometer (Fluoroskan Ascent FL, Thermo Labsystems). The remaining cells in the 35 mm dishes were further cultured for immunoblotting. For testing the effect of PERK knockdown on PC2-regulated cell proliferation (Fig. 4D), HEK293T cells were transfected with pEGFP-PC2 or vector pEGFP using 35 mm dishes. At 6 h post-transfection, cells were split into two equal fractions for subsequent transfection with PERK or control siRNA at 24 h post-transfection. Forty-eight hours after pEGFP-PC2 transfection, cells were cultured in a 96-well plate for the cell growth assay described above. MDCK stable cell lines were serum-starved overnight and then grown in the presence of serum for 48 h in a 96-well plate before measuring luminescence activity. The efficiency of siRNA knockdown and PC2 expression were assessed by immunoblotting and/or immunofluorescence microscopy.

Metabolic labeling
To measure protein translational rates, HEK293T cells were transfected with plasmid pEGFP-PC2 or vector pEGFP. At 40 h post-transfection, cells were starved for 1 h in the pre-labeling medium (L-methionine and L-cystin-depleted DMEM with 10% fetal bovine serum and penicillin/streptomycin) (Invitrogen), followed by pulse-labeling with 50 µCi of [35S]methionine/cystine (35S-Protein Labeling Mix, Perkin Elmer) for 10 min. Cell extracts were applied for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and autoradiography.

Data analysis
Student's t-test was used for statistical significance analyses. Data were expressed in the form of mean±SE (N), where SE is the standard error and N is the number of experiment repeats. Probability values (P) of less than 0.05 and 0.01 were considered significant and very significant, respectively.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research and the Kidney Foundation of Canada (to X.-Z.C.). X.-Z.C. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.


   ACKNOWLEDGEMENTS
 
Myc-tagged PERK plasmid, PERK+/+ and PERK–/– MEF cells were kindly provided by Dr David Ron (New York University).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

  1. Gabow P.A. Autosomal dominant polycystic kidney disease. Am. J. Kidney Dis. (1993) 22:511–512.[Web of Science][Medline]

  2. Grantham J.J. 1992 Homer Smith Award. Fluid secretion, cellular proliferation, and the pathogenesis of renal epithelial cysts. J. Am. Soc. Nephrol. (1993) 3:1841–1857.[Abstract]

  3. Nadasdy T., Laszik Z., Lajoie G., Blick K.E., Wheeler D.E., Silva F.G. Proliferative activity of cyst epithelium in human renal cystic diseases. J. Am. Soc. Nephrol. (1995) 5:1462–1468.[Abstract]

  4. Murcia N.S., Sweeney W.E.J., Avner E.D. New insights into the molecular pathophysiology of polycystic kidney disease. Kidney Int. (1999) 55:1187–1197.[CrossRef][Web of Science][Medline]

  5. Igarashi P., Somlo S. Genetics and pathogenesis of polycystic kidney disease. J. Am. Soc. Nephrol. (2002) 13:2384–2398.[Free Full Text]

  6. Qian F., Noben-Trauth K. Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2). Pflugers Arch. (2005) 451:277–285.[CrossRef][Web of Science][Medline]

  7. Bichet D., Peters D., Patel A.J., Delmas P., Honore E. Cardiovascular polycystins: insights from autosomal dominant polycystic kidney disease and transgenic animal models. Trends Cardiovasc. Med. (2006) 16:292–298.[CrossRef][Web of Science][Medline]

  8. The International Polycystic Kidney Disease Consortium. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. Cell (1995) 81:289–298.[CrossRef][Web of Science][Medline]

  9. Mochizuki T., Wu G., Hayashi T., Xenophontos S.L., Veldhuisen B., Saris J.J., Reynolds D.M., Cai Y., Gabow P.A., Pierides A., et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science (1996) 272:1339–1342.[Abstract]

  10. Clapham D.E. TRP channels as cellular sensors. Nature (2003) 426:517–524.[CrossRef][Web of Science][Medline]

  11. Koulen P., Cai Y., Geng L., Maeda Y., Nishimura S., Witzgall R., Ehrlich B.E., Somlo S. Polycystin-2 is an intracellular calcium release channel. Nat. Cell Biol. (2002) 4:191–197.[CrossRef][Web of Science][Medline]

  12. Bhunia A.K., Piontek K., Boletta A., Liu L., Qian F., Xu P.N., Germino F.J., Germino G.G. PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2. Cell (2002) 109:157–168.[CrossRef][Web of Science][Medline]

  13. Grimm D.H., Karihaloo A., Cai Y., Somlo S., Cantley L.G., Caplan M.J. Polycystin-2 regulates proliferation and branching morphogenesis in kidney epithelial cells. J. Biol. Chem. (2006) 281:137–144.[Abstract/Free Full Text]

  14. Li X., Luo Y., Starremans P.G., McNamara C.A., Pei Y., Zhou J. Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2. Nat. Cell Biol. (2005) 7:1202–1212.[Web of Science][Medline]

  15. Nishio S., Hatano M., Nagata M., Horie S., Koike T., Tokuhisa T., Mochizuki T. Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation. J. Clin. Invest (2005) 115:910–918.[CrossRef][Web of Science][Medline]

  16. Ellgaard L., Helenius A. Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. (2003) 4:181–191.[CrossRef][Web of Science][Medline]

  17. Rutkowski D.T., Kaufman R.J. A trip to the ER: coping with stress. Trends Cell Biol. (2004) 14:20–28.[CrossRef][Web of Science][Medline]

  18. Ron D. Translational control in the endoplasmic reticulum stress response. J. Clin. Investig. (2002) 110:1383–1388.[CrossRef][Web of Science][Medline]

  19. Brewer J.W., Diehl J.A. PERK mediates cell-cycle exit during the mammalian unfolded protein response. Proc. Natl Acad. Sci. USA (2000) 97:12625–12630.[Abstract/Free Full Text]

  20. Sequeira S.J., Ranganathan A.C., Adam A.P., Iglesias B.V., Farias E.F., Guirre-Ghiso J.A. Inhibition of proliferation by PERK regulates mammary acinar morphogenesis and tumor formation. PLoS One (2007) 2:e615.[CrossRef]

  21. Harding H.P., Zhang Y.H., Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase (vol 397, pg 271, 1999). Nature (1999) 398:90.[CrossRef][Web of Science]

  22. Marciniak S.J., Garcia-Bonilla L., Hu J., Harding H.P., Ron D. Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK. J. Cell Biol. (2006) 172:201–209.[Abstract/Free Full Text]

  23. Brewer J.W., Hendershot L.M., Sherr C.J., Diehl J.A. Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proc. Natl Acad. Sci. USA (1999) 96:8505–8510.[Abstract/Free Full Text]

  24. Hori O., Ichinoda F., Yamaguchi A., Tamatani T., Taniguchi M., Koyama Y., Katayama T., Tohyama M., Stern D.M., Ozawa K., et al. Role of Herp in the endoplasmic reticulum stress response. Genes Cells (2004) 9:457–469.[Abstract/Free Full Text]

  25. Kokame K., Agarwala K.L., Kato H., Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J. Biol. Chem. (2000) 275:32846–32853.[Abstract/Free Full Text]

  26. Kokame K., Kato H., Miyata T. Identification of ERSE-II, a new cis-actin element responsible for the ATF6-dependent mammalian unfolded protein response. J. Biol. Chem. (2001) 276:9199–9205.[Abstract/Free Full Text]

  27. Liang G., Li Q., Tang Y., Kokame K., Kikuchi T., Wu G., Chen X.Z. Polycystin-2 is regulated by endoplasmic reticulum-associated degradation. Hum. Mol. Genet (2008) 17:1109–1119.[Abstract/Free Full Text]

  28. Schulze A., Standera S., Buerger E., Kikkert M., van Voorden S., Wiertz E., Koning F., Kloetzel P.M., Seeger M. The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. J. Mol. Biol. (2005) 354:1021–1027.[CrossRef][Web of Science][Medline]

  29. Harding H.P., Novoa I., Zhang Y., Zeng H., Wek R., Schapira M., Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell (2000) 6:1099–1108.[CrossRef][Web of Science][Medline]

  30. Wu Y.L., Dai X.Q., Li Q., Chen C.X., Mai W.Y., Hussain Z., Long W.T., Montalbetti N., Li G.C., Glynne R., et al. Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin. Hum. Mol. Genet. (2006) 15:3280–3292.[Abstract/Free Full Text]

  31. Wek R.C., Jiang H.Y., Anthony T.G. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. (2006) 34:7–11.[Web of Science][Medline]

  32. Yoshida H., Haze K., Yanagi H., Yura T., Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins—involvement of basic leucine zipper transcription factors. J. Biol. Chem. (1998) 273:33741–33749.[Abstract/Free Full Text]

  33. Shillingford J.M., Murcia N.S., Larson C.H., Low S.H., Hedgepeth R., Brown N., Flask C.A., Novick A.C., Goldfarb D.A., Kramer-Zucker A., et al. The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease. Proc. Natl Acad. Sci. USA (2006) 103:5466–5471.[Abstract/Free Full Text]

  34. Hamanaka R.B., Bennett B.S., Cullinan S.B., Diehl J.A. PERK and GCN2 contribute to eIF2alpha phosphorylation and cell cycle arrest after activation of the unfolded protein response pathway. Mol. Biol. Cell (2005) 16:5493–5501.[Abstract/Free Full Text]

  35. Li Q., Montalbetti N., Shen P.Y., Dai X.Q., Cheeseman C.I., Karpinski E., Wu G.Q., Cantiello H.F., Chen X.Z. Alpha-actin in associates with polycystin-2 and regulates its channel activity. Hum. Mol. Genet. (2005) 14:1587–1603.[Abstract/Free Full Text]

  36. Li Q., Dai X.D., Shen P.Y., Cantiello H.F., Karpinski E., Chen X.Z. A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel. FEBS Lett. (2004) 576:231–236.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
G. Liang, G. Bansal, Z. Xie, and K. M. Druey
RGS16 Inhibits Breast Cancer Cell Growth by Mitigating Phosphatidylinositol 3-Kinase Signaling
J. Biol. Chem., August 7, 2009; 284(32): 21719 - 21727.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
17/20/3254    most recent
ddn221v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liang, G.
Right arrow Articles by Chen, X.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liang, G.
Right arrow Articles by Chen, X.-Z.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?