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Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2.

Felekkis KN, Koupepidou P, Kastanos E, Witzgall R, Bai CX, Li L, Tsiokas L, Gretz N, Deltas C - BMC Nephrol (2008)

Bottom Line: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation.This p57 reduction is accompanied by an increase in Cdk2 levels.Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Cyprus, Cyprus. deltas@ucy.ac.cy

ABSTRACT

Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.

Methods: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.

Results: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.

Conclusion: Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

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Expression of wild-type or mutant PC-2 does not affect proliferation or STAT-1/p21/Cdk2 activity in NRK-52E cells. (A). Whole cell lysates containing equal amounts of protein from NRK-52E cells trasnsiently transfected with vector-only, HA-WT PKD2, HA-R742X PKD2 and HA-1–702 PKD2 were analyzed by Western blotting for expression of p21, phosphorylated STAT-1, PCNA, tubulin, HA and PC-2. A non-specific band is detected in vector-only and WT PKD2 lanes in the HA blot and co-migrates with mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates from each transfectant were subjected into an in vitro Cdk2 kinase assay using Histone 1A as substrate. Equal amount of Cdk2 was confirmed by immunoblotting the precipitates with anti-Cdk2 antibody. Data are representative of three independent experiments performed. No statistically significant difference was detected.
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Figure 4: Expression of wild-type or mutant PC-2 does not affect proliferation or STAT-1/p21/Cdk2 activity in NRK-52E cells. (A). Whole cell lysates containing equal amounts of protein from NRK-52E cells trasnsiently transfected with vector-only, HA-WT PKD2, HA-R742X PKD2 and HA-1–702 PKD2 were analyzed by Western blotting for expression of p21, phosphorylated STAT-1, PCNA, tubulin, HA and PC-2. A non-specific band is detected in vector-only and WT PKD2 lanes in the HA blot and co-migrates with mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates from each transfectant were subjected into an in vitro Cdk2 kinase assay using Histone 1A as substrate. Equal amount of Cdk2 was confirmed by immunoblotting the precipitates with anti-Cdk2 antibody. Data are representative of three independent experiments performed. No statistically significant difference was detected.

Mentions: The rat kidney epithelial cell line, NRK-52E was transiently transfected with vector-only (CT), WT PKD2, R742X PKD2 and 1–702 PKD2 (a PKD2 mutant lacking the entire carboxy-terminal region of the protein). At 48 hours after transfection, cells were synchronized by serum starvation. Whole cell lysates were immunoblotted with anti-p21 and anti-phospho-STAT-1 antibodies. Neither p21 levels nor STAT-1 phosphorylation is affected by expression of wild-type or mutant PKD2 (Figure 4A). Similarly, the levels of active Cdk2 were comparable among the four transfectants. In addition to the JAK2/STAT-1/p21/Cdk2 pathway, the proliferation capacity of NRK-52E transfected with WT, R742X and 1–702 PKD2 appeared unaltered compared to vector only transfectants as judged by PCNA Western blot analysis. Good expression of the wild-type PC-2 and of the two truncated proteins was achieved as judged by anti-HA and anti-PC2 blotting. In summary, these results duplicate the observation in HEK293 that wild-type or mutant PKD2 expression do not modify the activity of the JAK2/STAT-1/p21/Cdk2 pathway.


Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2.

Felekkis KN, Koupepidou P, Kastanos E, Witzgall R, Bai CX, Li L, Tsiokas L, Gretz N, Deltas C - BMC Nephrol (2008)

Expression of wild-type or mutant PC-2 does not affect proliferation or STAT-1/p21/Cdk2 activity in NRK-52E cells. (A). Whole cell lysates containing equal amounts of protein from NRK-52E cells trasnsiently transfected with vector-only, HA-WT PKD2, HA-R742X PKD2 and HA-1–702 PKD2 were analyzed by Western blotting for expression of p21, phosphorylated STAT-1, PCNA, tubulin, HA and PC-2. A non-specific band is detected in vector-only and WT PKD2 lanes in the HA blot and co-migrates with mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates from each transfectant were subjected into an in vitro Cdk2 kinase assay using Histone 1A as substrate. Equal amount of Cdk2 was confirmed by immunoblotting the precipitates with anti-Cdk2 antibody. Data are representative of three independent experiments performed. No statistically significant difference was detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533650&req=5

Figure 4: Expression of wild-type or mutant PC-2 does not affect proliferation or STAT-1/p21/Cdk2 activity in NRK-52E cells. (A). Whole cell lysates containing equal amounts of protein from NRK-52E cells trasnsiently transfected with vector-only, HA-WT PKD2, HA-R742X PKD2 and HA-1–702 PKD2 were analyzed by Western blotting for expression of p21, phosphorylated STAT-1, PCNA, tubulin, HA and PC-2. A non-specific band is detected in vector-only and WT PKD2 lanes in the HA blot and co-migrates with mutated PC-2 in this cell line. (B) Cdk2 immunoprecipitates from each transfectant were subjected into an in vitro Cdk2 kinase assay using Histone 1A as substrate. Equal amount of Cdk2 was confirmed by immunoblotting the precipitates with anti-Cdk2 antibody. Data are representative of three independent experiments performed. No statistically significant difference was detected.
Mentions: The rat kidney epithelial cell line, NRK-52E was transiently transfected with vector-only (CT), WT PKD2, R742X PKD2 and 1–702 PKD2 (a PKD2 mutant lacking the entire carboxy-terminal region of the protein). At 48 hours after transfection, cells were synchronized by serum starvation. Whole cell lysates were immunoblotted with anti-p21 and anti-phospho-STAT-1 antibodies. Neither p21 levels nor STAT-1 phosphorylation is affected by expression of wild-type or mutant PKD2 (Figure 4A). Similarly, the levels of active Cdk2 were comparable among the four transfectants. In addition to the JAK2/STAT-1/p21/Cdk2 pathway, the proliferation capacity of NRK-52E transfected with WT, R742X and 1–702 PKD2 appeared unaltered compared to vector only transfectants as judged by PCNA Western blot analysis. Good expression of the wild-type PC-2 and of the two truncated proteins was achieved as judged by anti-HA and anti-PC2 blotting. In summary, these results duplicate the observation in HEK293 that wild-type or mutant PKD2 expression do not modify the activity of the JAK2/STAT-1/p21/Cdk2 pathway.

Bottom Line: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation.This p57 reduction is accompanied by an increase in Cdk2 levels.Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Cyprus, Cyprus. deltas@ucy.ac.cy

ABSTRACT

Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.

Methods: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.

Results: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.

Conclusion: Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

Show MeSH
Related in: MedlinePlus