TI - Discussion . AB - Various studies on renal cystic tissues and cell lines demonstrated that altered regulation of tubular epithelial cell proliferation is a key factor in the pathogenesis of ADPKD . What remains unclear is the timing of the misregulated growth as well as the pathways involved . Recently , in an attempt to answer these questions a number of groups provided evidence for the involvement of Cdk2 in the process of cystogenesis . Progression through the cell cycle is regulated by a family of cyclin-dependent kinases ( CKs ) whose activities are controlled by the relative ratio of cyclins and Cdk inhibitors ( CKIs ) [36 , 37] . There are two classes of CKIs in mammals , the p21CIP1 and p16INK4 families . Members of the p16NK4 family bind and inhibit only Cdk4 and Cdk6 kinases [38] . In contrast , members of the p21CIP1 family ( p21CIP1 , p27KIP1 and p57KIP2 ) inhibit all G1/S phase CDKs . The transition of cells from the G0/G1 into the S phase of the cell cycle involves the activities of Cdk2 , Cdk4 and Cdk6 [37] . Bhunia et .al.were the first to address the role of CDKs in PKD -induced proliferation . Specifically , they demonstrated that one of the functions of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and consequently control cellular proliferation . They showed that overexpression of wild-type polycystin-1 can activate JAK2/STAT-1 , a process that resulted in upregulation of the CKI p21waf1 . As expected , increase in p21 levels led to inhibition of Cdk2 and cell cycle arrest . The ability of polycystin-1 to modulate Cdk2 activity was dependent on polycystin-2 . These results implied that compromised polycystin-1 activity is expected to have the opposite effect , thus explaining the abnormal proliferation observed in ADPKD cystic cells . An independent study addressed directly the role of PC2 in cell cycle regulation and Cdk2 activity . It was demonstrated that PC2 could directly interact with Id2 , a member of the HLH family that is known to control cell proliferation and differentiation . The direct association of PC2 with Id2 was shown to regulate the nuclear translocation of Id2 and thus modulate the cell cycle through the Id2/p21/Cdk2 pathway [17] . Based on these results a model was proposed according to which PC1 can increase PC2 PHOSphorylation leading to enhanced Id2/PC2 interaction and reduced Id2 nuclear import . This in turn , prevents Id2 repression of E-box -dependent activation of transcription of genes such as p21 . Increased p21 will inhibit Cdk2 activity and arrest the cells at G0/G1 phase of the cell cycle . At the same time PC1 can lead to Cdk2 inhibition independent of Id2 through the JAK/STAT pathway . Based on this model mutations in either PC1 or PC2 can disrupt these pathways leading to abnormal cell proliferation [17] . A recent report also demonstrated reduced levels of p21 in human and animal PKD tissues as well as in affected cell lines implying a role of p21/Cdk2 in cystogenesis [39] . In this study we attempted to examine further this hypothesis . We generated stable clones expressing either wild-type or mutant R742X PKD2 in HEK293 . To our surprise , overexpression of wild-type PC2 did not affect proliferation of these cells . Cell cycle profile analysis , PCNA , p21 expression levels and Cdk2 activity remained unchanged among different transfectants . The reason for this discrepancy remains unclear given that the same cell line and similar experimental conditions were used in the previous studies [13,17] . In order to eliminate the possibility that the exogenously expressed wild-type PKD2 was not functional , we performed whole cell current measurements in vector-only , WT PKD2 and R742X PKD2 clones . As expected , HEK293 clones expressing wild-type PKD2 displayed an increase in the current amplitude of whole cell inward and outward currents recorded either in normal extracellular tyrode solution or symmetrical K+ . Such result excludes the possibility that an inactive PC-2 was expressed in HEK293 cells . In addition , absence of phenotype could not be attributed to the mislocalization of the expressed protein as determined by immunofluorescent analysis . In an attempt to clarify these contradictory results we utilized a different cell line system . The NRK-52E cells are "normal" rat tubular epithelial cells , thus we hypothesized that this is a more appropriate system to study PC-2 -induced proliferation and STAT-1/p21/Cdk2 activation . Nevertheless , similar results were obtained with the NRK-52E cells ( Figure 4 ) . The disparity of our results compared to previous studies is puzzling . Li et .al [17] , observed cell cycle arrest and Cdk2 inhibition in HEK293T cells after expression of wild-type PC-2 , and not in HEK293 cells used in our study . HEK293T cell line is a derivative of HEK293 that stably expresses the large T-antigen of SV40 . In these cells transfected plasmids that contain the SV40 origin are replicated to a copy number of 400-1000 plasmids/cell and therefore express the transgene at higher levels . However , this is unlikely to be the reason for the discrepancy given that high expression of wild-type and mutated PC-2 was achieved in our HEK293 clones ( Figure 1 ) and in NRK-52E cells ( Figure 4 ) after transient transfection . One of the unwanted side effects of cellular immortalization might be the alteration of basal proliferation rate in cells . This can be highly significant in proliferation studies . As a result we decided to switch to a primary cell culture system . We examined the ability of mutated PC-2 ( 1-703 ) to activate the STAT-1/p21/Cdk2 pathway in primary renal epithelial cells isolated from PKD2 ( 1-703 ) transgenic rat [19] . Isolation of TECs from the transgenic animals was performed using a sequential filtration method . Using this method we avoided any potential activation of surface receptors taking place during antibody -based isolation techniques . Purified tubular epithelial cells were cultured in low serum medium and on laminin-coated plates to avoid differentiation . The epithelial character of the cells was regularly evaluated by measuring epithelial ( cadherin ) and fibroblastic ( vimentin ) markers . TECs isolated from different animals showed augmented PCNA levels , a decrease of the G0/G1 phase cells and increase of the G2/M phase cells . This was the first time in our hands that we observed a higher proliferation activity in cells overexpressing a mutated PC-2 . These results indicated that indeed PC-2 can alter cellular proliferation in renal epithelial cells , but it also suggests that such process is complicated and possibly multifactorial and can not be easily recapitulated in in vitro cell line systems [40] . In support of this , a recent report focused on the dynamics of cyst formation by utilizing an inducible Pkd1 mouse model , demonstrated that proliferation was not appreciably higher in cystic specimens than in aged matched controls . Based on their results , the authors suggested that the relationship between cellular proliferation and cyst formation may be indirect [41] . Similar data were obtained from Zebrafish studies where it was shown that increased cell number in cyctic phenotype is a secondary consequence of tubule dilation rather than the leading cause of cyst formation [42] . In our study , it appears that mutated PC-2 -induced proliferation in primary cells proceeds independently of the STAT-1/p21 pathway since there is no change in the levels of p21 or on STAT-1 PHOSphorylation . Based on these results it is clear that in the rat system we investigated , PC-2 -induced proliferation proceeds through an alternative pathway other than STAT-1/p21 . Using gene expression profiling we were able to identify a candidate that may mediate the PC2 -induced proliferation in PKD2 ( 1-703 ) rat . Among all the cell cycle related genes , only two showed misregulation in TECs isolated from diseased rats , cyclin -dependent kinase inhibitor 1C (p57kip2) and Cdk2 . The p57 kip2 belongs to the p21WAF/Cip1 family . Studies have shown that p57 binds tightly to the G1 and S phase kinases , cyclin E/Cdk2 , cyclin D2/Cdk4 , cyclin A/Cdk2 and to a lesser extent to cyclin B/Cdc2 and effectively inhibits their activity [43] . An important difference between p57 and the other members of the family , is that p57 is not regulated by p53 but by p73 [44-46] . We observed a downregulation of p57 at both mRNA and protein levels in mutant cells with the absence of any change in p21 levels . This possibly signifies that PC-2 might alter cellular proliferation through p57/Cdk2 in these cells . It is possible that expression of mutant PC-2 can result in p57 downregulation by augmenting Id2 nuclear import and subsequent inhibition of p57 transcription [17] . This hypothesis is in agreement with experiments in neural cells where it was shown that Id2 could regulate cell cycle through p57 [47] . In addition to p57 downregulation , we observed an increase in Cdk2 protein level . This is interesting since it appears that Cdk2 activity might be augmented simultaneously in two different ways ( downregulation of the inhibitor and upregulation of the kinase ) . Whether Cdk2 increase is part of a positive feedback loop is still not known . Nevertheless , this simultaneous alteration in p57 and Cdk2 levels might result in a rapid increase in Cdk2 activity and subsequently to higher proliferation rate . A concern regarding our results might arise from the possibility that the isolated TECs are not equally representative of the various nephron segments in healthy and mutant rats , a concern however that cannot easily be addressed within the scope of this work . More specifically though , we addressed the issue of over-representation of the TECs from the proximal cysts by showing similar levels of a proximal tubule marker , megalin expression in normal and mutant TECs (Fig 5A) . In conclusion , the level of p57 contribution in the PC-2 -induced proliferation in renal epithelial cells is still unclear . Future experiments will focus on identifying the pathways leading to p57 reduction and whether this decrease is necessary for PC-2 -induced proliferation in renal tubular epithelial cells . We consider it of particular significance that no matter how these experiments pan-out , our study introduces a new pathway in ADPKD , through which PC-2 might lead to Cdk2 activation and increase in cellular proliferation , which is independent of STAT-1/p21 . Also , once again it should be emphasized that biological systems are unpredictably complex and may exert similar effects and end results through more than one pathway . Finally a word of caution should be expressed as regards the interpretation of experiments performed on grossly modified established cells lines , which are far from representing the complexity of whole organs or organisms .