Cdc42 Interacting Protein-4 Regulates Transforming Growth Factor-β1 Induced Renal Tubular Epithelial To Mesenchymal Transition Through The Polarity Protein Par6

Tubular interstitial fibrosis(TIF) is a common pathological characteristics and an inevitable outcome of all kinds of chronic kidney disease (CKD) progress to end-stage renal failure. Tubular epithelial-mesenchymal transition (TEMT) contributes significantly to the onset and pathogenesis of renal fibrosis. This phenotype has been shown to a complicated multistep process instead of simply a morphological transition, which consists of four key steps:loss of epithelial cell adhesion, de novo a-SMA expression and actin reorganization, disruption of tubular basement membrane, and enhanced cell migration and invasion, transforming growth factor-β1(TGF-β1), as a sole one, initiates and completes the entire course of EMT. TGF-β1 mediates the EMT process via intracellular pathways, including the canonical Smad pathway, phosphatidylinositol-3-kinase/Akt (PI3K/AKT) pathway, mitogen-activted protein kinase (MAPK) pathway, integrinlinked kinases(ERK) pathway, p38MAPK pathway, c-Jun N-terminal kinases (JNK) pathway and Rho-like GTPase signaling pathway. While the Smad, MAPK and PI3K/AKT pathways have been well described, less is known about Rho GTPase signaling molecular mechanism.Cdc42 interacting protein-4 (CIP4) is a small Rho GTPase, belongs to the Bin/ amphiphysin/Rvs (BAR) domain protein superfamily, which involved in membrane remodeling in various cellular pathways ranging from endocytosis and cytokinesis, T-tubule formation to cell migration and neuromorphogenesis. Thus, CIP4 plays important roles in the many aspects of biological fuctions such as the construction of cytoskeleton, cell adhesion, intracellular transportation, signal transduction and cell cycles. In human renal tumor cells, large amount of CIP4 splicing variant has been produced to promote the (3-catenin tyrosine phosphorylation and lead to renal tumor cells metastasis. The activated CIP4 can also induce cellular transformation and cell-cell interactions, the overexpression of CIP4 in pancreatic tumor cells and breast cancer cells have been demonstrated to be able to promote their migration and invasion. As the migration and invasion of cancer cells is a process analogous to that observed during renal epithelial-mesenchymal transition (EMT), the data above suggests that CIP4 may play as an important Rho GTPase signaling molecule to promote renal tubular EMT. In present study, we examined whether TGF-β1-induced EMT is mediated by CIP4. For this purpose, we measured CIP4 in the fibrotic renal tissue in a model of 5/6 nephrectomy, this model associated with EMT induced by over-expression of TGF-beta, and in vitro, we detected CIP4 in HK-2 cells treated with TGF-β1, and examined the effect of CIP4-siRNA on TGF-β1-induced EMT. As the polarity protein Par6 was identified as a key regulator in epithelial cell polarity and tight-junction, the interaction between CIP4 and Par6 was subsequently detected.Par I Relationship of CIP4 and Tubulointerstitial fibrosisObjective To study the expression and significance of Cdc42 interacting protein-4 (CIP4) in the model of chronic renal failure of 5/6 nephrectomized rats and the model of EMT of HK-2 cells induced by TGF-(31.Methods 20 SD rats were involved in this experiment cotaining 10 in the normal group. The model of chronic kidney disease was induced by a operation of 5/6 nephrectomy in rat. The expression of CIP4 was detected by Immunohistochemical SABC method at 12 weeks, and Western Blot was used to evaluate the proteins of CIP4. In vitro, CIP4 in HK-2 cells induced by TGF-β1 for 12,24,48,72 h were also detected by Western blot and RT-PCR.Results Immunohistochemical assay showed that rat CIP4 (rCIP4) as extensively expressed in renal tubules in 5/6-nephrectomized rats, whereas there was little staining in sham-operated rats at 12 weeks. The mRNA and proteins of CIP4 was also notably up-regulated in HK-2 cells induced by TGF-β1.Conclusion TGF-β1 could significantly improve the level of CIP4 in renal tubular epithelial cells both in vivo and in vitro.Partâ…¡CIP4 Regulates TGF-β1-Induced Renal Epithelial to Mesenchymal TransiformationObjective To observe the effect of CIP4 on human renal tubular epithelial to mesenchymal transition induced by TGF-β1 and to study the mechanism of its generation.Methods According to CIP4(human Cdc42 interacting protein-4) cDNA sequence in genebank (NM₀04240), Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the full-length CIP4 by utilizing two primers including restriction sites Not I. After purification by RT-PCR, the amplified cDNA was inserted into the pcDNA 3.1 plasmid at the Not I site. To carry on, the recombined plasmids pcDNA 3.1/CIP4 were transfected into HK-2 cells. Western blot or confocal microscope was performed to detect the phenotypic changes correlated to EMT in pcDNA3.1/CIP4 transfected HK-2 cells, as indicated the changes in epithelial and mesenchymal protein E-cadherin and a-SMA respectively. One set of siRNA oligos specific for CIP4 were designed based on the full CIP4 sequence in Genbank, Then HK-2 cells induced by TGF-β1 were transfected with CIP4-siRNA via Lipofactamine 2000.Western blot were used to evaluate the protein expressions of CIP4, E-cadherin andα-SMA respectively in control cells, TGF-β1 treated cells, siRNA transfected cells. The distribution of E-cadherin and a-SMA was observed by confocal microscope. After we interferenced HK-2 cells stimulated with TGF-β1 with specific inhibitor of PI3K/Akt (Wortmannin) 1μM for 48 hours, Western blotting was used to detect the CIP4 protein in control cells and Interferenced cells.Results HK-2 cells acquired phenotypic changes correlated with EMT by transfection of CIP4. The normal HK-2 and pcDNA3.1/Zeo transfected cells were similar in morphology, presenting typical morphology of epithelial cells of round or ovoid shape, and they grew by adhering to the flask wall. As compared with normal HK-2 and pcDNA3.1/Zeo transfected cells, the cells transfected with hCIP4 were altered in morphology, presenting long fusiform and widened inter-cellular space, which was similar to the morphology of myofibroblast. A cell-cell adhesion molecule, E-cadherin present in the plasma membrane of most epithelial cells, was decreased and formed zipper-like patterns at cell borders in CIP4-transfected cells. a-SMA, markers of mesenchymal cells, were increased in CIP4-transfected cells. It show that TGF-β1 can induce EMT, after transfected with CIP4-siRNA, the protein expression of E-cadherin was increased, and a-SMA was decreased, the EMT induced by TGF-β1 was effectively reversed. With treatment of Wortmannin, the expression of CIP4 was decreased.Conclusion TGF-β1 produced a large number of CIP4 via PI3K/Akt pathway in renal tubular EMT and CIP4 further participate in TGF-β1 induced EMT.Partâ…¢Cdc42 Interacting Protein-4 Regulates Transforming Growth Factor-β1-induced Renal Tubular Epithelial-Mesenchymal Transition through the Polarity Protein Par6Objective To observe the effect of CIP4 on the transdifferentiation of rat renal tubular epithelial cells (NRK-52E) induced by TGF-β1, and to search for the influence of the polarity protein Par6 on the TGF-β1-mediated transdifferentiation of NRK-52E cells.Methods In vitro, the rat tubular epithelial cells (NRK-52E) were cultured with 20μg/ L TGF-β1 for 72 h. Western blot or confocal microscope was used to detected the phenotypic changes associated with EMT in TGF-(31-treated NRK-52E cells, as illustrated by alterations in epithelial and mesenchymal protein E-cadherin and a-SMA, also as demonstrated the level of protein CIP4 and Par6 phosphorylation. To investigate whether CIP4 interacts with Par6, immunoprecipitation were used to evaluate the combination CIP4 and Par6. To further demonstrate the CIP4-induced EMT related to the activation of the polarity protein Par6, According to Par6 cDNA sequence (NM₀01003653) and CIP4 cDNA (NM 053920.1) in genebank, a Par6-siRNA and a CIP4-siRNA were devised and synthesised by Shanghai Invitrogen company. The Par6-siRNA and CIP4-siRNA were transfected into TGF-β1-treated cells respectively, and the phenotype changes associated with TGF-β1-induced EMT were then detected by confocal microscope or Western blot.Results TGF-β1 induced phenotypic alternations correlate with EMT in NRK-52E cells. E-cadherin molecule, a cell-cell adhesiontake on in the plasma membrane of most epithelial cells, was decreased and shaped zipper-like patterns at cell borders in TGF-β1-treated cells. a-SMA, markers of mesenchymal cells, were increased in TGF-β1-treated NRK-52E cells. With treatment of Par6-siRNA and CIP4-siRNA respectively, the phosphorylated Par6 was reduced and the losting E-cadherin was re-expressed and re-localized at cell boundary in TGF-β1-treated cells, on the other hand a-SMA were deceased.Conclusion The overexpression of CIP4 is directly involved in TGF-β1-induced tubular EMT through the polarity Par6.