Roles Of Scaffold Protein JLP In Regulation Of Epithelial-Mesenchymal Transition In Renal Tubular Epithelial Cells

Objective:JNK-associated leucine zipper protein(JLP)is a scaffold protein which can bring JNK and p38MAPK with a set of proteins,kinases and transcription factors.The execution of specific signalling pathway presents at cell migration,cytokinesis,apoptosis,myogenesis,neurite outgrowth,synaptic loss and CD40 receptor internalization.Classical cadherins such as E-cadherin,N-cadherin and R-cadherin attracted more attention for cell adhesion and transduction of signals functions.Recent reports described that N-cadherin was specially detected in the proximal tubules of human and HK-2 cells.N-cadherin is involved in aberrant activition of TGF-? and transdifferentiation in tubular epithelial cells.We have Previously demonstrated the expression of JLP in tubular epithelial cells in mice with unilateral ureteral obstruction(UUO)kidney and the progression of renal intersticial fibrosis with JLP deficiency.It is unclear whether JLP regulates epithelial mesenchymal transition(EMT).The interaction of JLP and N-cadherin in proximal tubule remains elusive.Additionally,the regulation of JLP through N-cadherin needs to solve.The present study will investigate the effects of JLP deficiency on the EMT in renal experimental models.Methods:1.Both jlp-wildtype(jlp+/+)mice and jlp-deficient(jlp-/-)mice were divided into sham-operated group and UUO-operated group respectively and sujected to the indicated operation.Mice were sacrificed at the 7th day after the operation.The kidney samples were collected.Immunohistochemistry staining,Western blotting and RT-PCR were performed to evaluate the expressions of JLP,E-cadherin,TGF-?1,?-SMA,COL-I and FN.2.The knock-down of jlp gene was constructed with jlp-shRNA in HK-2 cells.HK-2 cells were maintained in both negative control group and jlp-shRNA group with or without FGF-2 stimulation.The cells were collected and Western blotting and immunofluorescence were performed to evaluate the expressions of JLP,E-cadherin,TGF-?1,?-SMA,pP38-MAPK and N-cadherin.Immunoprecipitation of JLP and N-cadherin was performed in normal HK-2 cells.Immunofluorescence was performed to evaluate the expressions of JLP and N-cadherin.Results:1.Western blotting and RT-PCR analysis demonstrated that the expressi-on of E-cadherin in UUO groups was significantly down-regulated,while the expressi?ons of TGF-?1,?-SMA and COL-1,FN were significantly up-regulated in jlp-/-UUO group when compared with that in jlp+/+ UUO group.Immunohistochemistry staining displayed the similar expression pattern.Meanwhile,the expression of JLP protein and mRNA was notably decreased in jlp+4+ UUO group.2.Western blotting analysis show-ed that the expression of E-cadherin in FGF-2-treated cells was significantly down-reg-ulated,while the expressions of TGF-?1,?-SMA and pP38-MAPK in jlp-shRNA + FGF-2-treated cells were significantly up-regulated,when compared with that in FGF-2-treated cells.At same time,the expression of JLP protein was markedly decreased in FGF-2-treated cells.3.Western blotting and immunofluorescence analysis indicated that the expression of N-cadherin was significantly up-regulated in FGF-2-treated cells,while down-regulated in jlp-shRNA and jlp-shRNA +FGF-2-treated cells.Lysat-es of normal HK-2 cells of the indicated jlp genotype were immunoprecipitated with N-cadherin antibodies.Immunofluorescence staining indicated that JLP and N-cadher-in were co-localized in the cytoplasm of negative control group.Conclusion:Scaffolding Protein JLP deficiency promoted the progression of kidney fibrosis and EMT of tubular epithelial cells through TGF-?1 and p-P38 activation in UUO model and HK-2 cells.JLP deficiency results in loss of N-cadherin and aggravates EMT under profibrotic processes.