JAK2/STAT3 Signaling Pathway Is Involved In Transdifferentiation Of Renal Tubularepithelia Cells Mediated By CTGF

Objective: To investigate the possible role and mechanisms of JAK2/STAT3 pathway during the process of Epithelial-mesenchymal transdifferentiation of human renal tubular epithelial cells(HKCs) promoted by Recombinant Human Connective Tissue Growth Factor(CTGF).Methods: HK-2 were incubated with Dulbecco‘s Modified Eagle Media: Nutrient Mixture F-12 medium supplemented with 10% fetal bovine serum in vitro, and seeded on plastic plates to approximately 70%to 80% confluence, and then shifted to free serum medium for 24 hours. These cells were divided into three groups:(1)Control group:The HK-2 were cultured with 10% FBS DMEM/F12.(2) CTGF-1(5ug/L)group:The HK-2 were treated with5ug/LCTGF.(3) CTGF+JAK2/STAT3 inhibitor AG490 group:The HK-2 were co-incubated CTGF(5ug/L) and AG490(40 umol/L).According to the above were cultured 12 h,24h for 48 h respectively. The expression of a-SMA、JAK2/STAT3 and p-JAK2/STAT3 proteins were assessed by western-blotting(WB).After pre-treated with different doses of AG490(20、40 、60umol/L)respectively for one hour, and then adding the same concentration of CTGF(5ug/L) for48 hours. The morphological changes of cells were observed under inverted phase contrast microscope.The expressi of a-SMA and JAK2/STAT3 and p-JAK2/STAT3 proteins were assessed by western-blotting(WB). MTT was used to detect the effects on cells apoptosis and proliferation.Results:(1).The results of WB showed that when the 5ug/L CTGF at different times,the expression of a-SMA protein was up-regulated(P<0.05), JAK2/STAT3 protein was up-regulated(P<0.05),p-JAK2/STAT3 proteinwas up-regulate(P<0.05). All of them were in a time-dependent manner.(2).Compared with the CTGF(5ug/L)alone,CTGF(5ug/L) and AG490(40umol/L) co-interaction, the expression of a-SMA protein was down-regulated(P<0.05), JAK2/STAT3 protein was down-regulated(P<0.05), p-JAK2/STAT3 protein was down-regulated(P<0.05). Both of them were in a time-dependent manner.(3) HK-2 were incubated with CTGF(5ug/L), the expression of a-SMA m RNA was up-regulated(P<0.05), and p-JAK2/STAT3 protein was up-regulated(P<0.05).(4) The results of WB showed that different concentrations of AG490 could make the expression of a-SMA protein down-regulated(P<0.05), and p-JAK2/STAT3 protein down-regulated(P<0.05).Both of them were in a dosedependent manner.(5) CTGF stimulated the morphlolgical oval-to-fusiform transdifferentiation of the cells, and increased the cell proliferactive activity in HK-2cells.AG490 could inhibit these changes.(6)The results of DAPI showed that IGF-1can inhibit apoptosis and increase the cell proliferactive activity in HKCs cells. But AG490 induce apoptosis, all of them were in a dose- and time-dependent manner.Conclusin:(1)CTGF can cause renal tubular epithelial cell myofibroblast phenotype of a-SMA expression increased, suggesting that EMT occurrence。(2)JAK/STAT pathway through phosphorylation of JAK2/STAT3 activation, EMT is involved in the regulation of renal tubular epithelial cells; JAK2/STAT3 inhibitor AG490 can inhibit the conversion process.(3)Under the action of a certain concentration of AG490, the expression of a-SMA,p JAK2 protein and p STAT3 protein in renal tubular epithelial cells was down regulated with time.( 4) In a certain time range, with the increase of the concentration of AG490, the expression of p STAT3 m RNA, p JAK2 and a-SMA in renal tubular epithelial cells was down regulated.