The Expression And Significant Of C-Jun In The Blockage Of Tubular Epithelial-Myofibriblast Transition By Hepatocyte Growth Factor

Objective: To investigate the possible role and mechanisms of heptocyte growth factor (HGF) during the process of the human tubular epithelial-myofibriblast Transition (EMT) induced by transforming growth factor β1(TGF-β1) in cultured human renal tubular epithelial cell lines(HKC). Methods: The HKC cells were cultured in vitro with Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, and seeded on twenty-four-well culture plates to approximately 60% to 70% confluence, and then shifted to serum-free medium for 24 hours. These cells were divided into four groups: ①Control (C) group:The HKC cells were treated respectively with free serum medium(Cf),U0126 20umol/l(Cu),SB202190 20umol/l(Csb)or curcumin100umol/l(Ccu)for 48 hours.②TGF-β1 (T) group:The HKC cells were co-incubated TGF-β1 (10ng/ml) with free serum medium (Tf) ,U0126 20umol/l(Tu) ,SB202190 20umol/l(Tsb)and Curcumin 100umol/l (Tcu) respectively for 48 hours. ③HGF (H) group:The HKC cells were co-incubated HGF (100ng/ml) with free serum medium(Hf) ,U0126 20umol/l(Hu) ,SB202190 20umol/l(Hsb)and curcumin100umol/l(Hcu)respectively for 48 hours. ④HGF+TGF-β1(HT) group:The HKC cells were co-incubated HGF (100ng/ml) and TGF-β1 (10ng/ml) with free serum medium(HTf) ,U0126 20umol/l(HTu) ,SB202190 20umol/l(HTsb) and curcumin 100umol/l(HTcu) respectively for 48 hours. Cells were observed the morphological change by microscope, and were analyzed the expression of E-cadherin, alpha-smooth muscle actin(α-SMA) and c-Jun by indirect immunofluorescence. At the time, the analysis of mRNA expression of α-SMA and c-Jun were assessed by reverse transcriptase-ploymerase chain reaction(RT-PCR), and the expression of c-Jun protein were analyzed by Western blot. Results: Cell shape observation: ①C group: The shape of HKC cells showed a classic cobblestone morphology, U0126,SB202190 or curcumin could not induce cell morphologic changes alone. ②T group: TGF-β1 induced profound morphologic changes, with cells becoming elongated in shape, disassociating from neighboring cells, and losing their cobblestone monolayer pattern .U0126,SB202190 or curcumin could not affect HKC cells morphologic changes induced by TGF-β1. ③H group: HGF alone or HGF with inhibitors could not induce HKC cells morphologic changes, cells showed a classic cobblestone morphology. ④HT group: Simultaneous incubation of HGF with TGF-β1largely restored the epithelial morphology. Incubated with U0126 or SB202190, HGF also exhibited a remarkable ability to block this phenotypic transition. Incubated with curcumin, some cells becomed elongated, the others maintained cobblestone morphology. immunofluorescence assays:①α-SMA: α-SMA expression was negative in HKC cells, while TGF-β1 induced α-SMA expression and assembly in HKC cells, as shown by the presence of cytoplasmic α-SMA-positive microfilaments. HGF markedly blocked this induction of α-SMA in HKC cells, at a concentration of 100ng/ml, HGF completely blocked α-SMA expression induced by 10ng/ml TGF-β1. Incubated with U0126 or SB202190, HGF also exhibited a remarkable ability to block this induction of α-SMA. Incubated with curcumin, HGF only exhibited a partial ability to block this induction of α-SMA.②E-cadherin: E-cadherin expression was positive in normal HKC cells, treatment of TGF-β1 resulted in total loss of E-cadherin staining in the plasma membrane of HKC cells, while HGF largely restored the E-cadherin protein staining. Incubated with U0126 or SB202190, HGF also exhibited a remarkable ability to restore the E-cadherin protein staining. Incubated with curcumin, HGF only restored partially the E-cadherin protein staining.③c-Jun: There was a certain extent c-Jun staining in the karyon of normal HKC cells, HGF increased the expression of c-Jun, while curcumin exhibited a remarkable ability to decreased c-Jun protein staining in Ccu,Tcu,Hcu,and HTcu group. U0126 and SB202190 have no effect on the expression of c-Jun. RT-PCR assays: ①α-SMA mRNA: The expression of α-SMA mRNA were negative in HKC cells, while TGF-β1 strongly induced α-SMA mRNA expression. HGF markedly blocked this induction of α-SMA mRNA in HKC cells. Incubated with U0126 or SB202190, HGF also exhibited a remarkable ability to block this induction of α-SMA mRNA. Incubated with curcumin, HGF only exhibited a partial ability to block this induction of α-SMA mRNA ②c-Jun mRNA: There was acertain extent c-Jun mRNA expression in HKC cells, HGF increased the expression of c-Jun mRNA, while curcumin exhibited a remarkable ability to decreased the expression of c-Jun mRNA. U0126 or SB202190 have no effect on the expression of c-Jun mRNA. Western blot assays: There was a certain extent c-Jun protein expression in HKC cells. TGF-β1 increased the expression of c-Jun protein, while HGF signaficantly increased the expression of c-Jun protein. curcumin exhibited a remarkable ability to decreased c-Jun protein in Ccu,Tcu,Hcu,and HTcu groups. U0126 or SB202190 have no effect on the expression of c-Jun protein. Conclusions: 1. TGF-β1 (10ng/ml) induced EMT in HKC cells, while HGF (100ng/ml) markedly blocked this induction. 2. HGF showed the inhibition of EMT through JNK pathway in HKC cells. 3. Downregulation of c-Jun expression in HKC cells resulted in the increase of TGF-β1 sensitivity to induce EMT. 4. Upregulation of c-jun expression induced by HGF leaded to inhibition of TGF-β1-induced-EMT in HKC cells.