The Roles Of STAT3 And SOCS3 In The Proliferation And Differentiation Of Hepatic Progenitor Cell

ObjectiveThe liver has a remarkable and apparently almost unlimited capacity to regenerate. Preexisting hepatocytes may replace liver tissue lost as a consequence of less severe liver injury. If this pathway is impaired during liver failure, then survival depends on recruitment of either stem cell or hepatic progenitor cells (HPCs). There are HPCs in the adult mammalian liver, which can migrate into the liver, proliferate and then differentiate to both hepatocytes and cholangiocytes. The mechanisms that trigger HPC to proliferate and differentiate to hepatocytes and cholangiocytes remain poorly understood. Recent studies have shown that the signal transducer and activator of transcription3 (STAT3), which is normally associated with the cytokine signal transduction pathway, is induced by various growth factors and cytokines. Evidence suggests a link between STAT activation and cellular proliferation and differentiation. Suppressors of cytokine signal 3(SOCS3), which are induced by STAT3 and function to suppress STAT3, has been identified as negative feedback molecules of STAT3. A negative regulatory mechanism for the suppression of STAT3 signaling may be involved in hepatic regeneration. However, the roles of STAT3 and SOCS3 in the proliferation and differentiation of HPC remain poorly understood. In the present study, we investigate the proliferating features of HPC in liver failure and the relationship between the expressions of p-STAT3, SOCS3 and the levels of HPC in liver failure. Furthermore, we investigate the roles of STAT3 and SOCS3 in the proliferation and differentiation of rat hepatic stem-like cell line (WB-F344 cell) in vitro. Methods(1) Liver tissues taken from 76 cases of liver failure and acute or chronic common hepatitis were obtained by autopsy, surgical resection and liver biopsy in China Medical University from 1965 to 2005. All specimens were fixed in formalin and embedded in paraffin. Fourμm serial sections were used to detect the morphological changes with HE stain. The liver tissues were also tested for the the expression of HPC with OV6 and the expressions of (cytokeratin 19)CK19, p-STAT3 and SOCS3 by immunohistochemistry. To quantify the immunostaining observations, 100 cells over five high power fields (×400) randomly were counted and the percentage of positive cells was calculated. To quantify the immunostaining of OV6, cells were scored for positive staining using the following scale: Cases with no positive cells or seldom positive cells were considered (—); Cases with focus cells positive were considered (+); Cases with continuous positive cells (<50% portal area) were considered (++); Cases with continuous positive cells (>50% portal area) were considered (+++). The case with the results (—) was considered negative; and the case with the results (+), (++) and (+++) was considered positive. To quantify the immunostaining of CK19, p-STAT3 and SOCS3, following methods were used. Negative was considered if the numbers of positive cells were <25%, while positive was considered if the numbers of positive cells were >25%.(2) WB-F344 cells were exposed to HGF and AG490, the protein expressions of STAT3, p-STAT3 and SOCS3 were detected by Western blot and immunofluorescence, then SOCS3 mRNA was detected by RT-PCR. Cell cycle and apoptosis were detected by flow cytometry and MTT after WB-F344 cell was treated with STAT3 inhibitor AG490 and HGF.(3) WB-F344 cells were induced by HGF, EGF, Insulin and dexamethasone for 3 weeks. The morphological changes were studied with microscope. The differentiated cells were identified by reverse transcription polymerase chain reaction (RT-PCR) with albumin(ALB), alpha-1-fetoprotein(AFP) and CK19. The protein expressions of STAT3, p-STAT3 and SOCS3 were detected by Western blot and SOCS3 mRNA was detected by RT-PCR during the differentiation period of WB-F344.(4) The SPSS 13.0 software was employed to analyze the data. P<0.05 was considered as statistical significance.Results(1) There were a lot of proliferating biliary ducts in cases of subacute liver failure(SALF) and acute-on-chronic liver failure(ACLF) by immunohistostaining with CK19. The percentage of cases with positive CK19 in liver failure (62.5%)was significantly higher than that in common hepatitis(30%) (P<0.05). The percentage of cases with positive OV6 in liver failure (85.7%) was significantly higher than that in common hepatitis (35%)(P<0.05). The percentage of cases with positive p-STAT3 in liver failure (67.9%) was significantly higher than that in common hepatitis (25%))(P<0.05). The percentage of cases with positive SOCS3 in liver failure (60.7%) was significantly higher than that in common hepatitis (35%) (P<0.05). There was correlation between the expression of OV6 and the expressions of CK19, p-STAT3 and SOCS3 (r_s equal to 0.689, 0.239 and 0.322 respectively, P<0.05).(2) The expressions of Stat3 protein in WB-F344 cell treated with HGF did not show variations compared to the expressions in untreated WB-F344 cell (P>0.05 ). The protein levels of phosphorylated STAT3 were increased after HGF stimulation and peaking at 30 minutes after treatment (P<0.05). The expressions of SOCS3 protein and SOCS3 mRNA were increased after HGF stimulation and peaking at 10-30 minutes after HGF treatment (P<0.05). HGF exerts a proliferative and anti-apoptosis effect on serum starved WB-F344 cell in a dose-dependent manner, the count of G1 phase increased and that of S phase decreased, meanwhile that of apoptosis decreased after WB-F344 cell was stimulated by HGF(P<0.05). The proliferative and anti-apoptosis effect of HGF on WB-F344 can be inhibited when STAT3 pathway is abolished by the specific inhibitor AG490 (P<0.05). (3) When WB-F344 cells were cultured in the differentiation system (DMEM, HGF 10-50ng/ml, EGF 20ng/ml, Insulin 1μg/ml,Dexamethasone 1μmol/1), The cells showed obvious phenotype changes, including that cells were scattered and then differentiated into hepatic cell like shape. In addition, the treated cells increase the expression of mature hepatic cell marker ALB, but decrease that of hepatic stem cell marker AFP and cholangiocytes marker CK19 during differentiation period by RT-PCR(P<0.05). Furthermore, the expressions of phospho-STAT3 and SOCS3 were reduced during the differentiation by Western and RT-PCR (P<0.05).Conclusion(1) HPCs are frequently detected in livers of patients with liver failure. The expressions of p-STAT3 and SOCS3 were significantly higher in livers of patients with liver failure than those in common hepatitis and were associated with the proliferation of HPCs. Thus p-STAT3 and SOCS3 might participate in the proliferation of HPCs.(2) These in vitro data indicate that HGF can activate STAT3 and SOCS3. HGF exerts a proliferative and anti-apoptosis action on WB-F344 cell via activation of the expression of STAT3.(3) HGF, EGF, Insulin and dexamethasone can induce WB-F344 cell to differentiate into mature hepatocytes in vitro. The expressions of p-STAT3 and SOCS3 were reduced during the differentiation, which means that they might participate in inhibiting the differentiation of WB-F344 cell.