TEC Protein Tyrosine Kinase Involved In Signal Regulation Of Liver Regeneration And Hepatocyte Proliferation

TEC, a non receptor tyrosine kinase, was initially cloned from liver cancer cells. The rat homology of Tec cDNA was recently isolated by us from regenerating livers (30-240 min following partial hepatectomy) using representational difference analysis (RDA). Tec kinase is highly expressed in liver and hematopoietic tissue and plays an important role in hematopoietic cell proliferation and differentiation. However, its function in hepatocytes has not been well characterized. In our previous study we provide evidence that Tec kinase might be involve in liver regeneration and hepatocyte proliferation. The current study was designed to further investigate the role of TEC in liver regeneration and hepatocyte proliferation and to define the underlying molecular mechanisms of its function in this context.To confirm the potential involvement of TEC tyrosine kinase in hepatocyte proliferation and liver regeneration, TEC kinase gene expression in rat tissues and different stage embryonic fetal livers was determined by Northern blotting, RT-PCR and Western blotting analyses. We showed that TEC mRNA was up-regulated 0.5 hr after partial hepatectomy. Its expression level reached maximal at 2 hrs and then returned to basic level after 4 hrs. Tec was specifically distributed in rat liver and kidney. A highest expression of Tec was detected in embryonic day 15-19 fetal livers, while the expression level of TEC in adult and neonatal rat livers was significantly lower. Furthermore, in rat regenerating livers, increased phosphorylation of TEC kinase was observed within 1~2 hour after partial hepatectomy. Expression level of TEC protein was also up-regulated. The same results were obtained using the mouse liver regeneration model. Moreover, Tec expression in serum-starved primary rat hepatocytes was dramatically induced by hepatocyte growth factor (HGF) stimulation. Additionally,Tec expression was down-regulated in serum-starved liver tumor HepG2 cell line. However, its expression is significantly up-regulated after stimulation with 10% fetal bovine serum (FBS) and insulin but not epidermal growth factor (EGF). These results suggest that Tec is an inducible early response gene that might be involved in hepatocyte proliferation and liver regeneration.Next, a liver stem cell line WB F-344 was used to investigate the regulatory role of TEC kinase in the hepatocyte proliferative signaling pathway induced by HGF. Immunoprecipitation, Western blotting, and in vitro kinase assay were used to test whether TEC was activated after HGF stimulation. The results showed that TEC was quickly tyrosine phosphorylated in 10 min after stimulation with HGF in WB cells. This activation could be sustained for 1 hr. Consistent with this result, TEC kinase activity was greatly increased after HGF stimulation. Further studies using the reporter gene assay showed that TEC enhanced activity of Elk and serum response element (SRE) in response to HGF stimulation. Inhibition of MEKl, the upstream activator of the Erk pathway, significantly suppressed TEC phosphorylation, suggesting that TEC is activated downstream of MEKl. Blocking expression of TEC by TEC antisense oligo nucleotides resulted in a decrease in HGF-induced Erk activation. As a result, 3H-TdR incorporation of WB F-344 cells transfected with Tec antisense was dramatically decreased. Additionally, the eukaryotic expression vector of TEC kinase domain mutant (TECK/M) was constructed and a stably cell line (designated WB?) expressing TEC1^, was generated. The signaling molecular responses to HGF and cell proliferation were then examined in WB M cell line. Compared to wild type WB cell, a dramatically decreased phosphorylation of ERK and cell proliferation were observed in WB? cells. These results demonstrated that tyrosine phosphorylation and activation of TEC are induced by HGF stimulation in hepatocytes. Tec kinase is mainly involved in the HGF-induced Erk signal pathway, functioning downstream of MEKl and playing a positive role in hepatocyte proliferation and potentially liver regeneration.