Hepatitis B Virus X Protein Stabilizes Cyclin D1 and Increases Cyclin D1 Nuclear Accumulation through ERK-Mediated Inactivation of GSK-3β.

The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3 beta, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3 beta along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3 beta. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression.