Down-regulation of cytokeratin 14 gene expression by the polyoma virus middle T antigen is dependent on c-Src association but independent of full transformation in rat liver nonparenchymal epithelial cells

Polyoma virus middle T antigen (mT) transforms the T51B cell line and induces the loss of the cytokeratin 8 and 14 pair (CK8/CK14) present in these rat nonparenchymal liver epithelial cells (LECs), because of the selective down-regulation of CK14 gene expression. To identity the initial steps of the mT-induced signaling pathway(s) leading to this inhibition, T51B cells were transfected with vectors encoding the NG59, dI23, and 248M mT mutants, which are known to interact in a differential manner with c-Src, PI3-kinase, and Shc. Immunofluorescence microscopy and Northern blot analysis showed a loss of cytokeratins in dI23 or 248M but not in NG59 mT mutant-containing cells. An in vitro kinase assay demonstrated that only the dI23 and 248M mT mutants could associate with c-Src. This c-Src-mediated action of mT on CK14 gene expression was further confirmed by adding the v-src gene product in T51B cells. The assessment of the transforming capacity of the mT mutants demonstrated that the NG59 and dI23 mT mutants were nontransformant, whereas the 248M mT mutant expressed an appreciable transforming activity. These results show that the down-regulation of CK14 gene expression by mT in the LEC line T51B is dependent on the association with the c-Src tyrosine kinase, but interestingly, this c-Src-mediated action of mT can occur in the absence of transformation. Furthermore, when coupled with recent data on the plasticity of LECs, the present findings provide the first essential element in our definition of the signaling pathway(s) that link growth/differentiation events with CK gene regulation in typical simple epithelial cells.