Regulation of connexin32 and connexin43 gene expression by DNA methylationin rat liver cells

Gap junction proteins (connexins) are expressed in a cell-specific manner a nd expression is often reduced in neoplastic cells. We investigated the mec hanisms of connexin32 (Cx32) and connexin43 (Cx43) expression in hepatic ce lls using MH1C1 rat hepatoma cells and freshly isolated, adult rat hepatocy tes that express Cx32 but not Cx43 and WB-F344 rat liver epithelial cells t hat express Cx43 but not Cx32, Southern blotting after DNA restriction with MspI and HpaII indicated that two MspI/HpaII restriction sites in the Cx32 promoter (positions -147 and -847) were methylated in WB-F344 cells, but n ot in MH1C1 cells or hepatocytes. In contrast, an MspI/HpaII restriction si te in the Cx43 promoter (position 38) was methylated in MH1C1 cells, but no t in WB-F344 cells or hepatocytes, Transient transfection of the cell lines with connexin promoter-luciferase constructs indicated that the Cx32 promo ter was 7-fold more active in MH1C1 cells and the Cx43 promoter was 5-fold more active in WB-F344 cells. These results suggest that transcription of C x32 and Cx43 in hepatic cells is controlled by promoter methylation and by cell-specific transcription factors. Similar mechanisms may be involved in the reduced expression of these genes frequently observed in neoplastic cel ls.