LOCALIZATION AND FUNCTION OF THE CONNEXIN-43 GAP-JUNCTION PROTEIN IN NORMAL AND VARIOUS ONCOGENE-EXPRESSING RAT-LIVER EPITHELIAL-CELLS

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye tran sfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disru pted gap-junctional intercellular communication (GJIC) mediated by con nexin 43 (Cx43). Compared with normal rat liver epithelial cells, cell s neoplastically transformed by src, neu, ras, and myc/ras all display ed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further d emonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-tra nsformed cells, but not in the ras- and myc/ras-transformed cells. Nuc lei isolated from WB-neu cells showed substantially higher levels of C x43 on western blotting than did nuclei from WB-neo control cells, sup porting the idea that the nuclear-localized immunopositive material de tected by confocal microscopy was Cx43 protein. In a GJIC-deficient mu tant rat liver epithelial cell line containing normal numbers of plasm a membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overe xpressing myc, on the other hand, displayed a normal degree of GJIC, i ncreased levels of plasma membrane-localized Cx43 plaques, and hyperph osphorylation of the Cx43 protein. Cells expressing raf, previously sh own to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showe d a marked reduction in GJIC and plasma membrane-associated Cx43 immun ostaining. These data suggest that altered localization of the gap-jun ction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastica lly transformed rat liver epithelial cells. (C) 1996 Wiley-Liss, Inc.