CHANGES IN GAP-JUNCTION PERMEABILITY, PHOSPHORYLATION, AND NUMBER MEDIATED BY PHORBOL ESTER AND NON-PHORBOL-ESTER TUMOR PROMOTERS IN RAT-LIVER EPITHELIAL-CELLS

The effects of three tumor promoters on gap-junction permeability; con nexin 43 and 26 mRNA levels, protein levels, and phosphorylation; and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changer in th ese parameters correlated with the inhibition of gap-junction function . 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL), dieldrin (10 m u g/mt), and heptachlor epoxide (10 mu g/mL) inhibited gap-junctional intercellular communication (GJIC) assayed by fluorescent dye transfer by 80-90% after a 5-min exposure and by more than 90% within 1 h. Dec reases in steady-state connexin 43 mRNA levels were detected by northe rn blot analysis within 1 h and paralleled changes in steady-state bet a-actin mRNA, but these changes did not occur rapidly enough to accoun t for the rapid loss of gap-junction function. A substantial loss in t he number of connexin 43 immunostained gap-junctional membrane plaques was detected after a 15-min exposure to all three promoters, but litt le change had occurred at 5 min. Western blot analyses using connexin 43-specific antibodies showed changes in the degree of connexin 43 pho sphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43-immunoreactive band (P3) and a concomitant de crease in the relative intensity of the unphosphorylated (PO) band wit hin 5 min of treatment. P3, in addition to bands P1 and P2, disappeare d after treatment with alkaline phosphatase. In contrast, dieldrin and heptachlor epoxide induced loss of P2 with a concomitant increase in the relative staining intensity of PO within 1 h of exposure, but no c hanges were seen after 5 min. Connexin 43 phosphorylation levels recov ered in parallel with the recovery of GJIC for all three tumor promote rs. Connexin 26 mRNA levels showed little change after a l-h exposure to the three promoters, but reductions in connexin 26 immunofluorescen t staining were observed. These results suggest that (i) TPA-induced h yperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor epoxide modulated co nnexin phosphorylation in a manner different from TPA by promoting hyp ophosphorylation of connexin 43, (iii) redistribution of plasma membra ne gap-junctional plaques after treatment with phorbol ester and non-p horbol-ester tumor promoters occurred subsequent to changes in gap-jun ction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these p romoters. (C) 1994 Wiley-Liss, Inc.