Abstract
The effects of three tumor promoters on gap‐junction permeability; connexin 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 changes in these parameters correlated with the inhibition of gap‐junction function. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA; 10 ng/mL), dieldrin (10 μg/mL), and heptachlor epoxide (10 μg/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. Decreases in steady‐state connexin 43 mRNA levels were detected by northern blot analysis within 1 h and paralleled changes in steady‐state β‐actin mRNA, but these changes did not occur rapidly enough to account for the rapid loss of gap‐junction function. A substantial loss in the number of connexin 43 immunostained gap‐junctional membrane plaques was detected after a 15‐min exposure to all three promoters, but little change had occurred at 5 min. Western blot analyses using connexin 43‐specific antibodies showed changes in the degree of connexin 43 phosphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43‐im‐munoreactive band (P3) and a concomitant decrease in the relative intensity of the unphosphorylated (P0) band within 5 min of treatment. P3, in addition to bands P1 and P2, disappeared 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 P0 within 1 h of exposure, but no changes were seen after 5 min. Connexin 43 phosphorylation levels recovered in parallel with the recovery of GJIC for all three tumor promoters. Connexin 26 mRNA levels showed little change after a 1‐h exposure to the three promoters, but reductions in connexin 26 immunofluorescent staining were observed. These results suggest that (i) TPA‐induced hyperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor epoxide modulated connexin phosphorylation in a manner different from TPA by promoting hypophosphorylation of connexin 43, (iii) redistribution of plasma membrane gap‐junctional plaques after treatment with phorbol ester and non‐phorbol‐ester tumor promoters occurred subsequent to changes in gap‐junction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these promoters. © 1994 Wiley‐Liss, Inc.