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; 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 micrograms/mL), and heptachlor epoxide (10 micrograms/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 beta-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-immunoreactive 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 expoxide 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 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 expoxide 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.
Matesic
D F
DF
Department of Pediatrics/Human Development, Michigan State University, East Lansing 48824.
Rupp
H L
HL
Bonney
W J
WJ
Ruch
R J
RJ
Trosko
J E
JE
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D002450:Cell Communication / Q000187:drug effects; D002460:Cell Line; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics; D004026:Dieldrin / Q000494:pharmacology*; D017629:Gap Junctions / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D006534:Heptachlor Epoxide / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000187:drug effects; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002273:Carcinogens; D018031:Connexin 43; D017630:Connexins; C000607044:Gjb2 protein, rat; D012333:RNA, Messenger; D006534:Heptachlor Epoxide; D000072259:Connexin 26; D004026:Dieldrin; D013755:Tetradecanoylphorbol Acetate