Altered expression of connexin43 in the inhibition of gap junctional intercellular communication by chlorohydroxyfuranones in WB-F344 rat liver epithelial cells.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) promote foci formation in the two-stage cell transformation assay in vitro. These chlorohydroxyfuranones (CHFs) and their structural congener 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) inhibit gap junctional intercellular communication (GJIC) in Balb/c 3T3 mouse fibroblast cells. In the present study, the effects of MX, MCA, CMCF, and MCF on GJIC were evaluated in liver cells (WB-F344 rat liver epithelial cells), the target cells of MX-induced carcinogenicity, using the scrape-loading dye transfer technique. The CHFs inhibited GJIC after 1 h exposure in a concentration-dependent fashion. The order of potency was MX>CMCF approximately MCA>MCF. In terms of the lowest observed effective concentrations, the difference in the potency was about 27-fold (MX 1.875 microM, MCF 50 microM). After a prolonged exposure period (12 h), the inhibition of GJIC by MX and CMCF remained stable, but MCA and MCF exhibited increasing inhibitory effects. After removal of the CHFs, the GJIC slowly recovered. At the transcriptional level, CHFs caused essentially no change in the level of connexin43 (Cx43) mRNA. Preincubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 decreased substantially the inhibition of GJIC by all four CHFs. Activation of the mitogen-activated protein kinases (MAPKs) signaling pathway was necessary for inhibition of GJIC. CHFs did not increase the basal phosphorylation state of the Cx43 protein, but all CHFs caused a concentration-dependent degradation of the Cx43 protein. The results indicate that all the studied CHFs inhibit GJIC in WB-F344 cells by altering Cx43 expression.
Hakulinen
Pasi
P
National Public Health Institute, Laboratory of Toxicology, P.O.B. 95, FI-70701 Kuopio, Finland. pasi.hakulinen@ktl.fi
Rintala
Elina
E
Mäki-Paakkanen
Jorma
J
Komulainen
Hannu
H
D000818:Animals; D015153:Blotting, Western; D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line; D018031:Connexin 43 / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects*; D005419:Flavonoids / Q000494:pharmacology; D005663:Furans / Q000494:pharmacology*; D017629:Gap Junctions / Q000187:drug effects*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D018031:Connexin 43; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005663:Furans; C121895:chlorohydroxyfuranone; D011493:Protein Kinase C; D048369:MAP Kinase Kinase 1; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one