Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

Abstract

Transcriptional activation of the murine Cypla-l (cytochrome P1450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYPlAl mRNA induction by TCDD as well as the concomitant increase in CYPlAl enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cypla-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYPlAl mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYPlAl mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cypla-) gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the enzyme responsible for the phosphorylation is likely to be protein kinase C.The mammalian CYPJAl gene encodes an enzyme expressed endogenously during cell division, embryogenesis, and differentiation, suggesting that this gene as well as certain other P450 genes might be involved in distinct critical life processes (51,53). The CYPlAl enzyme also plays an important role in the detoxification of numerous polycyclic aromatic hydrocarbons, including benzo[a]pyrene, a major component in the production of cigarette smoke and other combustion processes. Moreover, CYPlAl is an essential enzyme in the detoxification of chemicals responsible for activation of the CYPIAI gene, and paradoxically, the enzyme also potentiates promutagens and procarcinogens by converting them into reactive intermediates (cf. references 53 and 60 and references therein from the transcriptional initiation site (14,19,26,28,40,55,56). As a consequence of gene activation, the rates of CYPlAl mRNA and protein synthesis are increased 20-to 100-fold (for a review, see reference 54). Posttrans...

Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis

“…This effect has been observed in multiple liver cell types [Puga et al, 1992;Enan et al, 1998b;Ashida et al, 2000], but not in all [Gohl et al, 1996]. In other cells, such as LPS-activated B cells, TCDD downregulates AP-1 expression [Suh et al, 2002], suggesting a cell type-dependent effect of AHR ligands on immediate-early protooncogene induction.…”

“…Exposure of multiple rodent cell lines to TCDD results in an essentially immediate increase in protein kinase C levels and activity in cellular membranes [Bombick et al, 1985Madhukar et al, 1988;Carrier et al, 1992]. TCDD specifically activates tyrosine kinases associated with the EGFR [Madhukar et al, 1984[Madhukar et al, , 1988 and induces the association of adaptor proteins such as SHC, GRB2, and SOS with EGFR [Park et al, 1998].…”

cAMP-dependent Negative Regulation of Rat Aldehyde Dehydrogenase Class 3 Gene Expression

“…Recently, a number of in vivo and in vitro studies have been focused on the effects of protein kinase activation on the specific signal transduction pathways in the control of cyp1A1 gene expression. Notably, protein kinase C-mediated phosphorylation (15,16) appears to be a determinant in the regulation of PAHinducible cyp1A1 gene expression. Our past experiments suggested that PKC-dependent phosphorylation of AhR appears to be required for PAH induction of all of the xenobiotic metabolizing enzymes in the Ah gene battery, not just for cyp1A1 (17).…”

“…Carrier et al (15) and Reiners et al (16) have provided evidence that some step in transactivation by the AhR is dependent upon phosphorylation by PKC. Our studies (17) with cultured rat hepatocytes demonstrated that the PKC inhibitors H7 and staurosporine concomitantly inhibited PAH induction of all five genes we tested, including aldh3, cyp1A1, gst1, qor, and ugt1.…”

The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells

“…The activated AhR complex then binds promoter regions of target genes at specific DNA-binding sites (aryl hydrocarbon response elements (AhREs)), thereby modulating gene transcription (Okey et al, 1994;Hankinson, 1995;Porter et al, 2001). A common outcome of AhR activation is the induction of genes such as those encoding the cytochrome P-4501A1, 1A2, and 1B1 monooxygenases (Poland et al, 1974;Whitlock, 1990;Carrier et al, 1992;Larsen et al, 1997). AhR-dependent upregulation of these enzymes results in increased metabolism of some AhR ligands such as PAH and the production of carcinogenic intermediates (Buters et al, 1999;Dertinger et al, 2001).…”