CAMP-DEPENDENT NEGATIVE REGULATION OF RAT ALDEHYDE DEHYDROGENASE CLASS-3 GENE-EXPRESSION

We investigated the inhibitory effects of intracellular cyclic adenosi ne monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrog enase (aldh3) gene expression using cultures of primary rat hepatocyte s and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of ma ny members of the Ah gene battery have been shown to be negatively reg ulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is tr anscriptionally inducible by polycyclic aromatic hydrocarbons (PAH), a nd this induction involving function of the arylhydrocarbon (Ah) recep tor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoqui noline-sulfonyl)-2-methylpiperaziae di-HCl (H7) and staurosporine, How ever, PAH induction of ALDH-3 activity, protein, and mRNA was potentia ted 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-( 2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-g uanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhib itors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 a nd HA1004 in a concentration-dependent manner, but not by H7, and ther e was an inverse correlation observed between potentiation of PAH-indu ced aldh3 gene expression and inhibition of specific PRA activity by t he PRA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cycla se activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction ansi H8 po tentiation of PAH induction of aldh3 expression but had no effect on i nduction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expres sion plasmid containing approximately 3.5 kilobase pairs of the 5'-fla nking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 mu M). cAMP analogs, activators of PRA activity, or protein phosphatase inhibitors diminis hed expression of the reporter gene in a manner identical to the nativ e gene in cultured rat hepatocytes. Using deletion analysis of the pAL DH3.5CAT construct, we demonstrated the existence of a negative regula tory region in the 5'-flanking region between -1057 and -991 base pair s which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two a pparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor w hich requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive proce ss which allows PKA activity to negatively regulate expression of aldh 3 under either basal or inducible conditions.