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

Abstract

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes.Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 M). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5-flanking region between ؊1057 and ؊991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.The aldehyde dehydrogenases (ALDH, 1 aldehyde NAD(P) ϩ oxidoreductase EC 1.2.1.3) are a family of NAD(P) ϩ -dependent enzymes that oxidize a broad class of alde...

Induction and Regulation of Xenobiotic-Metabolizing Cytochrome P450s in the Human A549 Lung Adenocarcinoma Cell Line

“…This is in agreement with findings that PKC inhibitors block the AHR-mediated induction of CYP1A1 (54,(57)(58)(59)(60)(61). The mechanism of PKC action on TCDD-elicited induction is controversial with conflicting reports in the literature.…”

“…Previously these inhibitors (3 nM calyculin A, 50 nM okadaic acid) were shown to enhance the AHR-mediated induction in mouse Hepa-1 cells by affecting a step subsequent to xenobiotic response element (XRE) binding (63). There is also a report where 20 nM okadaic acid had no effect on polycyclic aromatic hydrocarbon induction of CYP1A1 mRNA in rat primary hepatocytes (60). Phosphatase treatments of AHR/ARNT have been shown to abolish the ability of the receptor complex to bind at XRE (54,64,65).…”

Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89

“…That is, H89 inhibited ER export partially at 25 M, almost com- pletely at 50 M, and completely at 100 M (our unpublished results). In contrast, 60 M H7 (Reich and Pfeffer, 1990;Xiao et al, 1997), 120 M H8 Chuong, 1997), 9 M KT5720 (Linn et al, 1996), and 1 M chelerythrine (Herbert et al, 1990), at concentrations reported to inhibit PKA and PKC in vivo, had no detectable effect on ER export of VSVG.…”

“…However, the presence of 50 M H89 in the hypotonic medium prevented altogether the appearance of GPP130 tubules ( Figure 2D). Although H89 is a potent inhibitor of PKA (Chijiwa et al, 1990), H89 did not appear to exert its inhibitory effect on hypotonically induced Golgi resident redistribution through the inhibition of PKA or conventional PKC isozymes because other selective inhibitors of PKA (120 M H8 [Lee and Chuong, 1997;Figure 4 and 9 M KT5720 [Linn et al, 1996]) and PKC (60 M H7 [Reich and Pfeffer, 1990;Xiao et al, 1997] and 1-10 M chelerythrine [Herbert et al, 1990]), all used at concentrations equal to or greater than those previously reported to be effective in HeLa cells, did not inhibit the response (our unpublished results).…”

“…Inhibition of other kinases and ATP-binding proteins by H8 requires much higher concentrations; for example, 50-, 100-, 800-, and 600-fold higher for myosin light chain kinase, casein kinase I, casein kinase II, and myosin, respectively (Hidaka et al, 1984). In contrast to H8, H7 inhibits both PKA and PKC at 50 M (Reich and Pfeffer, 1990;Xiao et al, 1997), but like H8, it has little effect on other kinases except at very high concentrations (Hidaka et al, 1984). Thus, an examination of the sensitivities of a process of interest to a number of these inhibitors can provide a powerful tool for deducing the potential involvement of a certain protein kinase.…”