Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

Heme oxygenase-1 mRNA levels,metallothionein mRNA levels,lipid peroxidation and microsomal CYP1A activities in rats treated with 3,3-dichlorobenzidine and some other inducers of P450

The effect of 3,3-dichlorobenzidine (DCB), a potent inducer of CYP1A, on the levels of heme oxygenase-1 mRNA and metallothionein mRNAs was examined in the kidney, liver and lung of rats administered a single ip dose (157 μmol/kg) of the compound. DCB treatment increased heme oxygenase-I mRNA abundance in the kidney significantly from barely detectable levels in untreated animals; the maximum increase in the liver and lung was 24-fold and 4-fold, respectively. Hepatic microsomal heme oxygenase activity was also induced by DCB. In contrast with DCB, 2 other P450 inducers, β-naphthoflavone (β-NF) and phenobarbital did not elevate tissue HO-1 rnRNA levels. DCB pretreatment also elevated metallothionein mRNA levels in the kidney, liver and lung, with the effect in the lung being the least pronounced. In contrast with HO-1 mRNA, metallothionein mRNA was increased by the other P450 inducers examined. In vivo lipid peroxidation and in vitro NADPH-dependent microsomal lipid peroxidation were increased in the liver of DCB-treated rats but not in those of phenobarbital- or β-naphthoflavone-treated rats. Treatment with DCB or β-NF did not alter total hepatic microsomal P450 content, as measured spectrophotometrically, but induced the activity of CYP1A2. In contrast, the activity of CYP1A1 was induced to a lesser extent by DCB than by β-NF. The data show that DCB induces HO-1 as weD as P450 1A, confirm stimulation of lipid peroxidation by the compound, and suggest oxidative stress as a mechanism of HO-1 induction by the compound.     

Constitutively active protein kinase A qualitatively mimics the effects of follicle-stimulating hormone on granulosa cell differentiation

Activation of the protein kinase A (PKA) signaling system is necessary for FSH-induced granulosa cell differentiation, but it is not known whether activation of PKA is sufficient to account for the complex pattern of gene expression that occurs during this process. We addressed this question by infecting granulosa cells with a lentiviral vector that directs the expression of a constitutively active mutant of PKA (PKA-CQR) and compared the cellular responses to PKA-CQR with cells stimulated by FSH. Expression of PKA-CQR in undifferentiated granulosa cells resulted in the induction of both estrogen and progesterone production in the absence of cAMP. The stimulatory effects of both PKA-CQR and FSH on estrogen and progesterone production were suppressed by the PKA inhibitor H-89 and were mimicked by PKA-selective cAMP agonists. mRNA levels for P450scc and 3beta-HSD were induced to a similar extent by FSH and PKA-CQR, whereas mRNA levels for P450arom and the LHr were induced to a greater extent by FSH. Microarray analysis of gene expression profiles revealed that the majority of genes appeared to be comparably regulated by FSH and PKA-CQR but that some genes appear to be induced to a greater extent by FSH than by PKA-CQR. These results indicate that the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway during granulosa cell differentiation. However, optimal induction of aromatase, the LHr, and other genes by FSH appears to require activation of additional signaling pathways.     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

The role of heme metabolism during the induction of hepatic and renal cytochrome P-450 levels and drug-metabolizing enzymes in rats by a Prudhoe Bay crude oil

Administration of Prudhoe Bay crude oil (PBCO) to rats resulted in a dose-related increase in liver weight; rapid and marked increase in the activity of hepatic delta-aminolevulinate synthetase, the initial and rate-limiting enzyme in the heme biosynthetic pathway; rapid decline in the activity of hepatic heme oxygenase, the rate-limiting enzyme of heme catabolism; and more gradual increase in the levels of hepatic cytochrome P-450 and some mixed-function oxidase activities such as benzo[a]pyrene hydroxylase and 7-ethoxyresorufin-O-deethylase. PBCO treatment also increased renal cytochrome P-450 levels and mixed-function oxidase activities; however, delta-aminolevulinate synthetase and heme oxygenase activities were unchanged. This suggests that different regulatory mechanism(s) may be involved in renal heme metabolism and induction of monoxygenase system.