Silvia Vianello

Developmentally Regulated Expression and Activity of 17α-Hydroxylase/C-17,20-Lyase Cytochrome P450 in Rat Liver1

We have investigated the developmental pattern of expression and activity of 17α-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein·min) at a level comparable to that in the testis (188 pmol/mg protein·min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein·min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein·min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 ex...

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.

Trout GH promoter analysis reveals a modular pattern of regulation consistent with the diversification of GH gene control and function in vertebrates.

In vertebrates, growth hormone (GH) gene expression requires the pituitary-specific transcription factor Pit-1/GHF1 but is differently regulated by a variety of factors in different vertebrate species. Here, we have studied the transcriptional activity of the trout GH (tGH) promoter, which is synergistically stimulated by cAMP and glucocorticoid. Gel shift assays indicated that Pit-1 binds as a dimer to three high affinity sites in the -226/+24 tGH region, and that recombinant cAMP response element (CRE)-binding protein (CREB) binds to a CRE situated between the two distal Pit-1 sites. Deletional and mutational transfection experiments, performed in pituitary Pit-1-expressing GC cells, showed that the different Pit-1 sites play distinct roles and are obligatory elements in the mechanisms mediating cAMP and glucocorticoid responses. Remarkably, the results suggest a hierarchical modular model of regulation of the tGH promoter, according to which a critical module, triggered by Pit-1 bound to the proximal Pit-1 site, is necessary and sufficient to turn on and drive basal levels of transcription. The latter may be stimulated synergistically by two Pit-1-dependent reciprocally non-cooperative auxiliary modules, activated by cAMP and glucocorticoid, respectively. Such modularity explains, in evolutionary terms, the crucial role played by Pit-1 in transcriptional activation and the emergence of the wide variety of mechanisms regulating transcriptional levels of GH, prolactin and other Pit-1-target genes in vertebrates.

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17alpha-hydroxyprogesterone, androstenedione and testosterone. The transition from C21 to C19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17alpha-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-3H]progesterone and [1,2-3H]17alpha-hydroxyprogesterone as substrates. In the testis and male and female duodena, the Km values for progesterone 17alpha-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the Vmax values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the Km values for exogenous 17alpha-hydroxyprogesterone were 525, 675 and 637 nM, whereas the Vmax values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the Km values were calculated with respect to intermediate 17alpha-hydroxyprogesterone formed from progesterone, they were similar to the Km values for 17alpha-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the Vmax values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of Km values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17alpha-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17beta inhibited 17alpha-hydroxylation but not bond scission on intermediate 17alpha-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.

RETRACTED: Occurrence of steroid-converting enzymes in the gonads of the Manila clam, Ruditapes philippinarum (Adams and Reeve, 1850), and correlation with the gametogenic cycle.

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