P450scc plays a key role in carrying out the conversion of cholesterol to pregnenolone and is subject to many levels of regulation. In the adrenal, gonads, and placenta, P450scc is regulated by various tropic hormones, utilizing cAMP or calcium as intracellular messengers.12 Steroid hormone biosynthesis in the adrenals is stimulated by the pituitary hormone corticotropin, which binds to its receptor at the cell surface and activates adenyl cyclase to increase intra-cellular cAMP. The latter compound serves as an intracellular messenger to transduce both the acute and the long-term effects of corticotropin.
The acute effect of corticotropin increases the output of steroids within minutes of its admin-istration.12 This effect is due to increased synthesis of StAR protein, which accelerates the transport of cholesterol into the inner mito-chondrial membrane, where P450scc converts cholesterol to pregnenolone. The accelerated transport of substrate for P450scc action results in enhanced adrenal steroid hormone secretion. This rapid steroidogenic reaction is the method that cells use to respond to stress.
In contrast to the acute effect of corticotropin, its long-term action requires many hours to achieve and is due to stimulation of CYP11A1 gene expression.12 This relatively slow steroido-genic effect is important for maintenance of homeostasis in the body.
Another stimulator of CYP11A1 gene expression is the potent vasoconstrictor angiotensin II, which is formed from the inactive hormone an-giotensin I in a variety of tissues.12 Angiotensin II regulates mineralocorticoid biosynthesis in the zona glomerulosa of the adrenal cortex. Its mechanism of action involves binding to its membrane receptor and activation of protein ki-nase C, which in turn increases intracellular calcium levels and subsequent signal transduction and gene stimulation.
In the ovary, CYP11A1 expression is induced by follicle-stimulating hormone (FSH) and LH, resulting in biosynthesis of estrogens, progesterone, and androgens during the menstrual cycle.12 The major intracellular mediator for CYP11A1 stimulation by gonadotropins is cAMP.
Ferredoxin gene expression is also stimulated in the adrenal and ovary, using cAMP as intra-cellular messenger, in a manner similar to that described for CYP11A1.12 However, in contrast to the CYP11A1 andferredoxin genes, the mechanism of action of the ferredoxin reductase gene does not involve a change in cAMP or calcium levels.12 Instead, its mRNA levels are diminished, suggesting possible posttranscriptional regulation of the ferredoxin reductase gene.
Following the conversion of cholesterol to pregnenolone by the mitochondrial side-chain cleavage system, the adrenals and gonads can transform pregnenolone to either progesterone or 17a-hydroxypregnenolone. The formation of progesterone from pregnenolone is catalyzed by the enzyme 3jS-hydroxysteroid oxidoreductase [commonly referred to as 3j6-hydroxysteroid dehydrogenase (3jS-HSD)] in combination with A5,4-iso-merase, in the presence of the cofactor (NAD + ). In addition, 3jS-HSD catalyzes the formation of other A4-3-ketosteroids from corresponding A5-3j6-hydroxysteroids, which leads to the formation of androgens, estrogens, mineralocorticoids, and glucocorticoids. Thus, the reactions involving 3jS-HSD represent an obligatory step in the formation of highly biologically active steroids.
3jS-HSD is expressed in the adrenal, gonads, and placenta, as well as in other tissues such as the liver and kidney, where its function has not been elucidated.20-22 In cells, it is membrane-bound in the mitochondria and endo-plasmic reticulum.20-22 A family of closely related genes encode for 3jS-HSD, and the various 3jS-HSD isoforms are expressed in a tissue-specific manner involving separate regulatory mechanisms.
Presently, only two functional 3fi-HSD genes (type 1 and type 2) together with three pseudogenes have been characterized in the human genome.23-26 Both genes are located in the chromosome 1p13.1 region, are 7.84 and 7.88 kb in length, and consist of four exons and three introns.25,27 The type 1 and type 2 3jS-HSD proteins are 93.5% homologous in amino acid sequence.23-26 Type 1 gene expression occurs primarily in the placenta, mammary gland, and skin, whereas the type 2 isoform is expressed almost exclusively in the adrenals and gonads.
Formation of 17a-Hydroxypregnenolone, 17a-Hydroxyprogesterone, Dehydroepiandrosterone, and Androstenedione
After formation of pregnenolone and progesterone, each of these compounds can undergo hydroxylation at carbon 17 and subsequent cleavage of the side chain (carbons 20, 21) at carbon 17, forming dehydroepiandrosterone (DHEA) and androstenedione, respectively. 17a-Hydroxylation occurs through the action of 17a-hydroxylase, whereas Cx7,20-lyase catalyzes the side-chain cleavage. It is now well documented that both enzymatic activities are catalyzed by a single protein, P450c17 encoded by the CYP17 gene.28 Since both 17a-hydroxylation and cleavage of the bond between carbons 17 and 20 are obligatory reactions in the biosynthesis of androgens and estrogens, the P450c17 protein is of fundamental importance in reproductive biology.
Cloning of cDNA from human adrenals and gonads, and subsequent nuclease protection experiments, established that there was only one species of human P450c17 mRNA, which is identical in both the adrenals and gonads.29 Subsequent gene cloning confirmed that in humans there is only one CYP17 gene,30,31 which lies on chromosome 10q24.3.32-34 Additional functional genetic evidence supporting the single P450c17 concept was provided by studies showing ablation of both 17a-hydroxylase and Cx7,20-lyase activities due to mutations of this gene.35-37
The two activities of the CYP17 gene appear to be regulated independently.28 In testicular Leydig cells and ovarian theca cells, Cx7,20-lyase activity is very high so that the C21 steroid precursors are converted to C19 products, with little residual 17-hydroxylated C21 product. In contrast, the zona fasciculata of the adrenal produces large amounts of 17-hydroxylated C21 steroids and very little androgen. Increasing the molar ratio of electrons to P450c17 increases the ratio of Cx7,20-lyase activity to 17a-hydroxylase activity.28 In addition, P450c17 must be phos-phorylated on serine and threonine residues by a cAMP-dependent protein kinase to acquire Cx7,20-lyase activity. On the basis of these findings, it has been proposed that the ratio of C17,20-lyase to 17a-hydroxylase activity is regulated by the availability of electrons flowing to the en-zymes28. Electron flow can be increased by an increase in molar concentrations of the flavo-protein that carries electrons to P450c17. Alternatively the C17,20-lyase/17a-hydroxylase ratio may be regulated by alteration of serine/threo-nine phosphorylation.
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