Adrenarche: Physiology, Biochemistry and Human Disease

Richard J. Auchus; William E. Rainey

Disclosures

Clin Endocrinol. 2004;60(3) 

In This Article

Adrenarche and Steroidogenesis

CYP11A performs the first committed step common to all cells that synthesize steroid hormones: the conversion of cholesterol to pregnenolone. The acute regulation of this process is mediated by the steroidogenic acute regulatory protein (StAR) (Stocco, 1997), and the chronic maintenance of this activity is mediated by ACTH. Although CYP11A is essential for DHEA-S biosynthesis and for adrenarche, all steroid hormone production requires CYP11A as well. In addition, the expression of CYP11A appears to be similar in all three zones of the adrenal cortex, and there are no changes in adrenal reticularis expression of CYP11A seen at adrenarche (Suzuki et al., 2000). However, as CYP11A (with StAR) is the quantitative regulator of steroidogenesis, alterations in CYP11A activities may contribute to the genesis of premature and/or exaggerated adrenarche.

CYP17 catalyses the 17α-hydroxylation of both pregnenolone and progesterone and the 17,20-lyase reaction on their 17-hydroxy derivatives. Whereas CYP17 isoforms from various species all 17α-hydroxylate progesterone and pregnenolone with comparable efficiencies, the rates of the 17,20-lyase reactions can be very different for the two 17-hydroxysteroids. Human CYP17, as well as CYP17 from sheep, cow and other primates, executes the 17,20-lyase reaction efficiently only with 17α-hydroxypregnenolone (the Δ5-pathway) (Katagiri et al., 1995; Lee-Robichaud et al., 1995; Auchus et al., 1998). As discussed later, the substrate preferences for the human 17,20-lyase activity of CYP17 is an important component in the process of adrenarche.

Because CYP17 is poised at the key branch points in steroidogenesis, its two principal activities (or lack thereof) determine which classes of steroid hormones a given cell can produce. In the absence of CYP17 activities, only progesterone, mineralocorticoids such as 11-deoxycorticosterone and aldosterone, and the weak glucocorticoid corticosterone can be produced (i.e. human adrenal zona glomerulosa, rat and mouse adrenals). The 17α-hydroxylase activity is necessary for production of the glucocorticoid cortisol (human adrenal zona fasciculata), and both 17α-hydroxylase and 17,20-lyase activities are needed for C19 steroid production (adrenal zona reticularis, human foetal adrenal, Leydig and theca cells of the gonads). Therefore, CYP17 is recognized as one of the principal qualitative regulators of steroidogenesis (Miller, 1998). CYP17 expression is similar in the fasciculata and reticularis zones of the human adrenal, and therefore the regulation of CYP17 expression does not appear to be a cause of adrenarche. However, adequate amounts of CYP17 are certainly required for maximal DHEA production, so that CYP17 is an essential component of the adrenarche process.

An important feature of adrenarche is the increased conversion of 17α-hydroxypregnenolone to DHEA that results from an increase in the 17,20-lyase activity of CYP17. The relative activity of 17α-hydroxylase vs. 17,20-lyase activity appears to be regulated by multiple post-translational events, all of which could be influenced at adrenarche. CYP17, like all microsomal cytochromes P450, requires an electron transfer flavoprotein cytochrome P450 oxidoreductase (CPR) to shuttle electrons from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to its heme centre. Using CYP17 from porcine testes (Nakajin et al., 1981), Hall and colleagues showed that increasing the molar ratio of CPR to CYP17 increased the ratio of 17,20-lyase to 17α-hydroxylase activity (Yanagibashi & Hall, 1986) and that CPR is more abundant in porcine testis (which principally makes C19 steroids) than in adrenal (Yanagibashi & Hall, 1986). These data indicate that adequate amounts of CPR are essential for optimal 17,20-lyase activity. The expression of CPR has been examined in human adrenal glands throughout development (Suzuki et al., 2000). CPR is expressed in all three adrenal zones, with highest expression in the zona reticularis. At adrenarche, the expression of CPR increases in all zones of the adrenal, which should increase both activities of the CYP17 enzyme system, particularly 17,20-lyase activity, thus boosting the overall capacity to produce DHEA-S. Therefore, robust adrenal CPR expression is necessary for adrenal DHEA-S production, but increases in CPR content alone are not sufficient to explain the biochemical changes in the reticularis that occur during of adrenarche.

There is considerable evidence that the interaction of CYP17 with cytochrome b 5 profoundly influences the enzymatic activities of CYP17. Cytochrome b 5 (b 5) is a small haemoprotein with membrane-bound and soluble forms that perform specific functions in various tissues. Hall and colleagues were the first to show that addition of purified b 5 to the reconstituted CYP17 assay system selectively and dramatically enhanced the 17,20-lyase activity (Onoda & Hall, 1982). This effect of b 5 has been seen with CYP17 from many sources (Kominami et al., 1992; Katagiri et al., 1995; Lee-Robichaud et al., 1995; Auchus et al., 1998). In all cases, optimal molar ratios of b 5 to CYP17 increase the rate of the 17,20-lyase reaction over 10-fold, such that this rate approaches that of the 17α-hydroxylase reaction.

Immunohistochemical examination of the pattern of b 5 expression within the adrenal supports an important role for b 5 in DHEA-S biosynthesis. The adult adrenal reticularis of human beings and of rhesus macaques expresses high levels of b 5, much higher than those seen in the zona fasciculata (Mapes et al., 1999; Suzuki et al., 2000). This pattern is not seen in the preadrenarchal adrenal gland, in which low amounts of b 5 are present in all zones of the adrenal cortex. As adrenarche progresses, the expanding zona reticularis expresses high amounts of b 5 (Fig. 2).

The enzymatic activities of human CYP17 can also be regulated post-translationally through phosphorylation. Human CYP17 is phosphorylated on serine/threonine residues (Zhang et al., 1995), and dephosphorylation preferentially reduces 17,20-lyase activity. Although the kinase(s) responsible for CYP17 phosphorylation have not been identified, recent evidence suggests that protein phosphatase 2A (PP2A) inhibits 17,20-lyase activity by dephosphorylating CYP17 (Pandey et al., 2003). Nevertheless, alteration of CYP17 phosphorylation status is an attractive mechanism for driving the increased DHEA-S production during adrenarche, and kinases or phosphatases regulating this process are candidates for genes that are dysregulated in androgen excess states such as premature adrenarche and polycystic ovary syndrome (PCOS).

SULT2A1 catalyses the final step in the biosynthesis of DHEA-S (Fig. 3), and this enzyme is expressed at high levels in the human adrenal gland but not in the gonads. This restricted pattern is in contrast to all other enzymes involved in DHEA-S biosynthesis, which are expressed in both the adrenal and the gonads. SULT2A1 has a broad substrate specificity, which includes metabolism of pregnenolone, 17α-hydroxypregnenolone and DHEA to their respective sulphated products. Once sulphated by SULT2A1, pregnenolone and 17α-hydroxypregnenolone are no longer available as substrates for CYP17 or 3βHSD type 2 (see below). Therefore, SULT2A1 sulphation of pregnenolone and 17α-hydroxypregnenolone removes these pluripotent steroid precursors from the mineralocorticoid, glucocorticoid and adrenal androgen biosynthetic pathways. Thus balance and coordination of SULT2A1 and CYP17 activities must be maintained to optimize flux of pregnenolone to DHEA-S.

Steroidogenic pathway for the production of dehydroepiandrosterone sulphate (DHEA-S). Production of DHEA-S relies on the coordinated expression of steroidogenic acute regulatory (StAR) protein, cholesterol side-chain cleavage enzyme (CYP11A), 17α-hydroxylase/17,20-lyase (CYP17) and DHEA-sulphotransferase (SULT2A1). Also positively impacting DHEA-S biosynthesis is cytochrome P450 oxidoreductase (CPR), which enhances both CYP17 enzymatic activities, and cytochrome b5, which selectively enhances the 17,20-lyase activity of CYP17. Negatively impacting the production of DHEA-S is 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2), which irreversibly diverts DHEA-S precursors to mineralocorticoids and glucocorticoids in the Δ4-pathways.

To determine whether the SULT2A1 enzyme is specifically associated with cells that secrete DHEA-S, immunohistochemistry has been used to localize SULT2A1 in adrenals throughout the postnatal period from infancy to old age (Suzuki et al., 2000). Expression of SULT2A1 remains low through early childhood until the onset of adrenarche. As the adrenal reticularis begins to grow, adrenal expression of SULT2A1 increases (Fig. 2b). Zona reticularis expression of SULT2A1 continues into adulthood and is maintained into the later years of life. These data show that the increase in DHEA-S production occurring at adrenarche is associated with accelerated expression of SULT2A1 in the adrenal reticularis, which would increase the conversion of nascent DHEA into DHEA-S.

5/4
-Isomerases)

Another key regulatory process in human steroidogenesis is the flux of steroids from the Δ5-pathway to the Δ4-pathway. Pregnenolone, 17α-hydroxypregnenolone and DHEA are all substrates for the 3β-hydroxysteroid dehydrogenase/Δ5/4-isomerases (3βHSDs), which irreversibly transform these Δ5-steroids into their Δ4-congenors. In human beings there are two 3βHSDs: type 1 3βHSD is found in liver, skin, placenta and other peripheral tissues, and type 2 3βHSD (3βHSD2) is expressed in adrenal and gonads (Lorence et al., 1990; Rheaume et al., 1991; Labrie et al., 1992). Mutations in 3βHSD2 cause a rare form of congenital adrenal hyperplasia (Rheaume et al., 1994; Simard et al., 2002), and these patients have elevated circulating concentrations of Δ5-steroids, particularly 17α-hydroxypregnenlone (Lutfallah et al., 2002). However, 3βHSD2 is normal in most women with idiopathic hirsutism and premature adrenarche (Zerah et al., 1994; Sakkal-Alkaddour et al., 1996).

Inspection of the steroidogenic pathway illustrates the somewhat paradoxical role of 3βHSD2 in DHEA-S biosynthesis and why its downregulation in reticularis cells may be crucial for adrenarche to occur (Fig. 3). While 3βHSD2 activity is essential to the genesis of aldosterone and cortisol in the glomerulosa and fasciculata, 3βHSD2 expression in the reticularis would negatively impact DHEA biosynthesis. Thus 3βHSD2 activity in the zona reticularis would effectively drain steroid flux away from the Δ5-pathway leading to DHEA, as well as convert DHEA itself to androstenedione. Indeed, pharmacological inhibition of 3βHSD2 activity in isolated adrenal fasciculata cells greatly enhances DHEA production (Endoh et al., 1996). To determine whether 3βHSD expression correlates with the ability to produce DHEA(S), 3βHSD2 protein has been examined in adrenals in the postnatal period from infants through to old age by use of immunohistochemistry (Gell et al., 1998; Suzuki et al., 2000). Infants younger than 5 years old exhibit a poorly developed adrenal reticularis that expresses 3βHSD2. At adrenarche, the zona reticularis begins to expand, and 3βHSD2 content falls, restricting steroidogenesis to the Δ5-pathway. The content of 3βHSD2 protein in the reticularis remains low throughout adulthood, maintaining DHEA-S production (Fig. 2c). The content of 3βHSD2 mRNA also appears to be lower in adrenals from infants over 8 years of age compared to younger children (Dardis et al., 1999). In support of these observations, isolated adrenal reticularis cells express low amounts of 3βHSD2, while fasciculata cells have high 3βHSD2 expression (Endoh et al., 1996). In addition, the expression of 3βHSD2 correlates with the production of DHEA in certain adrenal carcinomas (Sakai et al., 1994). Taken together, these studies support the hypothesis that reduced expression of 3βHSD2 in the developing adrenal reticularis is a key determining factor driving DHEA(S) production during adrenarche.

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