Chapter 13. Regulation of Steroidogenesis€¦ · the so-called “backdoor pathway” of fetal andro-gen synthesis (see below). Based on their activi-ties, it is physiologically

C H A P T E R

13

Regulation of SteroidogenesisAndrew A. Bremer* and Walter L. Miller†

*Vanderbilt University, Nashville, TN, USA, †University of California, San Francisco, CA, USA

INTRODUCTION

Steroidogenesis is regulated at multiplelevels, principally by transcription of genesencoding steroidogenic enzymes and co-factors,and by their post-translational modification. Anunderstanding of steroidogenesis and its regula-tion first requires an understanding of the bio-chemistry and genetics of these enzymes and co-factors. The patterns of gland and cell type-specific steroidogenesis reflect variations inthese regulatory mechanisms. Understandingthe roles of steroidogenic factors has been facili-tated by identifying their genetic lesions, whichcause rare disorders of steroidogenesis.Understanding steroidogenesis and its regula-tion are important for understanding disordersof sexual differentiation, reproduction, fertility,hypertension, obesity, and physiologichomeostasis.

CLASSES OF STEROIDOGENICENZYMES

Most steroidogenic enzymes are either cyto-chrome P450s or hydroxysteroid dehydrogenases

(HSDs). These enzymes are functionally unidi-rectional, so the accumulation of product doesnot drive flux back to the precursor. All P450-mediated hydroxylations and carbonacarbonbond cleavage reactions are mechanisticallyirreversible. Alternatively, HSD reactions aremechanistically reversible and can run in eitherdirection under certain conditions in vitro, buteach HSD enzyme drives steroid flux predomi-nantly in either the oxidative or reductivemode in vivo.1 At least two HSD enzymes drivethe flux of a hydroxysteroid and its cognateketosteroid in opposite directions, favoringeither ketosteroid reduction or hydroxysteroidoxidation.

Cytochrome P450 Enzymes

Most steroidogenic enzymes are membersof the cytochrome P450 group of ezymes,2 ageneric term for a group of oxidases, all ofwhich have about 500 amino acids and containa single heme group. The enzymes are termedP450 (pigment 450) because they absorb lightat 450 nm in their reduced states. The genesfor these enzymes are formally termed CYPgenes, and a systematic nomenclature has

207Cellular Endocrinology in Health and Disease.

DOI: http://dx.doi.org/10.1016/B978-0-12-408134-5.00013-5 Copyright © 2014 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-408134-5.00013-5

been described (http://drnelson.uthsc.edu/cytochromeP450.html); the encoded proteinsmay be given the same name without italics.Cytochrome P450 enzymes bind their sub-strates and achieve catalysis in an active siteassociated with the heme group. The humangenome has 57 CYP genes; 7 encode “Type 1”P450s targeted to mitochondria; the other 50encode “Type 2” P450s targeted to the endo-plasmic reticulum. Although the liver metabo-lizes countless endogenous and exogenoustoxins, drugs, xenobiotics, and environmentalpollutants, these reactions are catalyzed byonly about eight P450s, and most P450s partici-pate in biosynthetic processes.3 Each P450enzyme can metabolize multiple substrates,catalyzing a broad array of oxidations.

Six P450 enzymes are involved in steroido-genesis (Figure 13.1). Mitochondrial P450scc(CYP11A1) is the cholesterol side-chain cleav-age enzyme catalyzing the series of reactions

formerly termed “20,22 desmolase.” Two distinctisozymes of P450c11, P450c11β (11β-hydroxylase;CYP11B1) and P450c11AS (aldosterone synthase;CYP11B2), also found in mitochondria, catalyze11β-hydroxylase, 18-hydroxylase, and 18-methyloxidase activities. P450c17 (CYP17A1), foundin the endoplasmic reticulum, catalyzes both17α-hydroxylase and 17,20-lyase activities, andP450c21 (CYP21A2) catalyzes the 21-hydroxy-lation of both glucocorticoids and mineral-ocorticoids. In the gonads and elsewhere, P450aro(CYP19A1) in the endoplasmic reticulum cata-lyzes aromatization of androgens to estrogens.

Hydroxysteroid Dehydrogenase Enzymes

The HSD enzymes have molecular masses ofB35�45 kDa, do not have heme groups, andrequire nicotine adenine dinucleotide or its phos-phate (NADH/NAD1 or NADPH/NADP1)

Cholesterol

Pregnenolone

StAR

P450c17POR

P450c17POR+b5

17βHSD1 17βHSD2 17βHSD3 17βHSD2 17βHSD1 17βHSD2

Cortisone

Cortisol

P450c11βFdx/FdR

11βHSD2 11βHSD1 H6PDH

Estradiol

Estrone

Testosterone

Dihydrotestosterone

5α-Reductasel 1 & 2

17βHSD5

Androstenediol

AndrostenedioneDHEA

P450c17POR

3βHSD

3βHSD

P450c17-PO4

3βHSD17OH-Progesterone170H-Preg

3βHSD

P450c21POR

P450c21POR

P450aroPOR

P450aroPOR

P450c11ASFdx/FdR

Aldosterone11-Deoxycorticosterone

11-Deoxycortisol

Progesterone

P450sccFdx/FdR

FIGURE 13.1 The major human steroidogenic pathways. Key enzymes and cofactors are shown near arrows indicat-ing chemical reactions. The steroids in the first column are Δ5-steroids, which constitute the preferred pathway to C19 ster-oids in humans. The dashed arrow indicates poor flux from 17α-hydroxyprogesterone to androstenedione via P450c17,and the three small arrows below P450c11AS emphasizes the three discrete steps with intermediates corticosterone and18-hydroxycorticosterone. Note: not all the intermediate steroids, pathways, and enzymes are shown.

CELLULAR ENDOCRINOLOGY IN HEALTH AND DISEASE

208 13. REGULATION OF STEROIDOGENESIS

http://drnelson.uthsc.edu/cytochromeP450.html

http://drnelson.uthsc.edu/cytochromeP450.html

as co-factors to either reduce or oxidize a steroidby two electrons via a hydride transfer mecha-nism. Unlike most steroidogenic reactions cata-lyzed by P450 enzymes, which are due to theaction of a single form of P450, each HSD reac-tion can be catalyzed by at least two, often verydifferent, isozymes. Members of this familyinclude the 3α- and 3βHSDs, the two 11βHSDs,and a series of 17βHSDs; the 5α-reductases areunrelated to this family.

The HSD enzymes are categorized into twogroups: the short-chain dehydrogenase reductase(SDR) family, characterized by a “Rossman fold,”and the aldo-keto reductase (AKR) family, char-acterized by a triosephosphate isomerase (TIM)barrel motif.1 The SDR enzymes include11βHSDs 1 and 2, and 17βHSDs 1, 2, 3, and 4; theAKR enzymes include 17βHSD5, which is impor-tant in extra-glandular activation of androgenicprecursors, and the 3αHSDs that participate inthe so-called “backdoor pathway” of fetal andro-gen synthesis (see below). Based on their activi-ties, it is physiologically more useful to classifythese enzymes as dehydrogenases or reductases:the dehydrogenases use NAD1 as their cofactorto oxidize hydroxysteroids to ketosteroids; thereductases mainly use NADPH to reduce ketos-teroids to hydroxysteroids. Although theseenzymes are typically bidirectional in vitro, theyphysiologically tend to function in only onedirection in vivo, with the direction determinedby the available intracellular cofactor(s).1

STEROID HORMONEBIOSYNTHESIS

Cholesterol Uptake, Storage, Transport,and Delivery to the Mitochondria

Steroidogenic cells can synthesize cholesterolde novo from acetate, but most of their choles-terol supply comes from dietary-derived low-density lipoproteins (LDL). The intracellularcholesterol pool is then regulated by the sterol

response element binding proteins (SREBPs), agroup of transcription factors involved in thebiosynthesis of cholesterol and fatty acids.4

Adequate concentrations of LDL suppress3-hydroxy-3-methylglutaryl co-enzyme A (HMGCoA) reductase, the rate-limiting enzyme incholesterol synthesis. However, tropic hor-mones that act via cyclic adenosine monopho-sphate (cAMP) (adrenocorticotropic hormone(ACTH) in the adrenal, luteinizing hormone(LH) in the gonad) stimulate the synthesis ofHMG CoA reductase and LDL receptors anduptake of LDL cholesterol (LDL-C). LDL-Cesters are taken up by receptor-mediated endo-cytosis, and are then stored directly or con-verted to free cholesterol for use in steroidhormone biosynthesis. Free cholesterol can beesterified by acyl-CoA:cholesterol transferase(ACAT) and stored in lipid droplets. Cholesterolis accessed by the NPC proteins (named for theircausative role in Niemann�Pick type C disease)and by hormone-sensitive lipase (HSL) in lateendosomes. ACTH stimulates HSL and inhibitsACAT, thus increasing the availability of freecholesterol for steroid hormone synthesis. Theunesterified “free” cholesterol may travel fromendosomes to the mitochondria by either vesicu-lar transport, involving membrane fusion, or bynon-vesicular transport, bound to cytosolicSTART (StAR-related lipid transfer) proteinsthat are structurally related to the steroidogenicacute regulatory protein, StAR. However, StARitself plays a minor role in cholesterol transportto the mitochondria, but instead functions tomove cholesterol from the outer mitochondrialmembrane (OMM) to the inner mitochondrialmembrane (IMM).5

The Steroidogenic Acute RegulatoryProtein, StAR

Whereas cells that produce polypeptide hor-mones can store hormone in secretory vesiclesfor immediate release in response to altered


209STEROID HORMONE BIOSYNTHESIS

membrane potentials, steroidogenic cells storevery little steroid, so that an acute demand formore steroid requires the rapid synthesis ofsteroid. ACTH, a proteolytic product of pitui-tary pro-opiomelanocortin (POMC), and possi-bly other POMC-peptides are trophic factorsthat stimulate adrenal cellular growth viafibroblast growth factor (FGF), epidermalgrowth factor (EGF), and insulin-like growthfactor 2 (IGF2). ACTH is also a tropic factorthat stimulates the chronic regulation of ste-roidogenesis by stimulating the transcriptionof genes for steroidogenic enzymes, principallyCYP11A1 encoding P450scc. The rapid induc-tion of steroidogenesis, such as in the classic“fight-or-flight” response or in response tointravenously administered ACTH, is medi-ated by regulating the movement of cholesterolfrom the OMM to the IMM.6 This mitochon-drial inflow of cholesterol is regulated by thesteroidogenic acute regulatory protein, StAR,which thus regulates steroidogenesis by regu-lating the access of cholesterol substrate to theP450scc enzyme residing on the IMM.7

Historically, when steroidogenic cells or intactrats were treated with inhibitors of protein syn-thesis such as cycloheximide, the acute steroido-genic response was eliminated, suggesting that ashort-lived protein acts at the level of the mito-chondrion as the specific trigger to the acute ste-roidogenic response. This factor was identified asshort-lived 30- and 37-kDa phosphoproteins thatwere rapidly synthesized when steroidogeniccells were stimulated with tropic hormones, thencloned from mouse Leydig MA-10 cells andnamed the steroidogenic acute regulatory pro-tein, StAR.6,7 The central role of StAR in steroid-ogenesis was proven by finding that StARmutations caused congenital lipoid adrenalhyperplasia.8,9 Thus, StAR is the acute triggerthat is required for the rapid flux of cholesterolfrom the outer to the inner mitochondrial mem-brane that is needed for the acute response of

aldosterone to angiotensin II, of cortisol toACTH, and of sex steroids to an LH pulse.

P450scc

The first and rate-limiting step in steroidogen-esis is the conversion of cholesterol to pregneno-lone by cytochrome P450scc (encoded byCYP11A1). The expression of this enzyme ren-ders a cell “steroidogenic” and determines its ste-roidogenic capacity, thus acting as the long-termchronic regulator of steroidogenesis.2,5 P450scccatalyzes three distinct chemical reactions, 20α-hydroxylation, 22-hydroxylation, and scission ofthe cholesterol side-chain to yield pregnenoloneand isocaproic acid. No other enzyme has beenfound that can produce pregnenolone from anysource. The dissociation constants for the 20-OH-cholesterol and 20,22-(OH)-cholesterol intermedi-ates are high, so that these steroids tend toremain bound to the active site, awaiting the nextreaction. The transcription of the single CYP11A1gene on chromosome 15 is regulated differentlyin different cell types. In the adrenal zona fascicu-lata and gonad, ACTH and LH act via cAMP uti-lizing specific cis-active promotor regions; in theadrenal zona glomerulosa, angiotensin II acts viathe calcium/calmodulin pathway utilizing whollydifferent cis-elements of the same promoter, butin both cases steroidogenic factor-1 (SF1) is essen-tial. By contrast, placental expression of CYP11A1,which is essential for placental synthesis of theprogesterone that suppresses uterine contractilityand hence maintains pregnancy, is wholly differ-ent. Placental CYP11A1 expression is independentof SF1, and utilizes TReP-132 and CP2 membersof the grainyhead family of transcription factorsthat act at wholly distinct cis-elements ofCYP11A1.2 Because placental progesterone isneeded to maintain pregnancy, it is counter-intuitive that there should be mutations in humanCYP11A1; nevertheless, at the time of writing, 19



such patients have been reported with mutationsranging from complete loss-of-function, to reten-tion of substantial activity.10

Transport of Electrons to P450scc:Ferredoxin Reductase and Ferredoxin

P450scc functions as the terminal oxidase in amitochondrial electron transport system.11

Electrons from NADPH are accepted by a flavo-protein termed ferredoxin reductase (alsoknown as adrenodoxin reductase) that is looselyassociated with the IMM. Ferredoxin reductasesubsequently transfers the electrons to an iron/sulfur protein termed ferrodoxin (also knownas adrenodoxin), which is found in the mito-chondrial matrix or loosely adherent to theIMM. Ferredoxin then transfers the electrons toP450scc (Figure 13.2). The same ferredoxin andferredoxin reductase also transfer electrons to

the P450c11β and P450c11AS isozymes of 11β-hydroxylase and to other mitochondrial P450ssuch as the vitamin D 1α- and 24-hydroxylases.Each of the three reactions catalyzed by P450sccrequires a pair of electrons transferred by thissystem; consequently, the rate of conversion ofcholesterol to pregnenolone is determined bythe diffusion of ferredoxin through the mito-chondrial matrix. Mutations have not beendescribed in ferredoxin or ferredoxin reductase.

3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase (3βHSD)

Once pregnenolone is produced from choles-terol, it may undergo 17α-hydroxylation byP450c17 to yield 17α-hydroxypregnenolone(17OH-Preg), or it may be converted to proges-terone, the first biologically important steroid inthe pathway. A single 42-kDa 3βHSD enzymecatalyzes both the conversion of the hydroxylgroup to a keto group on carbon 3 and theisomerization of the double bond from the B ring(Δ5 steroids) to the A ring (Δ4 steroids).2 Thissingle enzyme, 3βHSD, converts pregnenolone toprogesterone, 17OH-Preg to 17α-hydroxyproges-terone (17OHP), dehydroepiandrosterone(DHEA) to androstenedione, and androstenediolto testosterone, all with similar efficiency (Km

and Vmax). As is typical of HSD enzymes, thereare two isozymes of 3βHSD, encoded by separategenes (HSD3B1 and HSD3B2).12 These isozymesshare 93.5% amino acid sequence identity andare biochemically and enzymatically very simi-lar. The enzyme catalyzing 3βHSD activity in theadrenals and gonads is the type-2 enzyme, whilethe type-1 enzyme catalyzes 3βHSD activity inplacenta, breast, liver and other “extraglandular”tissues. Structural data show that 3βHSD can befound in both the endoplasmic reticulum and inmitochondria. It is not clear whether the subcel-lular distribution of 3βHSD differs in various

NADPH

P450

FAD

FeRedFedx

Fe

++

++

+

++––

––––

––

NADP+

c

FIGURE 13.2 Electron transport to mitochondrial

forms of cytochrome P450. The flavin group (FAD) of fer-redoxin reductase (FeRed), which is bound to the innermitochondrial membrane (IMM), accepts two electronsfrom NADPH, converting it to NADP1. These electronspass to the iron�sulfur (Fe2S2, diamond with dots) clusterof ferredoxin (Fedx), which is found either in the mito-chondrial matrix, as shown, or loosely associated with theIMM. Fedx then donates the electrons to the heme of theP450 (square with Fe). Negatively charged residues inFedx (2) guide docking and electron transfer with posi-tively charged residues (1) in both FeRed and the P450.For P450scc, three pairs of electrons must be transported tothe P450 to convert cholesterol to pregnenolone.



types of steroidogenic cells, but, this could be anovel mechanism in regulating the direction ofsteroidogenesis.2

P450c17: 17α-Hydroxylase/17,20-lyase

Microsomal P450c17 catalyzes both 17α-hydroxylase and 17,20-lyase activities in theadrenals and gonads, and hence is the princi-pal qualitative regulator of steroidogenesis,determining the class of steroid that will beproduced. In the absence of P450c17, the adre-nal zona glomerulosa, ovarian granulosa cells,and placenta produce 21-carbon (C21) 17-deoxysteroids such as progesterone and aldosterone;in the presence of 17-hydroxylase activity, theadrenal zona fasciculata produces the C21

17-hydroxy steroid, cortisol; and when both 17-hydroxylase and 17,20-lyase activities are pres-ent, the adrenal zona reticularis, ovarian thecacells, and testicular Leydig cells produce C19

androgens.2 Rodents fail to express P450c17 intheir adrenals, and consequently must utilizecorticosterone as their glucocorticoid. The 17α-hydroxylase reaction occurs more readily thanthe 17,20-lyase reaction. Pregnenolone andprogesterone may undergo 17α-hydroxylationto 17OH-Preg and 17OHP, respectively. 17OH-Preg may also undergo scission of the C17,20carbon bond to yield DHEA; but, since humanP450c17 catalyzes the conversion of 17OHP toandrostenedione with only 3% of the rate forconversion of 17OH-Preg to DHEA, this reac-tion makes a negligible contribution to humanandrogen synthesis.13 This strong “preference”for 17OH-Preg as the substrate for 17,20-lyaseactivity is typical of primates but not othermammals, and is consistent with the largeamounts of DHEA/S produced by primateadrenals. P450c17 is encoded by the CYP17A1gene on chromosome 10q24.3, which is struc-turally related to the CYP21A2 gene forP450c21 (21-hydroxylase). Adrenal transcrip-tion of CYP17A1 is principally regulated by

the transcription factors NF-1C, Sp1, Sp3 andGATA4/6.

Like all Type 2 P450 enzymes, P450c17 isbound to the smooth endoplasmic reticulumwhere it mediates catalysis with electrons fromNADPH donated by P450 oxidoreductase(POR); the efficiency of electron transport fromNADPH is the principal factor regulating the17,20-lyase reaction (Figure 13.1).14 Serum cor-tisol concentrations, an index of adrenal 17α-hydroxylase activity are fairly consistentthroughout life, whereas DHEA and DHEASconcentrations, which reflect adrenal 17,20-lyase activity, are low in early childhood butrise abruptly during adrenarche, which is con-temporaneous with, but independent of,puberty. While this suggested that distinctenzymes performed the hydroxylase and lyaseactivities, the cloning of P450c17 permitted therigorous demonstration that it catalyzed boththe 17α-hydroxylase and 17,20-lyase activi-ties.14,15 Thus, the distinction between 17α-hydroxylase and 17,20-lyase is functional andnot genetic or structural.

Transport of Electrons to P450c17: P450Oxidoreductase and Cytochrome b5

All microsomal P450 enzymes, includingsteroidogenic P450c17, P450c21, and P450aro,receive electrons from POR, a membrane-bound flavoprotein that is distinct from mito-chondrial ferrodoxin reductase.11 POR is abutterfly-shaped protein that has a flavin ade-nine dinucleotide (FAD) moiety in one“wing” and an flavin mononucleotide (FMN)moiety in the other “wing.” The FAD moietyreceives two electrons from NADPH, transfersthem to the FMN, which then transfers themone at a time to the P450 enzyme. ForP450c17, electron transfer for the 17,20-lyasereaction is promoted by the action of thehemoprotein cytochrome b5 as an allostericfactor rather than as an alternate electron



donor.13 The 17,20-lyase activity of P450c17also requires the phosphorylation of serineresidues on P450c17 by a cAMP-dependentprotein kinase (Figure 13.3).14 Thus the avail-ability of electrons determines whetherP450c17 performs only 17α-hydroxylation, oralso performs 17,20 bond scission; increasingthe ratio of POR or cytochrome b5 to P450c17in vitro or in vivo increases the ratio of 17,20-lyase activity to 17α-hydroxylase activity.Thus, the regulation of 17,20-lyase activity,and consequently of DHEA production,depends on factors that facilitate the flow ofelectrons to P450c17: high concentrations ofPOR, the presence of cytochrome b5, and ser-ine phosphorylation of P450c17.14,15

P450c21: Steroid 21-Hydroxylase

Microsomal P450c21 catalyzes the 21-hydroxylation of progesterone to deoxycorticos-terone (DOC) and 17OHP to 11-deoxycortisol inthe biosynthesis of mineralocorticoids and glu-cocorticoids, respectively (Figure 13.1).2 Thenature of the 21 hydroxylation reaction has beenof great clinical interest because 21-hydroxylasedeficiency causes about 95% of all cases of con-genital adrenal hyperplasia (CAH), which hasan incidence of about 1:15,000 births. The clini-cal symptoms associated with this commongenetic disease are complex and potentiallydevastating.16 Decreased aldosterone and corti-sol synthesis can lead to sodium loss, potassiumretention, and hypotension, which can causecardiovascular collapse and death in infancy ifnot treated appropriately. Decreased synthesisof cortisol in utero leads to overproduction ofACTH and consequent overstimulation of fetaladrenal steroid synthesis; as the 21-hydroxylasestep is impaired, 17OHP accumulates becauseP450c17 converts only miniscule amounts of17OHP to androstenedione. However, 17OH-Preg also accumulates and is efficiently con-verted to DHEA, and subsequently to andro-stenedione and testosterone, resulting in severeprenatal virilization of female fetuses.16

Clinical variations in this disease, especiallythe identification of patients without apparentdefects in mineralocorticoid activity, initiallysuggested that there were two separate 21hydroxylases that were differentially expressedin the adrenal zona glomerulosa and fasciculata.However, gene cloning has shown that there is asingle adrenal 21-hydroxylase encoded by a sin-gle functional gene (CYP21A2) on chromosome6p21.2,17,18 As this gene lies in the middle of themajor histocompatibility locus, disorders ofadrenal 21-hydroxylation are closely linked tospecific human leukocyte antigens (HLA) types.

P450c21 is found in smooth endoplasmicreticulum, where it employs the same POR usedby P450c17 to receive electrons from NADPH.

++

––

–

– +++

NADPH

FAD FMN

PORb5

–PO4

Fe

NADP+P450

c

FIGURE 13.3 Electron transport to microsomal forms

of cytochrome P450. NADPH interacts with P450 oxidore-ductase (POR), bound to the endoplasmic reticulum, andgives up a pair of electrons (e2), which are received by theFAD moiety. Electron receipt induces a conformationalchange, permitting the isoalloxazine rings of the FAD andFMN moieties to come close together, so that the electronspass from the FAD to the FMN. Following another confor-mational change that returns the protein to its original ori-entation, the FMN domain of POR interacts with theredox-partner binding site of the P450. Electrons from theFMN domain of POR then reach the heme group to medi-ate catalysis. The interaction of POR and the P450 is coor-dinated by negatively charged acidic residues on thesurface of the FMN domain of POR, and positivelycharged basic residues in the concave redox-partner bind-ing site of the P450, similar to the interaction of Fedx withmitochondrial P450s. The active site containing the steroidlies on the side of heme ring (Fe) opposite from the redox-partner binding site.



21-hydroxylase activity has also been describedin a broad range of extra-adrenal tissues, butextra-adrenal 21-hydroxylation is not catalyzedby P450c21. Hepatic 21-hydroxylation is medi-ated by several enzymes, notably CYP2C19 andCYP3A4, which are principally involved indrug metabolism.19 These enzymes can 21-hydroxylate progesterone, but not 17OHP, andhence may contribute to the synthesis of mi-neralocorticoids but not glucocorticoids andpossibly account for the diminished mineralo-corticoid requirements in adult patients. Thus,some 21-hydroxylase activity may be regulatedby the pattern of drug and xenobiotic exposure.

Isozymes of P450c11: P450c11β andP450c11AS

The final steps in the synthesis of glucocorti-coids and mineralocorticoids are catalyzed bytwo closely related mitochondrial enzymes,P450c11β and P450c11AS.2,20 These two iso-zymes have 93% amino acid sequence identityand are encoded by tandemly duplicated genes(CYP11B1 and CYP11B2, respectively) on chro-mosome 8q21-22. Like P450scc, these twoenzymes are found on the IMM, and use ferre-doxin and ferredoxin reductase to receive elec-trons from NADPH to mediate catalysis. By farthe more abundant of the two isozymes isP450c11β, which is the classical 11β-hydroxylasethat converts 11-deoxycortisol to cortisol and11-deoxycorticosterone (DOC) to corticosterone.The less abundant isozyme, P450c11AS, is foundonly in the zona glomerulosa, where it has 11β-hydroxylase, 18-hydroxylase and 18-methyl oxi-dase (aldosterone synthase) activities; thusP450c11AS is able to catalyze all the reactionsneeded to convert DOC to aldosterone. Bothenzymes can convert DOC to corticosterone andcorticosterone to 18OH-corticosterone, but onlyP450c11AS can synthesize aldosterone from18OH-corticosterone. Patients with disorders

in P450c11β have classical 11β-hydroxylase defi-ciency but can still produce aldosterone, whilepatients with disorders in P450c11AS have rareforms of aldosterone deficiency (so-called corti-costerone methyl oxidase deficiency) whileretaining the ability to produce cortisol.2,20

Transcription of the CYP11B1 gene encodingP450c11β is induced by ACTH via cAMP andis suppressed by glucocorticoids, whereas tran-scription of the CYP11B2 gene encodingP450c11AS is induced by potassium and angio-tensin II via the protein kinase C pathway.21

Isozymes of 17β-HydroxysteroidDehydrogenase (17βHSD)

The 17β-hydroxysteroid dehydrogenases(17βHSD), sometimes also termed 17-ketosteroidreductases, principally convert androstenedioneto testosterone, DHEA to androstenediol, andestrone to estradiol. The terminologies for theseenzymes vary depending on the direction of thereaction being considered,1,22 and there is confu-sion in the literature about the 17βHSDs because:i) there are several different 17βHSDs; ii) someare preferential oxidases, while others are prefer-ential reductases; iii) they differ in their substratepreference and sites of expression; iv) there isinconsistent nomenclature (especially withrodent enzymes); and v) some proteins termed17βHSD actually have very little 17βHSD activity,and are principally involved in other reactions.

17βHSD1, encoded by HSD17B1 is a cyto-solic reductive SDR enzyme first isolated andcloned from the placenta, where it producesestriol; it is expressed in ovarian granulosacells where it produces estradiol.1,22 17βHSD1uses NADPH as its co-factor to catalyze reduc-tase activity. It acts as a dimer and onlyaccepts steroid substrates with an aromatic Aring; thus, its activity is confined to activatingestrogens. To date, no genetic deficiency syn-drome for 17βHSD1 has been described.



17βHSD2, encoded by HSD17B2, is a micro-somal oxidase that uses NAD1 to inactivateboth estradiol to estrone and testosterone toandrostenedione. 17βHSD2 is found in the pla-centa, liver, small intestine, prostate, secretoryendometrium, and ovary. In contrast to17βHSD1, which is found in placental syncy-tiotrophoblast cells, 17βHSD2 is expressed inendothelial cells of placental intravillous ves-sels, consistent with its apparent role in defen-ding the fetal circulation from transplacentalpassage of maternal estradiol or testoster-one.1,22 No genetic deficiency syndrome for17βHSD2 has been reported.

17βHSD3, encoded by HSD17B3, is a micro-somal enzyme that is expressed in the testis, andis the principal androgenic form of 17βHSD.17βHSD3 is the enzyme that is disordered in theclassic syndrome of male pseudohermaphrodit-ism that is often termed 17-ketosteroid reductasedeficiency.1,22

17βHSD4 was initially identified as anNAD1-dependent oxidase with activities simi-lar to 17βHSD2, and thus given 17βHSD status.However, this protein is located in peroxi-somes and is primarily an enoyl-CoA hydra-tase and 3-hydroxyacyl-CoA dehydrogenase.2

Deficiency of 17βHSD4 causes a form ofZellweger syndrome, in which bile acid bio-synthesis is affected but steroidogenesis is not.

17βHSD5, originally cloned as a 3αHSD, isan AKR enzyme (in contrast to 17βHSD types1�4, which are SDR enzymes) termedAKR1C3. 17βHSD5/AKR1C3 catalyzes thereduction of androstenedione to testosterone.2,23

Interestingly, 17βHSD5/AKR1C3 is more highlyexpressed in the human fetal adrenal glandduring the time of sexual differentiation than17βHSD3; thus, it may participate in adrenaltestosterone production, particularly in viriliz-ing CAHs.24 The postnatal adrenal zona reticu-laris also expresses 17βHSD5/AKR1C3 at lowlevels, accounting for the small amount of tes-tosterone produced by the adrenal gland.25

P450aro: Aromatase

Estrogens are produced by the aromatiza-tion of androgens, including those producedby the adrenal gland, by a complex series ofreactions catalyzed by a single microsomal aro-matase, P450aro.26,27 This microsomal P450 isencoded by the CYP19A1 gene on chromosome15q21.1. This gene uses several different pro-moter sequences, transcriptional start sites,and alternatively chosen first exons to encodearomatase mRNA in different tissues underdifferent hormonal regulation. Aromataseexpression in “extraglandular” tissues, espe-cially adipose tissue, can convert adrenalandrogens to estrogens, and aromatase expres-sion in the epiphyses of growing bone convertstestosterone to estradiol. Males with aromatasedeficiency have tall stature, delayed epiphysealmaturation, and osteopenia, which are rapidlyreversed by estrogen replacement, whichshows that estrogen, not testosterone, is pri-marily responsible for epiphyseal maturation.Although estrogens were once considered nec-essary for embryonic and fetal development,fetuses with genetic lesions in CYP19A1, whocannot produce estrogens, and fetuses withgenetic lesions in CYP11A1, who cannot pro-duce any steroids, have normal fetal develop-ment and normal parturition showing thatfeto�placental estrogen is not essential.2,27

Isozymes of 5α-Reductase

Testosterone is converted to the more potentandrogen dihydrotestosterone (DHT) by twoforms of 5α-reductase. The type-1 enzyme,encoded by the SRD5A1 gene on chromosome5p15, is found in the scalp and other periph-eral tissues; the type-2 enzyme, encoded by thestructurally related SRD5A2 gene on chromo-some 2p23, is the predominant form found inmale reproductive tissues.28 Mutations in theSRD5A2 gene cause 5α-reductase deficiency, a



disorder of male sexual differentiation that hasbroad phenotypic variation, depending on thecausative mutation(s); mutations in SRD5A1have not been described. The 5α-reductasegenes exhibit distinct patterns of developmen-tal regulation. The type-1 gene is not widelyexpressed in the fetus, but is expressed in thefetal testis, where it participates in the alterna-tive “backdoor” pathway of androgen synthe-sis;29 it is then briefly expressed in newbornskin, and then remains unexpressed againuntil after puberty. The type-2 gene isexpressed in fetal genital skin, where it partici-pates in normal male sexual differentiation,and is also expressed in adult prostate andprostatic adenocarcinoma cells. Thus, the type-1 enzyme may be responsible for the pubertalvirilization seen in patients with classic 5α-reductase deficiency, and the type-2 enzymemay be involved in male pattern baldness.30

Isozymes of 11β-HydroxysteroidDehydrogenase (11βHSD)

Although certain steroids are typically catego-rized as glucocorticoids or mineralocorticoids, the“mineralocorticoid” (glucocorticoid type 2) recep-tor has equal affinity for both aldosterone andcortisol. However, because mineralocorticoid-responsive tissues (such as the kidney) convertcortisol to cortisone, which does not bind toreceptors, cortisol does not act as a mineralocorti-coid in vivo, even though its concentrations canexceed those of aldosterone by 100- to 1000-fold.The interconversion of cortisol and cortisone ismediated by two isozymes of 11β-hydroxysteroiddehydrogenase (11βHSD). Both isozymes can cat-alyze both oxidase and reductase activities,depending on the co-factor available (NADP1 orNADPH, respectively)31; the ratio of NADP1 toNADPH is regulated by hexose-6-phosphatedehydrogenase (H6PDH).32

The type-1 enzyme (11βHSD1), encoded bythe HSD11B1 gene, is expressed mainly inglucocorticoid-responsive tissues such as theliver, testis, lung, and proximal convolutedtubule. 11βHSD1 is located on the luminal sideof the endoplasmic reticulum, and is not incontact with the cytoplasm; in this unusual cel-lular location, 11βHSD1 receives NADPH pro-vided by H6PDH and links the enzyme to thepentose monophosphate shunt, providing adirect paracrine connection between local glu-cocorticoid production and energy storage asfat.33�35 11βHSD1 can catalyze both the oxida-tion of cortisol to cortisone using NADP1 asits co-factor (Km 1.6 μM), or the reduction ofcortisone to cortisol using NADPH as its co-factor (Km 0.14 μM); the direction of the reac-tion catalyzed depends on which co-factor isavailable, but 11βHSD1 can only function withhigh (micromolar) concentrations of steroid.By contrast, 11βHSD2, encoded by theHSD11B2 gene, only catalyzes the oxidation ofcortisol to cortisone using NADH, and canfunction with low (nanomolar) concentrationsof steroid (Km 10�100 nM). 11βHSD2 isexpressed in mineralocorticoid-responsive tis-sues and thus serves to “defend” the mineralo-corticoid receptor by inactivating cortisol tocortisone, so that only “true” mineralocorti-coids, such as aldosterone or DOC, can exert amineralocorticoid effect. Thus, 11βHSD2 pre-vents cortisol from overwhelming renal mineral-ocorticoid receptors and inactivates cortisol inthe placenta and other fetal tissues. Importantly,the placenta also has abundant NADP1 favoringthe oxidative action of 11βHSD1, so that in theplacenta both 11βHSD1 and 11βHSD2 protectthe fetus from high maternal concentrations ofcortisol (but not from maternally administeredbetamethasone or dexamethasone, which areoften used to enhance fetal lung developmentbecause they are ineffective substrates for11βHSD2).



Isozymes of 3α-HydroxysteroidDehydrogenase (3αHSD)

The four major human 3α-hydroxysteroiddehydrogenases (3αHSDs) are AKR enzymesof the AKR1C family; 3αHSD types 1, 2, 3, and4 are also termed AKR1C4, 1C3, 1C2, and 1C1,respectively.22 These enzymes are structurallyvery similar, are encoded by a gene cluster onchromosome 10p14�15, and catalyze a widearray of steroidal conversions and other reac-tions.2 The 3αHSDs are essential constituentsof the so-called “backdoor pathway” of ste-roidogenesis.36 This remarkable pathway (dis-cussed below), first discovered in studies ofsteroidogenesis in the marsupial fetal tes-tis,36,37 plays a central role in human male sex-ual differentiation.29 In this pathway, 17OHPis converted to DHT without going throughDHEA, androstenedione, or testosterone, andhence provides a mechanism by which 17OHPcan contribute to the virilization of femalefetuses with 21-hydroxylase deficiency.16,38

The “backdoor pathway” is characterized byboth reductive and oxidative 3αHSD activities;the reductive activity can be catalyzed byeither AKR1C2 (3αHSD3) or AKR1C4(3αHSD1),29 but the nature of the oxidativeenzyme(s) is uncertain.2 AKR1C3 (3αHSD2),which converts androstenedione to testoster-one in the adrenals, is also known as17βHSD5.

Steroid Sulfotransferase and Sulfatase

Steroid sulfates may be synthesized directlyfrom cholesterol sulfate or formed via the sulfa-tion of existing steroids by cytosolic sulfotrans-ferase (SULT) enzymes.39,40 At least 44 distinctisoforms of these enzymes have been identifiedbelonging to five families of SULT genes. Manyof these genes yield alternately spliced productsaccounting for the large number of enzymes,

and the obligatory sulfate donor for all theSULT enzymes is 30-phosphoadenosine-50-phos-phosulfate (PAPS). The SULT enzymes that sul-fonate steroids include SULT1E (which sulfatesestrogens), SULT2A1 (which sulfates non-aromatic steroids), and SULT2B1 (which sul-fates sterols). SULT2A1 is the principal SULTenzyme expressed in the adrenal gland, whereit sulfates the 3β-hydroxyl group of Δ5 steroids(pregnenolone, 17OH-pregnenolone, DHEA,and androsterone) but not of cholesterol.SULT2B1a will also sulfonate pregnenolone butnot cholesterol; alternatively, cholesterol is theprincipal substrate for SULT2B1b in the skin,liver, and elsewhere. Whether steroid sulfatesare simply inactivated forms of steroid orwhether they serve specific hormonal roles isunclear.

Knockout of the mouse SULT1E1 genecauses elevated estrogen levels, increasedexpression of tissue factor in the placenta, andincreased platelet activation, leading to placen-tal thrombi and fetal loss. Mutations ablatinghuman SULT enzymes have not beendescribed, but some single nucleotide poly-morphisms that alter the amino-acid sequencesand catalytic activity affect drug activity.African-Americans have a high rate ofSULT2A1 polymorphisms, which influencesplasma ratios of DHEA:DHEAS and may cor-relate with risk of prostatic and other cancers.

Steroid sulfates may also be hydrolyzed tothe native steroid by steroid sulfatase; dele-tions in the steroid sulfatase gene on chromo-some Xp22.3 cause X-linked ichthyosis, due tothe accumulation of steroid sulfates in the stra-tum corneum of the skin. The fact that maleshave a single copy of this gene also probablyaccounts for the higher DHEAS levels in malesthan females of the same age. In the fetal adre-nals and placenta, diminished or absent sulfa-tase deficiency reduces the pool of free DHEAavailable for placental conversion to estrogen,



resulting in low estriol concentrations in thematernal blood and urine.

TISSUE-SPECIFIC PATHWAYS OFSTEROIDOGENESIS

Adrenal Pathways

Diagrams of steroidogenic pathways, suchas the one shown in Figure 13.1 typically com-bine the pathways from multiple cell types toprovide an overview of all steroidogenic pro-cesses. However, such diagrams are mislead-ing, as the pathways differ in each cell type.The three major pathways of steroidogenesisin the human adrenal glands are shown inFigure 13.4. The adrenal zona glomerulosa (ZG)is characterized by three distinct features: i) itexpresses angiotensin II receptors; ii) itexpresses P450c11AS; and iii) it fails to expressP450c17. As a result, the ZG produces aldoste-rone under regulation by the renin/angiotensinsystem. By contrast, the adrenal zona fasciculata(ZF) does not express angiotensin II receptors orP450c11AS, but instead expresses MC2R (theACTH receptor) and P450c11β, which cannotconvert 18-hydroxycorticosterone to aldosteroneand has minimal capacity to convert corticoste-rone to 18-hydroxycorticosterone. Both the ZGand ZF express P450c21, but the ZF alsoexpresses P450c17, permitting cortisol synthesis.The ZF, however, expresses little (if any) cyto-chrome b5;

41 as a result, P450c17 in the ZFcatalyzes 17α-hydroxylation but very little 17,20-lyase activity. The ZF thus produces cortisol andcorticosterone under the influence of ACTH, butvery little DHEA. Patients with severe mutationsin P450c17 cannot synthesize cortisol but insteadincrease corticosterone production (as do rodentadrenals, which lack P450c17), which explainswhy they are not glucocorticoid-deficientdespite their lack of cortisol production(Figure 13.1). The adrenal zona reticularis (ZR)also expresses MC2R but very little P450c21 or

P450c11β, so that the ZR produces very little cor-tisol. By contrast, the ZR expresses largeamounts of P450c17 and cytochrome b5, maxi-mizing 17,20-lyase activity, so that DHEA is pro-duced, much of which is sulfated by SULT2A1to DHEAS. Furthermore, the ZR expresses rela-tively little 3βHSD2, and the Km of 3βHSD2 isB5 μM for pregnenolone and 17OH-Preg,whereas the Km for both the 17α-hydroxylaseand 17,20-lyase activities of P450c17 is B1 μM,favoring the production of DHEA.2 As DHEAaccumulates, small amounts are converted toandrostenedione; very small amounts of thisandrostenedione are then converted to testoster-one by AKR1C3/17βHSD5. The pattern of ste-roid products secreted by each adrenal zone istherefore determined by the enzymes producedin that zone and may be logically deduced froman understanding of their specific enzymaticproperties.2

Gonadal Pathways

The synthesis of testosterone in the testis fol-lows a pathway that is similar to C19-steroidproduction in the adrenal ZR, with thenotable exceptions that the stimulus for ste-roidogenesis is transduced by the LH receptorrather than MC2R and that Leydig cells expressabundant 3βHSD2 and 17βHSD3 but noSULT2A1. Thus, the DHEA produced in the tes-tis is not sulfated but is rather readily convertedto androstenedione and then testosterone(Figure 13.5A). As in the adrenal, the principalpathway to C19-steroids is via Δ5 steroids toDHEA; the Δ4 pathway from 17OHP to andro-stenedione makes only a minimal contribution.

Steroidogenesis in the ovary is partitionedbetween the granulosa and theca cells, whichsurround the oocyte and form a follicle. The pat-terns of steroidogenesis also vary during themenstrual cycle: Estradiol is the principal prod-uct in the follicular phase, whereas progesteroneis produced in the luteal phase (Figure 13.5B).



(A)

(C)

Zona GlomerulosaCholesterol

StAR

3βHSD2

P450sccFdx/FdR

Pregnenolone Progesterone 11-Deoxycorticosterone

P450c11ASFdx/FdR

P450c21POR

P450c11ASFdx/FdR

P450c11ASFdx/FdR

Corticosterone

Aldosterone

18-Hydroxycorticosterone

Zona Reticularis

Cholesterol

StAR P450sccFdx/FdR

P450c17POR

P450c17POR+b5

P450c17-PO4POR

Pregnenolone

17OH-Preg

DHEA

SULT2A1

3βHSD2 17βHSD5Androstenedione

DHEAS

PAPSS2

Testosterone

Cholesterol

stAR P450sccFdx/FdR P450c21

PORP450c11βFdx/FdR

P450c11βFdx/FdR

P450c21POR

P450c17POR

P450c17POR

11-Deoxycortisol

ProgesteronePregnenolone

Cortisol

Corticosterone

17OHP17OHP-Preg3βHSD2

3βHSD2 11-Deoxycorticosterone

Zona Fasciculata

(B)

FIGURE 13.4 Major steroidogenic pathways in the human adrenal cortex. The conversion of cholesterol to pregneno-lone by P450scc is common to all 3 zones of the adrenal cortex. (A) In the zona glomerulosa (ZG), 3βHSD2 converts preg-nenolone to progesterone. P450c17 is absent, but P450c21 produces 11-deoxycorticosterone, which is a substrate forP450c11AS. P450c11AS catalyzes 11-hydroxylation and two 18-oxygenations, which completes aldosterone synthesis. (B)The zona fasciculata (ZF) expresses P450c17, so pregnenolone is hydroxylated to 17α-hydroxypregnenolone, (or progester-one to 17-hydroxyprogesterone); but, the ZF contains little cytochrome b5, minimizing the 17,20-lyase activity of P450c17,


219TISSUE-SPECIFIC PATHWAYS OF STEROIDOGENESIS

The key point in ovarian steroidogenesis is thatgranulosa cells do not express P450c17. Thus, ingeneral, steroidogenesis is initiated in granulosacells under the influence of LH, which, viacAMP, stimulates the expression of P450scc.Pregnenolone and progesterone from granulosacells then diffuse into the adjacent theca cells,where they are converted to androstenedione byP450c17 and 3βHSD2. Small amounts of thisandrostenedione are secreted or converted to

testosterone (probably by AKR1C3/17βHSD5),but most of the androstenedione returns to thegranulosa cells where it is converted to estroneand then to estradiol by P450aro and 17βHSD1,under the influence of follicle-stimulatinghormone (FSH). Thus, as with the three zones ofthe adrenal, the patterns of gonadal steroid-ogenesis in the ovary are dictated by the cell-specific expression of specific steroidogenicenzymes.2

�

and little DHEA is produced. Instead, 3βHSD2 and P450c17 generate 17-hydroxyprogesterone, the preferred substrate forP450c21, yielding 11-deoxycortisol. P450c11β, which is unique to the ZF, completes the synthesis of cortisol.Corticosterone is normally a minor product (dashed arrows) derived from a parallel pathway without the action ofP450c17. (C) The zona reticularis (ZR) has large amounts of P450c17 and cytochrome b5 but little 3βHSD2, so that pregnen-olone is sequentially oxidized to 17-hydroxypregnenolone and then DHEA. SULT2A1, using PAPS synthesized byPAPSS2 (see text), sulfates DHEA, and DHEAS is exported to the circulation. Testosterone synthesis is a very minor path-way (dashed arrows).

Cholesterol(A) (B)

Leydig Cell

StARP450sccFdx/FdR

P450c17POR

P450c17POR+b5

P450aroPOR

P450aroPOR

P450c17-PO4POR

Pregnenolone

17OH-Preg

DHEA 3βHSD2

3βHSD2

17βHSD3 17βHSD3

Androstenedione Estrone

EstradiolTestosteroneAndrostenediol

Cholesterol Theca and Granulosa Cells

StAR P450sccFdx/FdR

P450c17POR

P450c17POR+b5 P450aro

POR

P450aroPOR

P450c17-PO4POR

Pregnenolone

17OH-Preg

Progesterone

Corpus Luteum

DHEA3βHSD2

3βHSD2

3βHSD2

17βHSD1 17βHSD5 17βHSD1

Androstenedione Estrone

EstradiolTestosteroneAndrostenediol

FIGURE 13.5 Major pathways of human gonadal steroidogenesis. (A) In testicular Leydig cells, cholesterol is con-verted to DHEA by the same enzymes using the same co-factors as in the adrenal zona reticularis. Leydig cells containabundant 17βHSD3, so that Leydig cells efficiently produce testosterone, via androstenedione and/or androstenediol. (B)Ovarian granulosa cells contain P450scc and convert cholesterol to pregnenolone. The ovarian theca cells express lowlevels of P450scc but high amounts of P450c17 and hence acquire C21 steroids from the granulosa cells and produce C19

precursors of sex steroids (the two-cell model of ovarian steroidogenesis). Theca cells do not express aromatase (P450aro);thus, androstenedione must return to the granulosa cells, which contain abundant aromatase and 17βHSD1, completingthe synthesis of estradiol. In the luteal phase, 3βHSD2 in the corpus luteum metabolizes nascent pregnenolone to proges-terone, the final product. Minor pathways are shown with dashed arrows.



The “Backdoor Pathway” to DHT

The alternative or “backdoor” pathwayleads from 17OHP to DHT without goingthrough androstenedione or testosterone asintermediate steroids. This pathway is initiatedby the 5α-reduction of either progesterone or17OHP by type-1 5α-reductase. The resulting5α-reduced C21 steroids, dihydroprogesterone(5α-pregnane-3,20-dione) and 5α-pregnane-17α-ol-3,20-dione, are then readily catalyzedby reductive 3αHSDs to yield allopregnano-lone (5α-pregnan-3α-ol-20-one; Allo) and17α-hydroxylated Allo (5α-pregnane-3α,17α-diol-20-one; 17OH-Allo). Dihydroprogesteroneand Allo are excellent substrates for the 17α-hydroxylase activity of P450c17, and 17OH-Allo is the most efficient substrate known forthe 17,20-lyase activity of human P450c17.Furthermore, unlike the conversion of 17OH-Preg to DHEA, the cleavage of 17OH-Allo toandrosterone is minimally dependent on cyto-chrome b5. The resulting androsterone maythen be 3α-oxidized to DHT by retinol dehy-drogenase (RoDH), the microsomal 3αHSD, 3(α-β)-hydroxysteroid epimerase (also knownas 17βHSD6) (Figure 13.6). The presence of5α-reductases in steroidogenic cells does notpreclude the production of C19 steroids, butrather paradoxically enhances the productionof DHT by directing steroid flux to 5α-reduced precursors of DHT. The “backdoorpathway” thus enables production of C19 ster-oids from 17OHP, despite the poor 17,20-lyaseactivity of human P450c17 for 17OHP, byusing 17OH-Allo as the substrate for the17,20-lyase reaction. The “backdoor pathway”is also relevant to normal and abnormalhuman steroidogenesis. In normal male sex-ual development, both the conventional path-way of androgen production (via DHEA,androstenedione, and testosterone to DHT)and the fetal testicular “backdoor pathway”are required for development of normal maleexternal genitalia.29 Similarly, when 17OHP

accumulates in 21-hydroxylase deficiency andPOR deficiency, the “backdoor pathway” isresponsible for a portion of the overproduc-tion of androgens.38,42

CHRONIC REGULATION OFSTEROIDOGENESIS

Whereas the acute regulation of steroido-genesis is determined by the action of StAR,P450scc is the enzymatic rate-limiting step insteroidogenesis. Thus, the chronic regulationof steroidogenesis is quantitatively (i.e., howmuch steroid is produced) determined byP450scc gene expression and qualitatively (i.e.,which steroids are produced) determined bythe expression of downstream enzymes, espe-cially P450c17. Patients with inactivating muta-tions in the ACTH receptor (MC2R)43 or theLH/hCG receptor44 make negligible steroidsfrom the adrenals or gonads. Conversely, acti-vating mutations of the Gαs protein, whichcouples G protein-coupled receptor (GPCR)binding to cAMP generation, and activatingmutations of the LH receptor cause hyperse-cretion of steroids,45 and cAMP-responsive ele-ments have been identified in the genes formost of the human steroidogenic P450enzymes. However, the regulation of cAMPgeneration alone does not explain the diversityof steroid production observed in the variouszones of the adrenal cortex and the gonads ofboth sexes. Other transcription factors, includ-ing AP2, SP1, SP3, NF1C, NR4A1, NR4A2,GATA4, and GATA6, also participate in regu-lating the basal- and cAMP-stimulated tran-scription of each gene. SF1 coordinates theexpression of steroidogenic enzymes in theadrenals and gonads. By contrast, steroidogen-esis in the brain and placenta is independentof SF1. Disruption of the mouse SF1 gene dis-rupts steroid biosynthesis and blocks thedevelopment of the adrenals, gonads, and


221CHRONIC REGULATION OF STEROIDOGENESIS

ventromedial hypothalamus. The action of SF1is further modified by other transcription fac-tors (e.g., WT1 and DAX1) and by phosphory-lation or sumoylation. Thus, the developmentof steroidogenic organs is intimately related tothe capacity to produce steroids, and multiplefactors acting on the genes for steroidogenicenzymes yield both common features anddiversity among the steroidogenic tissues.2

ACUTE REGULATION OFSTEROIDOGENESIS

Unlike cells that produce polypeptide hor-mones, which store large amounts of hormone

in secretory vesicles ready for rapid release, ste-roidogenic cells store very little steroid. Thus, arapid steroidogenic response (e.g., secretion ofaldosterone and cortisol in response to stress orthe “pulsing” of sex steroids in response to anLH surge) requires rapid synthesis of new ste-roid. ACTH promotes steroidogenic cell growthand maintains the steroidogenic machinery atthree distinct levels (LH probably acts similarlyon gonadal steroidogenic cells, but has not beenstudied as thoroughly). First, acting over weeksor months, ACTH promotes adrenal growth,primarily by ACTH-stimulated synthesis ofIGF-2, basic fibroblast growth factor, and EGF.These growth factors subsequently stimulateadrenal cellular hypertrophy and hyperplasia

P450sccFdx/FdR

P450c17POR

P450c17 POR+b5

P450c17 POR

170H-Preg

P450c17- PO4POR

3βHSD2

3βHSD2

Pregnenolone

StAR

Cholesterol

Classic vs. Backdoor Pathway

Androstenediol

DHEA Androstenedione

170HP5αRed 1

5αRed 2Androstanediol

170H-Allopregnanolone

Androsterone

AKRIC25α-Pregnan-17α-ol-3,20-dione

17βHSD3

Testosterone

17βHSD6 (RoDH)

DHT3βHSD2

17βHSD5 (adrenal)17βHSD3 (testis)

17βHSD5 (adrenal)17βHSD3 (testis)

FIGURE 13.6 Synthesis of dihydrotestosterone via the classic and alternative (“backdoor”) pathways. The classicpathway of steroidogenesis leading to dihydrotestosterone (DHT) is shown on the left; the alternative (“backdoor”) path-way is shown on the right. The factors in the classic pathway are CYP11A1 (cholesterol side-chain cleavage enzyme,P450scc), StAR (steroidogenic acute regulatory protein), CYP17A1 (17α-hydroxylase/17,20-lyase, P450c17), HSD3B2 (3β-hydroxysteroid dehydrogenase, type 2), HSD17B3 (17β-HSD3 (17β-hydroxysteroid dehydrogenase, type 3)), and 5α-reduc-tase, type 2 (5α-reductase 2, encoded by SRD5A2). The alternative (“backdoor”) pathway is characterized by the presenceof additional enzymes: 5α-reductase, type 1 (5α-reductase 1, encoded by SRD5A1), AKR1C2 3 (3α-reductase, type 3) andpossibly AKR1C4 (3α-reductase, type 1), and RoDH (3-hydroxyepimerase, encoded by HSD17B6). Most steroids are iden-tified by their common names; 17-hydroxy-dihydroprogesterone (17OH-DHP) is 5α-pregnane-17α-ol-3,20-dione;17-hydroxy-allopregnanolone (17OH-allo) is 5α-pregnan-3α,17α-diol-20-one; 5α-dihydroprogesterone (5α-DHP) is 5α-preg-nane-3,20-dione, and allopregnanolone is 3α-hydroxy-dihydroprogesterone (3α-OH-DHP) or 5α-pregnane-3α-ol-20-one.



and thus regulate the amount of steroidogenictissue. Second, acting over days, ACTH(through the cAMP pathway) and angiotensinII (through the calcium/calmodulin pathway)promote the transcription of genes encodingvarious steroidogenic enzymes and electron-donating co-factor proteins, thus determiningthe amount of steroidogenic machinery in thecell. Third, ACTH rapidly stimulates StAR genetranscription and the post-translational phos-phorylation of Ser195 in existing StAR toincrease the flow of cholesterol from the OMMto the IMM where it becomes substrate forP450scc.5,7

Some adrenal steroidogenesis occurs indepen-dent of StAR. When non-steroidogenic cells aretransfected with StAR and the P450scc system,they convert cholesterol to pregnenolone atB14% of the StAR-induced rate. Furthermore,the placenta utilizes mitochondrial P450scc toinitiate steroidogenesis but does not expressStAR. The specific mechanism(s) of StAR-independent steroidogenesis is unclear; it mayoccur without a triggering protein, or some otherprotein may exert StAR-like activity to promotecholesterol flux, but without StAR’s rapid kinet-ics; for example a protein called N-218MLN64 isfound in mitochondria and has StAR-like activ-ity.5 The exact mechanism of StAR’s action isalso unclear, but it is well-established that StARacts on the OMM, does not need to enter themitochondria to be active, and undergoes con-formational changes on the OMM that arerequired for its activity.5�7 Mechanistically,StAR functions as a component of a molecularmachine termed a “transduceosome” on theOMM that consists of StAR, TSPO (the transloca-tor protein formerly known as the peripheralbenzodiazepine receptor), TSPO-associated pro-tein 7 (PAP7; ACBD3 for acyl-CoA-binding-domain 3), the voltage-dependent anion channel(VDAC-1), and protein kinase A regulatory sub-unit 1α (PKAR1A).46 The mechanism by whichthese proteins interact and move cholesterol

from the outer mitochondrial membrane toP450scc, and the means by which cholesterol isloaded into the OMM, remains unclear.

POST-TRANSLATIONALDIFFERENTIAL REGULATION OF

STEROIDOGENIC ENZYMES

Contemporary work is focused on identify-ing post-translational mechanisms regulatingsteroidogenesis. The phosphorylation of StAR,phosphorylation and sumoylation of SF1, andglycosylation of P450aro are well-describedexamples. The increase in 17,20-lyase activityof P450c17, but not its 17-hydroxylase activityby phosphorylation of P450c17 is an especiallyinteresting example.

Adjusted for body surface area, the humansecretion of cortisol remains fairly constantfrom infancy to old age. However, the secre-tion of C19 adrenal steroids is low in childhoodbegins to rise just before puberty, reaches max-imum levels in young adulthood (well afterthe completion of puberty), and then fallsslowly to childhood levels in the elderly.47 Theperipuberal rise in DHEA, DHEAS, and andro-stenedione48,49 is referred to as “adrenarche.”Although these C19 steroids are commonlyreferred to as “adrenal androgens,” they areandrogen precursors rather than true andro-gens because they do not activate the androgenreceptor. Primates are unique among mammalsin having high concentrations of adrenal C19

steroids; the pattern of human age-related riseand fall in C19 steroids is also unique.49

Adrenarche is of interest for many reasons:i) evolutionarily, because it appears to be arecent innovation; ii) physiologically, becausethe function, if any, of adrenal C19 steroids iscontroversial; iii) endocrinologically, becauseits regulatory mechanisms are unknown; and iv)biochemically, because it represents an unusual


223POST-TRANSLATIONAL DIFFERENTIAL REGULATION OF STEROIDOGENIC ENZYMES

example of post-translational differential regula-tion of P450c17’s two enzymatic activities.

The traditional questions concerning adre-narche have concerned its regulation, intracel-lular mechanisms, and role in humanphysiology. Investigators have long searchedfor a hypothetical, specific Adrenal AndrogenStimulating Hormone that would regulate C19

steroid synthesis by the adrenal ZR, analogousto angiotensin II acting on the ZG or ACTHacting on the ZF; however, no such factor hasbeen identified. Adrenarche that begins earlierand that results in higher concentrations of C19

steroids also frequently precedes the develop-ment of polycystic ovary syndrome (PCOS),which is characterized by adrenal and ovarianhyperandrogenism, insulin resistance, and obe-sity, although many affected women have onlysome of these features;50 many now regardpremature exaggerated adrenarche as an earlyform of PCOS.51�53 Because of the many con-nections between adrenal C19 steroid produc-tion and metabolic regulation, factors notspecific to the adrenal glands, including nutri-tion, insulin, insulin-like factors, leptin, andFGF have also been considered as potentialtriggers of adrenarche.54

Because Ser/Thr phosphorylation of P450c17augments its 17,20-lyase activity, and becauseSer phosphorylation of the beta-chain of theinsulin receptor causes insulin resistance, it hasbeen proposed that the hyperandrogenism andinsulin resistance in PCOS may be connectedby a signal transduction pathway that ulti-mately increases the Ser/Thr phosphorylationof both P450c17 and either the insulin receptoror its substrate.55,56 Rho-associated, coiled coil-containing protein kinase 1 (ROCK1) can pro-mote the 17,20-lyase activity of P450c17, buthow ROCK1 promotes this activity is unclear.Recent work shows that P450c17 can be phos-phorylated by p38α in a fashion that selectivelyaugments its 17,20-lyase activity, thus providinga proof-of-principle for the regulation of 17,20-lyase activity by P450c17 phosphorylation.57

p38α is a mitogen-activated kinase (MAPK14)that is the terminal component of a typicalthree-component MAP kinase cascade; thephosphorylation of P450c17 by p38α can bereversed by protein phosphatase 2A, which inturn can be regulated by phosphoprotein SET,permitting the modeling of the relevant cellularpathways (Figure 13.7). Hormones (e.g., insulin,IGFs, leptin), environmental agents, and dietaryfactors could potentially activate a pathway thatmay involve ROCK1, which might act as anupstream scaffolding protein in a MAPK path-way.57 The identification of this pathway isincomplete, but promises to help define themechanisms regulating adrenal C19 steroid syn-thesis, and may also reveal links to PCOS.

CONCLUSION

Steroidogenesis involves the conversion ofcholesterol to glucocorticoids, mineralocorti-coids, and sex steroids, and is regulated at mul-tiple levels, principally by transcription of genesencoding steroidogenic enzymes and co-factors,and by their post-translational modification, ina tissue-specific fashion. Most steroidogenicenzymes are either HSD or cytochrome P450enzymes, the activities of which are modulatedby post-translational modifications and co-fac-tors, especially electron-donating redox part-ners. The first, rate-limiting step insteroidogenesis is catalyzed by P450scc in allsteroidogenic tissues, determining steroidogeniccapacity; the qualitative regulation of steroido-genesis, determining the class of steroid pro-duced, is determined by the expression ofdownstream enzymes, principally P450c17.Steroidogenesis regulates development andphysiology, and its understanding is necessaryto fully understand disorders of sexual differen-tiation, reproduction, fertility, hypertension,obesity, and physiologic homeostasis. Moreover,an understanding of steroidogenesis is essentialfor rational steroid therapies.



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HormonesEnvironmental

factors

ROCK1 (?)

M3K Kinase

SET

??

PP2A

17α-hydroxylase 17α-hydroxylaseand

17,20-lyase

MKK (3, 4, or 6?)

p38α

P450c17 p450C17-PO4

FIGURE 13.7 A hypothetical model of signaling leading to

P450c17 phosphorylation. We propose that hormones and otherfactors activate an intracellular pathway that may includeROCK1, eventually leading to a MAP kinase kinase kinases(M3K); the number of steps involved and potential sites of cross-talk remain undetermined. An activated M3K would activate aMAP kinase kinase, such as MKK3, 4, or 6, which would thenactivate p38α, permitting phosphorylation of P450c17 and theacquisition of 17,20-lyase activity. P450c17 is dephosphorylatedby protein phosphatase 2A (PP2A), which in turn can be inhib-ited by phosphoprotein SET. Potential cross-talk with other sec-ond messenger signaling pathways is not shown. Steps that havebeen established experimentally are shown with closed arrows;hypothetical steps are shown with open arrows; inhibitory stepsare shown with dashed lines.


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Documents

Chapter 13. Regulation of Steroidogenesis€¦ · the so-called “backdoor pathway” of fetal andro-gen synthesis (see below). Based on their activi-ties, it is physiologically