Steroidogenic Acute Regulatory Protein: The Principal Regulator of Gonadal and Adrenal Steroidogenesis

Translocation of cholesterol from the outer mitochondrial membranes to the relatively sterol-poor inner membranes is the critical step in steroidogenesis. This translocation process occurs at modest rates in the absence of specific effectors. It is markedly enhanced by STARD1, a protein with a short biological half-life. The following evidence established STARD1 as the key mediator of substrate flux to the cholesterol side-chain cleavage system:

• 1.
  

  Expression of STARD1 is directly correlated with steroidogenesis.

  
  
• 2.
  

  Coexpression of STARD1 and the cholesterol side-chain cleavage enzyme system in cells that are not normally steroidogenic results in substantial pregnenolone synthesis above that produced by cells expressing the cholesterol side-chain cleavage enzyme system alone.

  
  
• 3.
  

  Mutations that inactivate STARD1 cause congenital lipoid adrenal hyperplasia, a rare autosomal recessive disorder in which the synthesis of all adrenal and gonadal steroid hormones is severely impaired before the cholesterol side-chain cleavage step.

  
  
• 4.
  

  Targeted deletion of the murine Stard1 gene results in a phenotype in nullizygous mice that mimics human congenital lipoid adrenal hyperplasia.

Human STARD1 is synthesized as a 285 amino acid protein. The N-terminus of STARD1 is characteristic of proteins synthesized in the cytoplasm and then imported into mitochondria, with the first 26 amino acid residues predicted to form an amphipathic helix. Newly synthesized STARD1 preprotein (37 kDa) is rapidly imported into mitochondria and processed to the “mature” 30-kDa form. The preprotein has a very short half-life (minutes), but the mature form is longer-lived (hours). The START domain consists of a unique structure that accommodates a cholesterol molecule. However, the binding of one sterol molecule is not sufficient to explain STARD1 action, and it is evident that STARD1 must be a catalyst for the transfer of multiple cholesterol molecules.

STARD1 contains two consensus sequences for cAMP-dependent protein kinase phosphorylation at Ser57 and Ser195. Ser195 of human STARD1 must be phosphorylated for maximal steroidogenic activity in model systems.

Tissues that express STARD1 at high levels perform trophic hormone–regulated mitochondrial sterol hydroxylations through the intermediacy of cAMP. STARD1 messenger RNA (mRNA) and protein are not present in the human placenta, an observation that is consistent with the fact that pregnancies hosting a fetus affected with congenital lipoid adrenal hyperplasia go to term. Although estrogen production is impaired in these pregnancies because of diminished fetal adrenal androgen production, placental progesterone synthesis is not significantly affected, indicating that the trophoblast cholesterol side-chain cleavage reaction is independent of STARD1.

The abundance of STARD1 protein in steroidogenic cells is determined primarily by the rate of STARD1 gene transcription, which is influenced by transcription factors that control other genes encoding proteins involved in cholesterol metabolism (e.g., SREBPs, NR5A1, NR5A2, LXRs). STARD1 mRNA stability and translational mechanisms (in some species determined by the 3′-untranslated region of the transcripts) may also contribute. In differentiated cells, the STARD1 gene is activated by the cAMP signal transduction cascade within 15 to 30 minutes. In differentiating cells (e.g., luteinizing granulosa cells), the induction of STARD1 transcription takes hours and requires ongoing protein synthesis.

STARD1 was initially thought to stimulate cholesterol movement from the outer to the inner mitochondrial membrane as it was imported into the mitochondria. However, STARD1 protein lacking the mitochondrial targeting sequence is as effective as native STARD1 in stimulating steroidogenesis. Other STARD1 constructs engineered for prolonged tethering to the surface of the mitochondria were very active in stimulating pregnenolone production, suggesting that the residency time of the protein on the mitochondrial surface determines the duration of the steroidogenic stimulus. Recombinant human STARD1 lacking the mitochondrial targeting sequence enhanced pregnenolone production by isolated ovarian mitochondria in a dose- and time-dependent fashion, with significant increases in steroid production observed within minutes. Collectively, these findings strongly suggest that STARD1 acts on the outer mitochondrial membrane to promote cholesterol translocation. This perspective implies that import of the protein into the mitochondrial matrix, rather than being the trigger to steroid production, is actually the “off” mechanism, because it removes STARD1 from its site of action ( Fig. 4.4 ). Consequently, ongoing production of STARD1 preprotein is required to sustain steroidogenesis.

Structure of steroidogenic acute regulatory protein (STARD1) and model for its mechanism of action on intramitochondrial cholesterol translocation.

StAR preprotein is targeted to the outer mitochondrial membrane where it acts to promote cholesterol movement to the cholesterol side-chain cleavage enzyme (P450scc) . StAR preprotein is subsequently imported by the translocator complex into the mitochondrial matrix where it is proteolytically processed to the mature 30-KDa form. TIM , Inner mitochondrial membrane translocators; TOM , outer mitochondrial membrane protein translocators; START , STAR-related lipid transfer/domain.

The findings of the previously described experiments are most consistent with the idea that STARD1 enhances desorption of cholesterol from the sterol-rich outer mitochondrial membrane to the relatively sterol-poor inner membranes. The desorption process may involve a pH-dependent conformational change (molten globule transition). Even though STARD1 contains a hydrophobic pocket that binds cholesterol, sterol binding is not required for steroidogenic activity.

The molecule or structure on the mitochondrial outer membrane that STARD1 acts on to promote cholesterol movement to the inner membrane has not been definitively identified. The STARD1 “receptor,” which could be a protein or a lipid, is not specific for mitochondria of steroidogenic cells, since STARD1 works in the context of COS-1 cells, which are not normally steroid hormone-producing cells. Thus the specificity of the mechanism of mitochondrial cholesterol translocation is determined by whether STARD1 is expressed. Initially the translocator protein (TSPO; also known as the peripheral benzodiazepine receptor ), which is found in the outer mitochondrial membrane of many cell types, was postulated to be a target of STARD1 because molecules that bind to TSPO stimulate steroidogenesis and knockdown of TSPO expression in cultured cells reduced steroid synthesis in the presence of STARD1. However, the subsequent phenotyping of TSPO-deficient mice revealed that TSPO is dispensable for steroidogenesis. Despite ongoing debate in the literature over the latter findings, the weight of evidence indicates that TSPO is not required for STARD1 action. Another potential candidate for the STARD1 effector molecule is the mitochondrial voltage-dependent anion channel 2 (VDAC2), which is also expressed by many different cell types and has been proposed to be a cholesterol “pore” through which outer mitochondrial membrane cholesterol could flow to the inner membrane.

As noted above, mutations in the STARD1 gene cause congenital lipoid adrenal hyperplasia, a rare autosomal recessive disease. Exceptions occur in Japan and Korea, however, where the mutation accounts for at least 5% of all cases of congenital adrenal hyperplasia. The pathophysiology of the disease entails a two-step process in which impaired use of cholesterol for steroidogenesis leads to accumulation of sterol esters in lipid droplets. These droplets ultimately compress cellular organelles, causing damage through the formation of lipid peroxides. This damage occurs prominently in the adrenal cortex and Leydig cells. Mutations inactivating NR5A1 (SF-1) also cause cholesterol accumulation in Leydig cells because STARD1 and cholesterol side-chain cleavage enzyme expression are impaired.

Mutations found in the STARD1 gene, which is composed of seven exons, include frameshifts caused by deletions or insertions, splicing errors, and nonsense and missense mutations. These mutations lead to the absence of STARD1 protein or the production of functionally inactive protein. Several nonsense mutations were shown to result in C-terminus truncations of STARD1. One of these mutations, Gln258Stop, results in the deletion of the final 28 amino acids of the STARD1 protein and accounts for 80% of the known mutant alleles in the affected Japanese population. Known point mutations that produce amino acid substitutions occur in exons 5 to 7 of the gene, the exons that encode the C-terminus. Mutations that cause partial loss of STARD1 activity (usually 20% to 30% of normal) are associated with a milder disease phenotype or “nonclassical” disease.

Although affected XY subjects are pseudohermaphrodites (46,XY disorder of sexual development [DSD]) because of an inability to generate sufficient fetal testicular testosterone to masculinize the external genitalia, 46,XX subjects have normal external genitalia, develop female secondary sexual characteristics, and experience menarche. They are, however, anovulatory and unable to produce large amounts of estradiol and progesterone in a cyclic fashion. The fact that some ovarian estradiol synthesis occurs reflects the existence of STARD1-independent substrate movement to the cholesterol side-chain cleavage system.

Cholesterol Side-Chain Cleavage Enzyme (P450scc Encoded by CYP11A1 )

Cholesterol side-chain cleavage is catalyzed by cytochrome P450scc and its associated electron transport system, consisting of a flavoprotein reductase (ferredoxin or adrenodoxin reductase) and an iron sulfoprotein (ferredoxin or adrenodoxin), encoded by the FDX1 gene, which shuttles electrons to cytochrome P450scc. The side-chain cleavage reaction involves three catalytic cycles: the first two lead to the introduction of hydroxyl groups at positions C-22 and C-20, and the third results in scission of the side chain between these carbons. Each catalytic cycle requires one molecule of NADPH and one molecule of oxygen so that the formation of one mole of the cleavage products, pregnenolone and isocaproaldehyde, uses three moles of NADPH and three moles of oxygen.

The slowest step of the reaction is the binding of cholesterol to the hydrophobic pocket of P450scc where the heme resides. The sterol substrate remains bound to a single active site on cytochrome P450scc for all three cycles because of the tight binding of the reaction intermediates. The dissociation constant (K _d ) for binding of cholesterol, a measure of the enzyme’s affinity for its substrate, is approximately 5000 nM, whereas the K _d for the binding of the intermediate product 22-hydroxycholesterol is 4.9 nM; the K _d for 20,22-dihydroxycholesterol is 81 nM. The estimated K _d for pregnenolone, the end product, is 2900 nM, which permits its dissociation from the enzyme at the end of the reaction.

Reducing equivalents are shuttled to cytochrome P450scc by ferredoxin in cycles of reduction and oxidation, facilitated by differential affinities of the proteins, depending on their state of oxidation or reduction. Ferredoxin forms a 1 : 1 complex with ferredoxin reductase, which catalyzes reduction of the iron-sulfur protein. The reduced ferredoxin then dissociates and forms a 1 : 1 complex with cytochrome P450scc and is subsequently oxidized when it donates its electrons to P450scc. Oxidized ferredoxin returns to ferredoxin reductase for electron recharging. This recharging is facilitated by the fact that ferredoxin reductase has a greater affinity for oxidized over reduced ferredoxin. The binding of cholesterol to cytochrome P450scc increases its affinity for reduced ferredoxin, which enhances the shuttle of electrons to substrate-loaded enzyme.

The rate of formation of pregnenolone is determined by:

• 1.
  

  Access of cholesterol to the inner mitochondrial membranes

  
  
• 2.
  

  The quantity of cholesterol side-chain cleavage enzyme, and secondarily, its flavoprotein and iron-sulfur protein electron transport chain

  
  
• 3.
  

  Catalytic activity of P450scc, which can be influenced by posttranslational modification

Acute alterations in steroidogenesis generally result from changes in the delivery of cholesterol to P450scc, whereas long-term alterations involve changes in the quantity of enzyme proteins as well as cholesterol delivery.

Mutations in the CYP11A1 gene that result in significantly diminished cholesterol side-chain cleavage activity have been reported in association with adrenal insufficiency and 46,XY DSD (sex reversal), phenotypes that are similar to those associated with inactivating mutations in the STARD1 gene.

The discovery of mutations causing severe P450scc deficiency in some humans born at term challenges the notion that absence of P450scc activity in the fetus and placenta is incompatible with pregnancy progressing beyond the usually limited period of luteal progesterone support. Therefore, compensatory mechanisms appear to exist to maintain sufficient progestational activity, perhaps sustained corpus luteum function, to support pregnancy and fetal viability in women hosting a fetus with CYP11A1 mutations. Another surprising and distinguishing feature of some subjects with CYP11A1 mutations is the absence of adrenal hypertrophy, as seen in STARD1 mutations.

17α-Hydroxylase/17,20-Lyase (P450c17; CYP17A1)

P450c17 is an endoplasmic reticulum enzyme that catalyzes two reactions: hydroxylation of pregnenolone and progesterone at carbon 17 and conversion of pregnenolone into C19 steroids (in the case of the human enzyme, progesterone is also converted but to a much lesser extent). The 17α-hydroxylation reaction requires one pair of electrons and molecular oxygen. The lyase reaction requires a second electron pair and molecular oxygen. The reducing equivalents are transferred to the P450c17 heme iron from NADPH by NADPH cytochrome P450 reductase (POR), an enzyme functioning in the endoplasmic reticulum. The hydroxylase and lyase reactions are both believed to proceed through a ferryl oxene mechanism with the substrate bound to the enzyme in the catalytic pocket in the same orientation. P450c17 also catalyzes 16α-hydroxylation of progesterone and DHEA.

The importance of POR in steroid metabolism catalyzed by endoplasmic reticulum cytochrome P450 enzymes has been illuminated by the phenotype of individuals with POR deficiency, which causes a form of congenital adrenal hyperplasia. The autosomal recessive disorder results in a steroid profile suggestive of combined 21-hydroxylase and 17-hydroxylase/17,20-lyase deficiency, which presents as a range of phenotypes, including adrenal insufficiency, ambiguous genitalia (DSD), and Antley-Bixler syndrome-like skeletal malformation syndrome.

Several factors determine whether substrates only undergo 17α-hydroxylation or subsequent scission of the 17,20 bond, including:

• 1.
  

  The nature of the substrate

  
  
• 2.
  

  The amount of POR and flux of reducing equivalents

  
  
• 3.
  

  Allosteric effectors such as cytochrome b ₅

  
  
• 4.
  

  Posttranslational modification of P450c17 ( Fig. 4.5 )

  
  

  
  

  
  

  

  
  

  
  

  Factors modulating 17α-hydroxylase and 17,20-lyase activities of P450c17 (CYP17A1) .

  
  

  Phosphorylation of serine/threonine residues and cytochrome b5 stimulate (designated by plus symbol) lyase activity, with pregnenolone being the preferred substrate for the lyase reaction. Dephosphorylation of the serine/threonine residues by protein phosphatase 2A reduces lyase activity. Flow of reducing equivalents ( e ^– , electrons) from cytochrome P450 reductase (POR) stimulates both 17α-hydroxylase and lyase activities. NADP , Nicotinamide adenine dinucleotide phosphate.

  
  

Collectively, these factors determine the nature of the products produced by the enzyme, which, in the gonads and zona reticularis, favor androgens through augmentation of lyase activity. In contrast, 17α-hydroxylation required for glucocorticoid and mineralocorticoid synthesis is favored in the zona fasciculata and glomerulosa.

Human P450c17 preferentially uses Δ ⁵ substrates for 17,20 bond cleavage. Cytochrome b ₅ (CYB5A) promotes electron transfer via POR for the lyase reaction acting as an allosteric effector and not as an electron donor per se, because the apo b ₅ protein is also effective. Cytochrome b ₅ also increases the use of 17α-hydroxyprogesterone as a substrate for androstenedione synthesis. The distribution and regulation of cytochrome b ₅ expression in the adrenal cortex, being greatest in the adult zona reticularis, supports a role for this protein in the regulation of lyase activity. The product of a second cytochrome b ₅ gene (type 2 cytochrome b ₅ , CYB5B), found in human testis and expressed in the adrenals, also increases lyase activity.

Phosphorylation of P450c17 at serine and threonine residues by a yet-to-be-identified protein kinase appears to be necessary for maximal 17,20-lyase activity. The phosphorylated P450c17 protein is evidently a substrate for protein phosphatase 2A (PP2A), since inhibitors of PP2A enhance lyase activity in cultured adrenal tumor cells.

Adrenarche, the increased production of adrenal androgens in the absence of increased production of cortisol or levels of ACTH, may result from enhanced P450c17 lyase activity due to increased expression of cytochrome b ₅ or the state of P450c17 phosphorylation.

A P450c17-independent mechanism for conversion of pregnenolone into DHEA in glial tumor cell homogenates promoted by FeSO ₄ has been described. This conversion presumably results from the fragmentation of tertiary hydroperoxides, which are probably derived from pregnenolone molecules oxygenated at carbons 17 and 20. The physiological significance of this pathway, if any, to the formation of C19 steroids in the brain or other tissues is unknown.

Mutations in the CYP17A1 gene cause combined or isolated deficiency states for each activity of P450c17. Individuals with combined deficiency have a marked diminution in the production of C19 and C18 steroids, low levels of cortisol that result in elevated ACTH secretion, and excess production of steroids proximal to the P40c17 reaction. Hypertension from sodium retention and hypokalemia are a consequence of the increased production of 11-deoxycorticosterone. The inability to produce sex steroid hormones prevents adrenarche, puberty in females, and results in incomplete or absent development of male genitalia (46,XY DSD). Isolated 17,20-lyase deficiency is very rare. The documented cases result from point mutations that permit pregnenolone or progesterone to be bound and undergo 17α-hydroxylation, but prohibit the efficient receipt or usage of a second pair of electrons provided by POR to support the 17,20-lyase reaction.

Aromatase (P450aro, CYP19A1)

Aromatase, an endoplasmic reticulum enzyme, catalyzes three sequential hydroxylations of a C19 substrate by using three molecules of NADPH and three molecules of molecular oxygen to produce one molecule of C18 steroid with a phenolic A ring. The first hydroxylation yields a C19 hydroxyl derivative, which is converted in a second hydroxy­lation to a gem diol that collapses to yield a C19 aldehyde. The final hydroxylation involves the formation of a 19-hydroxy-19-hydroperoxide intermediate that results in the elimination of the C19 methyl group as formic acid and concurrent aromatization. This sequence of reactions occurs at a single active site on the enzyme, with reducing equivalents transferred to P450aro by POR. The crystal structure of aromatase complexed with androstenedione revealed hydrogen bonding and tight packing of hydrophobic side chains that explain the enzyme’s specificity for androgen substrates.

The aromatase protein is encoded by a single large gene, CYP19A1 . This gene produces cell-specific transcripts from different promoters ( Fig. 4.6 ). The promoter driving ovarian aromatase expression lies adjacent to the exon encoding the translation start site (promoter IIa). In granulosa cells, FSH stimulates transcription of the genes encoding both aromatase and POR. A separate promoter lying approximately 100 kb upstream from the start of translation controls placental CYP19A1 transcription. Expression of aromatase in adipose tissue, skin, and brain is driven from other promoters. Cytokines (including interleukin-11, interleukin-6, oncostatin-M, and leukemia-inhibiting factor) increase P450arom expression in adipose tissue via the I.4 promoter.

Structure of the human CYP19A1 gene showing the protein coding exons indicated by Roman numerals and the location of the different 5′-UTR-coding exons and tissue-specific promoters.

The P450arom heme-binding region and polyadenylation signals in exon 10 are indicated. ATG , Methionine codon; HBR , heme-binding region.

(Modified from Kamat A, Hinshelwood MM, Murry BA, Mendelson CR: Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans. Trends Endocrinol Metab 13:122, 2002.)

Several cases of aromatase deficiency have been described. Pregnancies in which the fetus is affected with aromatase deficiency are characterized by low maternal urinary estrogen excretion, maternal virilization, and ambiguous genitalia or female pseudohermaphroditism (46,XX DSD) in affected genetic females. The maternal and fetal virilization in the absence of placental aromatase activity highlights the importance and efficiency of the placenta in converting maternal and fetal androgens into estrogens.

Among the mutations identified in the CYP19A1 gene are an 87-bp insertion at the splice junction between exon 6 and intron 6, causing the addition of 29 in-frame amino acid residues, with the other mutations being mainly missense or nonsense mutations in exons 4, 9, and 10. The mutant protein with the 29 in-frame amino acid residues displayed less than 3% of the activity of normal aromatase. Compound heterozygous mutations in coding sequences found in patients with aromatase deficiency have also been shown to have minimal activity. When aromatase activity has been measured in placenta from offspring with CYP19A1 mutations, activities have been found to be markedly reduced.

The aromatase-deficient (ArKO) mice show many of the features of human aromatase deficiency, and the consequential lack of estrogens, including a profound bone phenotype with reductions in all indices of bone mineralization.

Families with autosomal dominant estrogen excess resulting from overexpression of aromatase have been reported. The phenotypes include severe prepubertal gynecomastia in males and macromastia and premature puberty in females. The aromatase overexpression is caused in some families by heterozygous genomic rearrangements. In some cases an inversion results in a constitutively active cryptic promoter placed in control of the CYP19A1 gene, driving excess aromatase production.

Inappropriate expression of aromatase in neoplastic and nonneoplastic tissue has also been found. In these pathological conditions, exemplified by breast cancer, there appears to be a shift in promoter use to favor the stronger gonadal (promoter IIa or I.3) over the weaker adipose tissue promoter (promoter I.4). This shift allows for activation of a cAMP-dependent signaling pathway, accounting for excessive aromatase expression and the resulting increase in estrogen synthesis.

11β-Hydroxylases (P450c11β and P450c11AS)

The human genome contains two genes that encode related mitochondrial enzymes involved in 11β-hydroxylation and aldosterone synthesis, respectively: P45011β, encoded by CYP11B1 , and P450c11AS (also referred to as “P450aldo,” “P450c18,” or “P450cmo”), encoded by CYP11B2. The encoded proteins differ in only 33 amino acid residues. Both enzymes display 11β-hydroxylase activity, but P450c11AS can also perform the two oxygenation steps at carbon 18 to produce aldosterone. They require molecular oxygen and reducing equivalents for catalysis, and the latter is shuttled to the enzymes by the adrenodoxin reductase–adrenodoxin system.

CYP11B1 , a gene whose transcription is stimulated by ACTH-triggered cAMP signaling pathways, is expressed in the zonae fasciculata and reticularis of the adrenal cortex. CYB11B1 is also expressed in human Leydig and theca cells and is responsible (along with type II 11β-hydroxysteroid dehydrogenase) for the production of 11-ketotestosterone, a molecule that activates androgen receptors. In contrast, CYP11B2 expression is restricted to the zona glomerulosa. Transcription of this gene is stimulated by protein kinase C signaling pathways, which are turned on by angiotensin II.

Mutations in the CYP11B1 gene cause 11β-hydroxylase deficiency, whereas mutations in CYP11B2 cause 18-hydroxylase or corticosterone methyl oxidase I deficiency and 18-oxidase or corticosterone methyl oxidase II deficiency. Unequal crossover of the adjacent CYP11B1 and CYP11B2 genes creates a third hybrid gene, in which the cAMP-regulated promoter of the CYP11B1 gene drives expression of a chimeric protein with aldosterone synthase activity. This leads to a state of glucocorticoid-suppressible aldosteronism.

11β-hydroxylase deficiency is characterized by high levels of 11-deoxycortisol and deoxycorticosterone, resulting in salt retention and hypertension. Affected females are virilized by the excess production of adrenal androgens driven by elevated ACTH levels. CYP11B1 mutations causing 11β-hydroxylase deficiency include those resulting in nonsynonymous amino acid substitutions and a premature stop codon.

Corticosterone methyl oxidase I deficiency results from the complete absence of P450c11AS activity. Aldosterone synthesis is absent, but the production of corticosterone and cortisol is retained. Corticosterone methyl oxidase II deficiency is caused by mutations that inactivate 18-methyl oxidase while retaining 18-hydroxylase activity. This causes elevated 18-hydroxycorticosterone and low aldosterone levels.

21-Hydroxylase (P450c21,CYP21A2)

P450c21 is an adrenal endoplasmic reticulum enzyme that catalyzes the 21-hydroxylation of progesterone and 17α-hydroxyprogesterone in the pathway of mineralocorticoid and glucocorticoid biosynthesis. The Michaelis constant (K _m ) for 17α-hydroxyprogesterone (1.2 µM) is lower than that for progesterone (2.8 µM), and the apparent maximum velocity (V _max ) for the former substrate is twice that for progesterone. The enzyme requires 1 mole of molecular oxygen and reducing equivalents (generated from NADPH through POR) to accomplish the hydroxylation of carbon 21. Mutations inactivating POR cause a partial deficiency in 21-hydroxylase activity and a partial deficiency in 17α-hydroxylase/17,20-lyase activity. The primary regulator of CYP21A2 expression in the zona fasciculata is ACTH.

The CYP21A2 gene is adjacent to a pseudogene (CYP21A1P) , separated by the complement C4B gene. These genes are embedded in the human leukocyte antigen region on band 6p21.1. Frequent unequal crossovers and gene conversions make 21-hydroxylase deficiency one of the most common autosomal recessive metabolic diseases, occurring in 1 : 10,000 to 1 : 15,000 births. Unequal crossover, with the complete loss of the C4B gene and a net deletion of CYP21A2 , along with gene conversion events in which mutations in the pseudogene are introduced into the expressed gene, result in reduced 21-hydroxylase enzyme levels or impaired catalytic activity ( Fig. 4.7 ). Large-scale deletions/gene conversions may extend into the adjacent gene encoding tenascin-X, which when mutated in both alleles, causes a form of Ehlers-Danlos syndrome.

Structure of the CYP21A2 gene and selected mutations causing phenotypes of 21-hydroxylase deficiency.

(Modified from White PC, Speiser PW: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 21:245, 2000.)

The signs and symptoms of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency reflect deficits in cortisol (because of inability to convert 17α-hydroxyprogesterone into 11-deoxycortisol) and aldosterone (because of inability to convert progesterone into deoxycorticosterone). Another contributing factor is the accumulation of adrenal androgens that results from elevated ACTH levels due to the absence of cortisol-negative feedback on the hypothalamic–corticotrophin axis.

The clinical phenotypes are, however, variable and dependent on the severity of the 21-hydroxylase deficiency. The non–salt-wasting, salt-wasting, and nonclassic forms are associated with certain mutations that affect the amount of residual 21-hydroxylase activity, with the salt-wasting form being characteristic of severe enzyme deficiency (deletions and large gene conversions). The simple virilizing (non–salt-wasting) form is associated with mutations that substantially reduce activity (e.g., the missense mutation resulting in a nonsynonymous amino acid substitution Ile172Asp), and the nonclassic (or late-onset ) form is caused by mutations that do not severely impair the level of expression or activity of P450c21 (e.g., Val28Leu, Pro30Leu).

Hydroxysteroid Dehydrogenases and Reductases

Hydroxysteroid dehydrogenases (HSDs) or oxidoreductases catalyze the interconversion of alcohol and carbonyl functions in a position- and stereospecific manner on the steroid nucleus and side chain, using oxidized (+) or reduced (H) nicotinamide adenine dinucleotide, NAD(H), or NADP(H) as cofactors. In some instances, HSDs display bifunctionality (e.g., they oxidize or reduce 17β and 20α oxy functions), such as HSD17B2. Although the HSDs can catalyze both the oxidation and reduction reactions in vitro under different conditions (e.g., substrate, pH and cofactor), in vivo they catalyze reactions in one direction and can be classified as dehydrogenases or reductases. The enzymes are members of the short chain dehydrogenase/reductase (SDR) and aldo-keto reductase (AKR) superfamilies.

The importance of cofactor availability to HSDs is exemplified by inactivating mutations in the hexose-6-phosphate dehydrogenase (H6PDH) gene, which regenerates NADPH in the endoplasmic reticulum required for the HSD11B1 reaction. Individuals with these mutations have apparent cortisone reductase deficiency due to impaired HSD11B1 activity.

Multiple isoforms of HSDs exist, and, coupled with their tissue-specific expression, account for the ability of specific enzymes to act predominantly as reductases (ketone reduction) or dehydrogenases (alcohol oxidation). In steroidogenic tissues, HSDs catalyze the final steps in progestin, androgen, and estrogen biosynthesis. In steroid target tissues, HSDs can regulate the occupancy of steroid hormone receptors by converting active steroid hormones into inactive metabolites or relatively inactive steroids to molecules with greater binding activity.

This is best exemplified by the human type 2 11β-HSD (HSD11B2), which controls mineralocorticoid activity in the kidney by converting cortisol (which has high affinity for both the glucocorticoid and mineralocorticoid receptors) to cortisone (which does not bind to the mineralocorticoid receptor). Thus the specificity of mineralocorticoid receptor activation is not determined by the receptor, but by activity of the HSD that removes the more abundant potential mineralocorticoid receptor ligand, leaving aldosterone as the controlling activator ( Fig. 4.8 ). Because of their tissue-specific roles in controlling the bioavailability of steroids, HSDs are interesting targets for pharmacologic manipulation.

The roles of 11β-HSD types 1 (HSD11B1) and 2 (HSD11B2) in controlling levels of bioactive glucocorticoids.

HSD11B1 reduces inactive cortisone into cortisol in liver and other tissues, whereas HSD11B2 oxidizes cortisol to cortisone. Cortisone cannot activate the mineralocorticoid receptor, thus allowing aldosterone (a steroid that is less abundant than cortisol) to specifically regulate the mineralocorticoid receptor. The supply of NADPH to HSD11B1 is a major determinant of the reaction and inactivating mutations in the H6PDH gene, which generates NADPH, causes cortisone reductase deficiency. CNS , Central nervous system; HSD , hydroxysteroid dehydrogenases; NADPH , nicotinamide adenine dinucleotide phosphate.

(Modified from Seckl J, Walker B: 11β-Hydroxysteroid dehydrogenase type I-A tissue-specific amplifier of glucocorticoid action. Endocrinology 142:1371, 2001.)

3β-Hydroxysteroid Dehydrogenase/Δ ^5-4 -Isomerases

The 3β-HSD/Δ ^5-4 -isomerases are membrane-bound enzymes localized to the endoplasmic reticulum and mitochondria that use nicotinamide adenine dinucleotide (NAD+) as a cofactor. These enzymes catalyze dehydrogenation of the 3β-hydroxyl group and the subsequent isomerization of the Δ ⁵ olefinic bond to yield a Δ ⁴ ketone structure. They convert pregnenolone into progesterone, 3-, 17α-hydroxypregnenolone into 17α-hydroxyprogesterone, and DHEA into androstenedione.

The dehydrogenase and isomerase reactions are performed at a single bifunctional catalytic site that adopts different conformations for each activity. The 3β-hydroxysteroid dehydrogenase step is rate-limiting in the overall reaction sequence, and the NADH formed in this reaction is believed to alter the enzyme conformation to promote the isomerase reaction.

The human genome has two active 3β-HSD/Δ ^5-4 -isomerase genes: one encodes a protein predominantly expressed in the placenta, liver, breast, and brain (HSD3B1), while the other encodes a protein expressed in the adrenal cortex and gonads (HSD3B2). These two genes, each consisting of four exons, lie 100 kb apart on band 1p13.1. The human genome also contains five unprocessed pseudogenes closely related to HSD3B1 and HSD3B2 on band 1p13.1, with two of them lying between the expressed genes. The DNA sequences of the exons of the two active genes are very similar, and the encoded proteins differ in only 23 amino acid residues. HSD3B1, however, has a lower K _m for substrate than HSD3B2, which facilitates metabolism of lower concentrations of Δ substrate. Electron microscope cytochemistry has localized HSD3B2 activity to the perimitochondrial endoplasmic reticulum. In some cell types, the enzyme appears to be localized in the inner mitochondrial membrane, positioned to act on pregnenolone produced by the cholesterol side-chain cleavage system.

Because most steroidogenic cells have a large capacity to generate progesterone when presented with exogenous pregnenolone, the 3β-HSD/Δ ^5-4 -isomerases are not believed to be rate-determining enzymes. However, mutations causing deficiency of HSD3B2 cause a form of congenital adrenal hyperplasia characterized by impaired adrenal and gonadal steroidogenesis with accumulation of Δ ⁵ steroids in the circulation. The presence of active HSD3B1 in extraadrenal and extragonadal tissues results in the formation of some Δ ⁴ steroids (e.g., 17α-hydroxyprogesterone).

In its severest form, HSD3B2 deficiency is associated with salt wasting because of insufficient mineralocorticoid production. Kinetic analysis of mutant proteins associated with the salt-wasting and non–salt-wasting forms of the disease showed a 4- to 40-fold reduction in catalytic efficiency for the conversion of pregnenolone into progesterone. The salt-wasting form of the disease is associated with frameshift mutations resulting in protein truncation and a variety of missense mutations that affect affinity for the cofactor and protein stability. The greater instability of the mutant proteins found in subjects with salt-wasting disease compared with those proteins found in the non–salt-wasting form appears to account, in part, for the different clinical phenotypes.

A so-called attenuated, or late-onset, form of 3β-HSD deficiency, diagnosed by steroid measurements, has been described in the literature. However, no mutations have yet been discovered in the genes encoding HSD3B1 and HSD3B2 in subjects with this clinical diagnosis. Mutations in the distal promoter or epigenetic factors that might alter enzyme expression cannot be excluded. The apparent reduced 3β-HSD activity could also be the result of alterations in the membrane environment that affect catalytic activity or posttranslational modifications to the enzyme that diminish its function. Mutations in HSD3B1 have not been detected, although several sequence variants of uncertain significance have been described.

11β-Hydroxysteroid Dehydrogenases: Key Regulators of the Activity of Glucocorticoids

The biological activity of cortisol in target tissues is controlled by the action of two different 11β-hydroxysteroid dehydrogenases that are members of the short-chain dehydrogenase/reductase family (see Fig. 4.8 ). These enzymes catalyze the interconversion of active glucocorticoids and their inert 11-keto metabolites. The type 1 enzyme (HSD11B1) is an endoplasmic reticulum protein with reversible oxidoreductase activity in vitro but preferentially catalyzes the reduction of the 11-keto group, using NADPH as a cofactor in vivo. This enzyme is expressed in the liver, lung, adipose tissue, brain, vascular tissue, and gonads, where it regenerates cortisol from 11-ketosteroids. In the case of the placenta, the activity ensures transport of biologically active cortisol to the fetus in the first half of pregnancy.

Targeted deletion of the type 1 enzyme gene (Hsd11b1) in mice results in animals with lower blood glucose levels in response to overfeeding and stress, impaired activation of gluconeogenesis, and blunted sensitivity to natural glucocorticoids. This finding substantiates a role for HSD11B1 in the amplification of cortisol and corticosterone action. HSD11B1 also plays a key role in the pharmacology of glucocorticoids. Hepatic HSD11B1 converts cortisone and prednisone, inactive prohormones, into active cortisol and prednisolone.

As noted above, mutations in the H6PDH gene cause a syndrome characterized by high ratios of cortisone to cortisol; impaired cortisol negative feedback resulting in elevated ACTH secretion; and increased production of adrenal androgens leading to hyperandrogenism, sexual precocity, and a polycystic ovary syndrome-like phenotype. Mice with targeted mutations in H6pdh share many of the phenotypes of mice with Hsd11b1 mutations. Heterozygous mutations in the HSD11B1 gene cause a mild form of cortisone reductase deficiency.

HSD11B2, also an endoplasmic reticulum enzyme, has a higher affinity for its substrate than HSD11B1 and catalyzes the oxidation of cortisol with NAD ⁺ as a cofactor. It shares only modest amino acid sequence identity (21%) with HSD11B1. HSD11B2 is highly expressed in the kidney, colon, salivary glands, and placenta, all tissues that respond to aldosterone, or in the case of the placenta, tissues that act to separate the maternal and fetal endocrine systems (which is important in the third trimester). By converting cortisol and corticosterone to 11-keto compounds, HSD11B2 protects the renal mineralocorticoid receptors, which cannot distinguish cortisol or corticosterone from aldosterone, from inappropriate activation by the glucocorticoids.

Mutations that inactivate HSD11B2 produce a syndrome of apparent mineralocorticoid excess in humans, which is mimicked in the mice deficient in the enzyme that display hypertension, hypokalemia, and renal structural abnormalities. Glycyrrhizic acid, a component of licorice, and its metabolite carbenoxolone are competitive inhibitors of HSD11B2, but they also cause reduced expression of the HSD11B2 mRNA when administered in vivo. As a result, a drug-induced syndrome of apparent mineralocorticoid excess is produced.

17β-Hydroxysteroid Dehydrogenases: Multiple Enzymes With Specific Biosynthetic and Catabolic Roles

The adrenals, gonads, and placenta reduce 17-ketosteroids into 17β-hydroxysteroids (which have greater biological potency), whereas target tissues usually oxidize 17β-hydroxysteroids, inactivating them. In humans, these metabolic processes are mediated by at least 7 of the 14 known mammalian 17β-HSDs, designated types 1 through 14, according to the chronological order in which they were identified ( Fig. 4.9 ). They are all members of the short-chain dehydrogenase/reductase family, except the type 5 enzyme, which is an AKR. They have different cofactor and substrate specificities, including molecules that are not steroids, subcellular locations, and tissue-specific patterns of expression.

The family of 17β-HSDs and some of their roles in androgen and estrogen synthesis and metabolism.

DHEA , Dehydroepiandrosterone; DHT , dihydrotestosterone; HSD , hydroxysteroid dehydrogenase.

(Modified from Luu-The V: Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase. Steroid Biochem Mol Biol 76:143, 2001.)

The structures of the genes encoding the 17β-HSDs differ, and their nucleotide sequence homology is low. They can be grouped into enzymes that catalyze NAD ⁺ -dependent oxidation (types 2, 4, 6, 8, 9, 10, 11, and 14) and those that catalyze NADPH-dependent reduction (types 1, 3, 5, and 7). Because of the broad substrate specificities, the primary roles of several of these enzymes are in basic metabolic pathways unrelated to steroid metabolism, and deficiencies of these enzymes cause metabolic disease.

The type 1 enzyme (HSD17B1) is referred to as the estrogenic 17β-HSD, because it catalyzes the final step in estrogen biosynthesis by preferentially reducing the weak estrogen estrone to yield the potent estrogen 17β-estradiol. The enzyme is a cytosolic protein that uses either NADH or NADPH as a cofactor. It has 100-fold higher affinity for C18 steroids than for C19 steroids. It also converts 16α-hydroxyesterone into estriol and shows modest 20α-HSD activity.

The HSD17B1 gene is expressed in granulosa cells of the ovary and the placental syncytiotrophoblast. HSD17B1 is also expressed at higher levels in breast cancer cells relative to HSD17B2, which converts estradiol into estrone. Amplification of HSD17B1 in estrogen receptor-positive breast cancers is associated with lower survival compared with subjects without amplification. The crystal structure of HSD17B1 has been determined to a resolution of 2.2 Å with and without bound substrates, providing a molecular framework for the design of specific inhibitors which might be employed in chemotherapy of estrogen-responsive cancers.

HSD17B2 is an endoplasmic reticulum enzyme that inactivates hormones; it preferentially oxidizes testosterone into androstenedione, and it oxidizes estradiol into estrone using NAD+ as its cofactor. However, HSD17B2 can also convert 20α-hydroxyprogesterone into progesterone.

The gene encoding HSD17B2 is expressed in liver, secretory endometrium, and the fetal capillary endothelial cells of the placenta, as well as the endothelial cells of the larger vessels. This expression pattern is consistent with its role of inactivating testosterone and estradiol. HSD17B2 in the fetal capillary endothelium protects the fetal compartment from estradiol formed in the syncytiotrophoblast and from any testosterone escaping aromatization. Its expression in the secretory endometrium permits the conversion of estradiol to estrone, whereas 20α-hydroxyprogesterone is converted back into progesterone, resulting in progestational dominance. In normal breast tissue, HSD17B2 expression predominates over HSD17B1 expression.

HSD17B3 is referred to as the androgenic 17β-HSD, because it catalyzes the final step in testosterone biosynthesis in the Leydig cells, reducing androstenedione to testosterone using NADPH as cofactor. It also can reduce estrone to estradiol. HSD17B3, an endoplasmic reticulum enzyme, is not expressed in the ovary, requiring androgen-producing cells of the ovary to employ another enzyme, probably HSD17B5, to synthesize testosterone.

In the absence of HSD17B3 activity, the testes are unable to convert androstenedione into testosterone, resulting in male pseudohermaphroditism (46,XY DSD) associated with 10- to 15-fold elevations in the ratio of blood androstenedione to testosterone. Females with mutations in the HSD17B3 gene are asymptomatic and produce normal amounts of estrogens and androgens. Molecular analysis of the HSD17B3 gene in affected individuals revealed mutations that affect splicing, cause amino acid replacements in exons 9 and 10, and a small deletion leading to a frameshift. Many of the missense mutations result in proteins that are devoid of catalytic activity when expressed in eukaryotic cells.

HSD17B5, located on chromosome 10p15-14, is a member of the AKR family (AKR1C3). It has 3α-HSD and 20α-HSD activity in addition to 17β-HSD activity that produces testosterone from androstenedione. It is expressed in the adrenal cortex and may serve as the androgenic 17β-HSD of ovarian theca cells. HSD17B5 is also expressed in prostate, mammary gland, and Leydig cells.

HSD17B6 (also known as 11-cis retinol dehydrogenase, RODH) has 3α-HSD activity and catalyzes the conversion of androstanediol to dihydrotestosterone in the prostate. It is also a 3α,3β-epimerase.

Human HSD17B7 is involved in cholesterol synthesis but also thought to produce active estrogens and inactivate androgens. It transforms estrone into estradiol and has 3-keto reductase activity for dihydrotestosterone. The gene is expressed in the ovary, breast, placenta, testes, prostate, and liver cells.

3α- and 20α-Hydroxysteroid Dehydrogenase Activities

A number of HSDs catalyze reactions at both the 3 and 20 positions of steroid hormones (e.g., AKR1C1). The enzymes that are 3α-reductases (e.g., AKR1C2 and AKR1C4) are encoded by tandemly duplicated genes on chromosome 10p14-p15. They are expressed in liver, prostate, breast, and uterus, but some are also expressed in the testis and adrenals. Recessive mutations in one of these genes, AKR1C2, were discovered in 46,XY individuals with DSD, implicating this enzyme in a pathway of bioactive androgen biosynthesis through conversion of androsterone to androstanediol, followed by HSD17B6-mediated conversion of androstanediol to dihydrotestosterone. This finding suggests that two pathways are needed for testicular androgen synthesis to support normal male sexual differentiation.

The enzymes with 20α-HSD activity that are members of the AKR family reduce progesterone to yield the inactive steroid, 20α-hydroxyprogesterone (AKR1C1). They are cytosolic proteins with a molecular weight of approximately 34 kDa, exemplified by genes expressed in human keratinocytes and cells of the liver, prostate, testis, adrenal gland, brain, uterus, and mammary gland. These enzymes prefer NADPH to NADH as a cofactor.

Enzymes with 3α-hydroxysteroid oxidative activity that are members of the short-chain dehydrogenase/reductase family also have 20α-HSD activity and preferentially oxidize 20α-hydroxyprogesterone into progesterone.

Δ ^4-5 -Reductases

The Δ ^4-5 -reductases are membrane-associated enzymes that reduce the Δ ^5-4 double bond in steroid hormones by catalyzing direct hydride transfer from NADPH to the carbon 5 position of the steroid substrate. They produce either 5α or 5β-dihydrosteroids.

Sulfotransferases

A family of enzymes introduce the sulfonate ( ) anion from an activated donor, 3 ′ -phosphoadenosine-5 ′ -phosphosulfate (PAPS), to a steroid hydroxyl acceptor, inactivating the hormone. The major enzymes performing this reaction include estrogen sulfotransferase (SULT1E1), an enzyme that sulfonates the 3-hydroxyl function of phenolic steroids, and the hydroxysteroid sulfotransferases encoded by the closely linked SULT2A1 and SULT2B1 genes on chromosome 19q13.4.

The SULT2A1 enzyme, also known as DHEA sulfotransferase, has a broad substrate range including the 3α-, 3β-, and 17β-hydroxy functions of steroid hormones. SULT2A1 enzyme is expressed at high levels in the fetal zone of the adrenal cortex, as well as in the zona reticularis after adrenarche, and in the liver, gut, and testes. This enzyme is developmentally regulated in the adrenal cortex, with expression increasing between ages 5 and 13 years in association with adrenarche. The GATA-binding factor 6 (GATA6) transcription factor, which is known to increase transcription of other genes involved in androgen biosynthesis, also activates expression of the SULT2A1 gene.

The SULT2B1 gene gives rise to two protein isoforms through alternative splicing. The SULT2B1a isoform sulfonates pregnenolone, whereas the SULT2B1b isoform preferentially sulfonates cholesterol. They both sulfonate dihydrotestosterone. Unlike the SULT2A1 gene that is expressed in a limited number of tissues, SULTB1 isoforms are present in a variety of hormone-producing and hormone-responsive tissues, including the placenta, ovary, uterus, and prostate.

SULT1E1 is expressed in many tissues, including the adrenal gland, liver, kidneys, muscle, fat, and uterus. In the endometrium, progesterone increases its activity, contributing to the inactivation of estradiol in the secretory phase. The importance of estrogen sulfotransferase in modulating local levels of bioactive estrogens has been shown in mice with targeted deletions of the gene. Male mice with estrogen sulfotransferase deficiency have Leydig cell hyperplasia and become infertile with age due to elevated testicular levels of bioactive estrogen. Adipose tissue mass also increases. Hydroxylated metabolites of polychlorinated biphenyls are potent inhibitors of estrogen sulfotransferase with IC50 values in the picomolar range. The blockade of estradiol inactivation by these compounds may account for the reported estrogenic activity of polychlorinated biphenyls.

Mutations in the gene that encodes one of the enzymes that synthesizes the sulfate donor, PAPS (PAPS synthase type 2 [PAPSS2]), results in increased free DHEA levels, with the subsequent conversion of DHEA into biologically active androgens.

Steroid Sulfatase

The sulfonate function on steroids is cleaved by steroid sulfatase, an enzyme encoded by the STS gene on chromosome Xp22.3. This enzyme plays a key role in controlling the generation of biologically active steroids from the inactive sulfated molecules (e.g., estrone sulfate and DHEA sulfate). The sulfated steroid substrates, which are hydrophilic and not membrane-permeable, are taken up by cells via specific transport proteins, including a sodium-dependent anion transporter (SLC10A6) .

The syncytiotrophoblast is enriched in steroid sulfatase, which plays a key role in placental estrogen synthesis by liberating sulfonated androgen precursors produced in the fetal compartment prior to aromatization. The enzyme is also expressed in skin where it metabolizes cholesterol sulfate and sulfated estrogens.

Sulfatase deficiency is associated with marked impairment of placental estrogen synthesis during pregnancy and ichthyosis developing after birth. It occurs mostly in males because of the X chromosome location of the sulfatase gene at a frequency of 1 : 2000 to 1 : 6000 live born males. The majority of subjects with steroid sulfatase deficiency have a deletion of the entire gene that results from recombination of repetitive elements that flank the locus. The large deletions of the STS gene occur in association with mutations in the adjacent Kallmann syndrome gene (KAL1) . Partial deletions in the STS gene causing enzyme deficiency have also been described.

Characteristically, in pregnancies hosting an affected fetus, maternal plasma estriol and urinary estriol excretion are quite low at approximately 5% of the levels found in normal pregnancies. Excretion of estrone and estradiol are also reduced at approximately 15% of normal. Maternal serum 16α-hydroxydehydroepiandrosterone levels are elevated, and intravenous administration of DHEA sulfate to the mother does not lead to an increase in estrogen excretion, whereas administration of DHEA does lead to increased estrogen excretion.

Steroid sulfatase is expressed in estrogen target tissues, including endometrium, bone, and breast. Increased sulfatase expression may contribute to greater bioavailability of estradiol in certain tumors, including breast and endometrial cancers. Inhibitors of sulfatase have been developed and tested in clinical trials (Irosustat).

UDP-Glucuronosyl Transferases

Glucuronidation, catalyzed by a family of uridine 5′-diphospho UDP-glucuronosyl transferases, is part of the metabolic clearance mechanism for steroid hormones by the liver and extrahepatic tissues. There are 18 known UDP-glucuronosyl transferases that have been divided into three subfamilies: UGT1A, UGT2A, and UGT2B. UGT1A enzymes are encoded by a single gene that gives rise to alternatively spliced products capable of acting on estrogens. The UGT2 enzymes are products of separate genes UGT2A and UGT2B . UGT2A is expressed in olfactory epithelium, and UGT2B is expressed in the liver, kidney, breast, lung, and prostate. At least seven members of the UGT2B family have been identified with different steroid substrate specificities. Three UGT2B enzymes, UGT2B7, UGT2B15, and UGT2B17, appear to be responsible for glucuronidation of 5α-dihydrotestosterone, androsterone, and androstane-3α-17β-diol. Of these, UGT2B15 and UGT2B17 are expressed in the prostate where they are proposed to modulate steroid action. Polymorphisms in genes encoding UDP-glucuronosyl transferases have been associated with estrogen levels, suggesting a role for these phase II biotransformation enzymes in regulating the bioactive steroid economy.