Hsd enzymes

Hsd enzymes DEFAULT

Recently generated mouse genetic model for overexpression of17β-HSD1 (HSD17B1-TG mice) by Saloniemi et al [10] provided valuable data about common female reproductive disorders like Polycystic Ovarian Syndrome (PCOS), ovarian carcinogenesis and endometiosis. Overexpression of hHSD17B1 leads to increased androgen exposure during embryonic development that caused androgen-dependent phenotypic alterations in female, such as increased anogenital distance, lack of vaginal opening and combination of vagina with urethra. These alterations observed in the HSD17B1-TG females were effectively rescued by prenatal anti-androgen (flutamide) treatment, further confirming the dependence of these phenotypes on androgens. Interestingly, the androgen exposure during pregnancy in the HSD17B1-TG mice resulted in benign ovarian serous cystadenomas in adulthood. As ovarian serous borderline tumours are positively associated with a history of PCOS, thus with a history of (foetal) hyperandrogenism, 17β-HSD1 may promote ovarian carcinogenesis via increased estrogen concentration, but also via enhanced androgen production. Endometrial hyperplasia in HSD17B1-TG mice closely resembled human disease and it was efficiently reversed by 17β-HSD1 inhibitor treatment. The data concerning the expression of 17β-HSD1 in normal and diseased human endometrium are not fully conclusive. However, in most of the studies, the 17β-HSD1 expression is detected in normal endometrium, endometriosis specimens and endometriotic cancer. Other 17β-HSD enzymes including 17β-HSD2, 17β-HSD5, 17β-HSD7 and 17β-HSD12 have also been detected in the endometrium under different pathological conditions like endometriosis and PCOS [10]. Collectively, the data suggest that 17β-HSD1 inhibition is one of the several possible approaches to reduce estrogen production both in eutopic and in ectopic endometrial tissue.

17β-HSD type-2: 17-HSD/KSR2 converts 17β-hydroxy forms of estrogens and androgens (estradiol, testosterone and 5α-dihydrotestosterone) to their less active 17-keto forms (estrone, androstenedione and 5α-androstanedione).The enzyme also possesses 20α-HSD activity, thereby activating 20α-hydroxyprogesterone to progesterone. The 17β-HSD2 enzyme is widely and abundantly expressed in both adult and fetal tissues such as placenta, uterus, liver, the gastrointestinal and urinary tracts. Due to its expression pattern and enzymatic characteristics, it has been suggested that the 17β-HSD2 enzyme protects tissues from excessive steroid action [251]. 17β-HSD2 is localised in the endoplasmic reticulum, and it is widely expressed in various estrogen and androgen target tissues both in human and in rodents including breast endometrium, placenta and prostate. Furthermore, the 17β-HSD2 expression in the placenta and in foetal liver and intestine, together with the observed oxidative 17β-HSD2activity, are the basis for the hypothesis, suggesting a role for the enzyme in lowering the sex steroid exposure of the foetus.

Phylogenic analyses have indicated that 17β-HSD2 is a close homologue of retinoid-converting enzymes and has a high sequence similarity to retinol dehydrogenase type 1. In addition, studies have shown that retinoic acid (RA) induces expression of 17β-HSD2 in a dose- and time-dependent manner in human endometrial epithelial and placental cells [10]. Recent data from transgenic mice (HADS17B2-TG) provide evidence for importance of 17β-HSD2 for prenatal eye morphogenesis and eye development [10]. These TG mice overexpressing human 17β-HSD2 showed growth retardation, disrupted spermayogenesis, female masculinization, delayed eye opening, squint appearance of the eyes and some of these defects closely resembeled those identified in retinoid receptor mutant mice. The most notable changes in the HSD17B1TG mice are well explained by alterations in sex steroid action, whereas in the HSD17B2-TG mice the connection to sex steroids is weaker. The opposite mouse model of deficiency of 17β-HSD2 provide evidence for the essential role of 17β-HSD2. Embryonic death in the HSD17B-KO mice is reported, related to lack of action of 17β-HSD2 enzyme in placenta. Furthermore, the treatment of pregnant female mice with an anti-estrogen or with progesterone did not prevent the foetal loss of the HSD17B2-KO mice, thus indicating that embryonic deaths is likely not due to the lack of progesterone or due to an increased action of estrogens.

Osteoporosis is well known to occurs in elderly people when the level of active sex steroids decreases. Estrogen replacement therapy is beneficial for the treatment of osteoporosis but it is no longer recommended because of adverse effects (breast, endometrial and ovarian cancers, stroke, thromboembolism). Since 17β-HSD2 oxidizes E2 into E1, decreasing the amount of E2 in bone cells, inhibition of this enzyme is a promising approach for the treatment of this disease [225]. Ovariectomized cynomolgus monkeys were used as an osteoporosis model to evaluate the efficacy of 17β-HSD2 inhibitors. Decrease in bone resorption and maintenance of bone formation was achieved in this experimental model.

17β-HSD type-3: 17-HSD/KSR3 17β-HSD3 converts ∆4-androstenedione into testosterone and it is essential for testosterone biosynthesis. The enzyme is present exclusively in the testis and the deficiency of the active enzyme results in male pseudohermaphroditism [252]. In addition to the conversion of androstenedione to testosterone, the enzyme is capable of catalyzing conversion of 5α-androstanedione to 5α- dihydrotestosterone as well as estrone to estradiol [108]. Messender RNA for 17β-HSD3 are over-expressed in prostate cancer tissues. As T is known to be responsible for cell proliferation in androgen dependent diseases, 17β-HSD3 inhibitors (exerting effects equivalent of chemical castration) could be therapeutics for the treatment of such diseases [225]. Day et al. [253] developed the first xenograft model in castrated mice to evaluate 17β-HSD3 inhibitors and strong suppression of tumor growth by 81% was found, suggesting that 17β-HSD3 inhibition might be an efficient strategy for the treatment of hormone dependent prostate cancer.

There are only few observations in human male deficient in 17β-HSD as rare mutation associated with 46XY disorder of sexual development [254]. Patients with 17β-HSD deficiency are usually classified as female at birth (although abdominal testes) but developed secondary male features at pubery with diminished virilization [255].

17β-HSD type-4: Among 17-HSD/KSRs, type 4 is an unique multifunctional enzyme consisting of 17-HSD/KSR-, hydratase- and sterol carrier 2-like domains. 17β-HSD4 is ubiquitously expressed, but in some tissues it shows cell-specific expression. In the brain it is present only in Purkinje cells, in the lung only in bronchial epithelium and in the uterus in luminal and glandular epithelium. The deficiency of 17β-HSD4 leads to disease known as Zellweger syndrome [251].

17β-HSD type-5: 17-HSD/KSR5 is also known as type 2 3α-HSD, and diferently from other 17-HSD/KSRs it belongs to the AKR (aldo-keto reductase) family. With other members of the AKR family (type 1 3α-HSD, type 3 3α-HSD and 20α-HSD), 17β-HSD5 shares 84%, 86% and 88% identity, respectively. Both human and mouse 17β-HSD5 catalyze the conversion of androstenedione to testosterone, and additionally possess 3α-HSD activity. Human 17β-HSD5 has been previously identified predominantly as 3α-HSD. Human, but not mouse, 17β-HSD5 also converts progesterone to 20α-dihydroprogesterone effectively. 17β-HSD5 appears to be involved in the formation of androgens in the testis and several peripheral tissues. Using specific probes and antibodies, human 17β-HSD5 has been localized in liver, adrenal, testis, basal cells of the prostate, and in prostatic carcinoma cell lines [251]. Recently, up-regulation of 17β-HSD5 was found in breast and prostate cancer [256].

17β-HSD type-6: 17-HSD/KSR6 is part of the catabolic cascade of 5α-dihydrotestosterone (DHT). The 17β-HSD6 shows low dehydrogenase activity with DHT, testosterone and estradiol and possesses a weak oxidative 3α-HSD activity. The 17β-HSD6 enzyme shares 65% sequence identity with retinol dehydrogenase type 1 and it is most abundantly expressed in liver and prostate, at least in rodent tissues [251].

17β-HSD type-7: 17β-HSD7 is expressed in the developing follicles and in luteinized cells, being the enzyme of ovarian estradiol biosynthesis. Both rodent and human 17β-HSD7 catalyze exclusively the conversion of estrone to estradiol. The 17β-HSD7 is abundantly expressed in corpus luteum during pregnancy and the enzyme is considered to be important in E2 production, especially during pregnancy. In addition, 17β-HSD7 mRNA has been detected in placental, mammary gland and kidney samples [251]. The 17β-HSD7 enzyme was first characterised as a prolactin receptor-associated protein in the rat corpus luteum, although its role in prolactin signalling has remained unknown.

A role for mouse 17β-HSD7 in cholesterol biosynthesis was also suggested by the studies, showing a similar expression pattern of 17β-HSD7 and cholesterogenic enzymes during mouse embryonic development. Data from HSD17B7-KO mouse embryos evidently showed the essential role of 17β-HSD7 for cholesterol biosynthesis in vivo. The lack of 17β-HSD7 resulted in a marked blockage in foetal de novo cholesterol synthesis. Histological analysis revealed that the 17β-HSD7 deficiency results in defects in the development of nerve system, vasculature, heart, associated with defect in cholesterol synthesis. HSD17B-KO deficient mice exhibit embryonic lethal phenotypes Tese data suggest a possible role of 17β-HSD7 in cholesterol biosynthesis in mice, while its role in E2 production in vivo needs further clarification [10].

17β-HSD type-8: The gene product has been characterized as a protein whose abnormal regulation is linked to the development of recessive polycystic kidney disease in mice and later it was discovered to be a 17βHSD8. In conditions, 17β-HSD8 converts most eficiently estradiol to estrone and, to some extent, it also catalyses oxidative reactions of androgens and the reduction from estrone to estradiol. The 17β-HSD8 is abundand in kidney, liver and gonads. Interestingly, in the ovary, 17β-HSD8 is present in cumulus cells and not in granulosa or luteal cells like 17βHSD1 and 7, respectively [251].

17β-HSD type-10: The 17β- -HSD10 has a very broad substrate profile. Interestingly, it has been proposed that this enzyme plays an important role in the pathological processes of Alzheimer’s disease (AD), mainly because 17β-HSD10 binds to amyloid-β peptide and appears to be up-regulated in patients suffering from this disease [225]. The mechanism by which 17β-HSD10 contributes to the pathology of AD is still not completely understood. The protein-protein interaction of 17β-HSD10 with amyloid-β appears to inhibit the enzymatic activity of 17β-HSD10. In vitro studies with a potent 17β-HSD10 inhibitor [257] have shown that inhibition of this enzyme can prevent its interaction with the amyloid-β peptide, suggesting 17β HSD10 as a potential target for the treatment of AD.Transgenic mice over-expressing human 17β-HSD10 suggesting that inhibition of 17β-HSD10 could protect from cerebral infarction and ischemia [258].

17β-HSD type-12: The mammalian 17β-HSD12 was initially characterised as a 3-ketoacyl-CoA reductase, involved in the long-chain fatty acid synthesis, particularly essential for brain arachidonic acid synthesis. Both the human and the mouse 17β-HSD12 share 40% sequence similarity with 17β-HSD3, and the data indicate that 17β-HSD12 is an ancestor of 17β-HSD3. In human and rodents, 17β-HSD12 is expressed universally and the highest expression of 17β-HSD12 is detected in tissues involved in the lipid metabolism, including the liver, kidney hearth, and skeletal muscle. In mice, the expression has also been detected in brown and white adipose tissue. 17β-HSD12 expression is also regulated by sterol regulatory element binding proteins, identically to that shown to be involved in fatty acid and cholesterol biosynthesis. Interestingly, a reduced expression of 17β-HSD12 in cultured breast cancer cells results in significant inhibition of cell proliferation that is fully recovered by supplementation of arachidonic acid. In addition to its putative role in fatty acid synthesis, human 17β-HSD12 has been shown to catalyse the conversion of E1 to E2 in cultured cells, and the enzyme was suggested to be a major enzyme converting E1 to E2 in postmenopausal women [10]. Analysis of the HSD17B12-KO embryos indicated that the embryos initiated gastrulation but further organogenesis was severely disrupted. The mutant embryos exhibited severe defects in the neuronal development (ectoderm-derives), they failed to grow several mesoderm-derived structures. Therefore, the embryos at the age of E8.5–E9.5 were avoid of all normal embryonic structures that caused their death.

13. Conclusion

HSD enzymes are broadly expressed in all steroidogenic organs as different isoforms with differential localization and function. HSD are key enzymes involved in growth and reproduction and they are considered as suitable targets to modulate the concentration of the potent steroids in case of steroid-dependent diseases. As they could act selectively in an intracrine manner, inhibitors of these enzymes might be superior to the existing endocrine therapies regarding the off-target effects. Although commont mechanisms operate in regulation of steroidogenesis, there are some differences/specificities between rodent and human, in particular the susceptibility of fetal testicular stereoidogenesis to environmental chemicals with estrogenic/antiandrogenic activity. As the latter appeared to be devoid of effect on fetal human testis, this should be taken into account when dial with risk assessment of endocrine disruptors for human reproductive health. Species specific diffences in steroiodogenesis cause real obstacles in investigation of HSD inhibitors. Some of the most active and selective inhibitors were investigated in vivo in animal disease-oriented models. They showed efficacy, but none of them reached the clinical trial stage. One reason for this might be the difficulty to identify an appropriate species to conduct the functional assays, as very potent inhibitors of the human enzyme show little activity toward HSD of other species (rodents). In this respect, experiments by using xenograft approach (human tissue xenografting in immunocompromised nude mice) would enable us to develop our studies for better understanding of regulatory mechanisms of the expression of HSD enzymes. Elucidation of molecular events involved in transcription control of HSD is of great importance for molecular desigh of new HSD inhibitors and development of new strategies for appropriate treatment of steroid-dependent deceases without use of invasive techniques.

Acknowledgement

The authors thank to Professor Richard Sharpe for providing samples from experimental models for hormonal manipulations, Chris McKinnel for technical expertise in immunohistochemistry. We are also grateful to Professor Michail Davidoff and Assoc. Professor Mariana Bakalska for studies on EDS experimental model. Authors’ work was supported in part by Grant DEER # 212844 funded by FP7-ENV-CP and Grant # DO 02/113 funded by NF “Scientific Research” of Ministry of Education Youth and Science in Bulgaria.

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Steroid hormones and role of hydroxysteroid dehydrogenases in steroidogenesis: steroidogenic pathways and general regulatory mechanisms",level:"1"},{id:"sec_3",title:"3. 3β-HSD gene family – function, tissues distribution, regulation and clinical importance",level:"1"},{id:"sec_4",title:"Clinical. importance of 3β-HSD genetic deficiency: ",level:"1"},{id:"sec_5",title:"4. 11β-hydroxysteroid dehydrogenase – biological role in the regulation of glucocorticoid metabolisms and cortisol levels",level:"1"},{id:"sec_6",title:"4. 11β-hydroxysteroid dehydrogenase in developing testis- marker for differentiation of the Leydig cells",level:"1"},{id:"sec_7",title:"6. 11β-hydroxysteroid dehydrogenase in aging testis- role in the response of Leydig cells to the glucocorticoids",level:"1"},{id:"sec_8",title:"7. 11β-hydroxysteroid dehydrogenase in the adrenal gland - expression profile under conditions of testosterone withdrawal",level:"1"},{id:"sec_9",title:"8. 11β –hydroxysteroid dehydrogenase in the ovary – cellular localization/distribution and relation to Polycistic Ovaries Syndrome and obesity in women",level:"1"},{id:"sec_10",title:"9. 11β –hydroxysteroid dehydrogenase in adipose tissue – relation to obesity and metabolic syndrome",level:"1"},{id:"sec_11",title:"10. 11β-HSD and metabolite syndrome - clinical importance ",level:"1"},{id:"sec_12",title:"11. 11β –hydroxysteroid dehydrogenase and pregnancy – role of 11b-HSD type 2 as a protective barrier for fetus to overexposure to glucocorticoids; implication in intrauterine growth retardation",level:"1"},{id:"sec_13",title:"12. 17β-HSD dehydrogenase and multifunstional izoforms: localization, function and relevance to clinical therapeutic strategies",level:"1"},{id:"sec_14",title:"13. 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  • Inst. Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria
'},{corresp:null,contributorFullName:"Yvetta Koeva",address:null,affiliation:'
  • Dept. Anatomy and Histology, Medical University, Plovdiv, Bulgaria
Sours: https://www.intechopen.com/books/dehydrogenases/hydrohysteroid-dehydrogenases-biological-role-and-clinical-importance-review

Structure, function and tissue-specific gene expression of 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) catalyses an essential step in the transformation of all 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3β-HSD cDNA clones and the corresponding genes which encode type I and II 3β-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3β-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3β-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3β-HSD activity than type II. We elucidated the structures of three types of rat 3β-HSD cDNAs as well that of one type of 3β-HSD from bovine and macaque ovary λgt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3β-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3β-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3β-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3β-HSD proteins convert 3β-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3β-hydroxy and 3-keto-5α-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3β-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing rat type I 3β-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD⁺ as co-factor, 5α-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3β-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3β-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3β-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3β-HSD mRNA accumulation accompanied by a similar decrease in 3β-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3β-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3β-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.

Sours: https://pubmed.ncbi.nlm.nih.gov/22217825/
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  3. Dust extraction attachment

11 beta-Hydroxysteroid dehydrogenases: key enzymes in determining tissue-specific glucocorticoid effects

Recent studies have demonstrated that the interconversion of active and inactive glucocorticoids plays a key role in determining the specificity of the mineralocorticoid receptor and controlling local tissue glucocorticoid receptor activation. Two distinct isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) have been identified. 11 beta-HSD1 is NADPH-dependent and at its major site of action (the liver) is a reductase, converting cortisone to cortisol (11-dehydrocorticosterone to corticosterone in the rat). 11 beta-HSD2 is NAD-dependent, is present in tissues such as the kidney and placenta, and converts cortisol to cortisone (corticosterone to 11-dehydrocorticosterone in the rat). Congenital or acquired deficiency of 11 beta-HSD2 produces the syndrome of apparent mineralocorticoid excess (SAME) in which cortisol gains access to the unprotected nonspecific mineralocorticoid receptor. The congenital deficiency is associated with mutations in the gene encoding the kidney isoform of 11 beta-HSD2; the acquired form results from inhibition of the enzyme by licorice, carbenoxolone, ACTH-dependent steroids in the ectopic ACTH syndrome, and possibly circulating inhibitors of the enzyme. This paper focuses on recent evidence, which suggest that low levels of placental 11 beta-HSD2 result in increased exposure of the fetus to maternal glucocorticoid and low birth weight. In animal studies using the rat we have shown that birth weight is correlated positively and placental weight negatively with the level of placental 11 beta-HSD. Thus animals with low birth weight and large placentae were those likely to be exposed to the highest level of maternal glucocorticoid. In man a similar relationship was found with birth weight being significantly correlated either with placental 11 beta-HSD activity or with the extent of cortisol inactivation by isolated perfused placental cotyledons. Administration of dexamethasone (which is poorly metabolized by placental 11 beta-HSD2) to pregnant rats resulted in decreased birth weight and the development of hypertension in the pups when adult. The same results were obtained when pregnant rats were given carbenoxolone, an inhibitor of placental 11 beta-HSD2. Low protein diet during pregnancy in the rat resulted in low birth weight of the pups, increased placental weight but decreased placental 11 beta-HSD activity, and adult hypertension. Thus increased glucocorticoid exposure of the fetus secondary to a failure of the normal inactivation of maternal glucocorticoid by the placental may be an important mechanism linking changes in the in utero environment and common adult diseases.

Sours: https://pubmed.ncbi.nlm.nih.gov/8733012/
Enzymes (Inhibition \u0026 Regulation; Part 3/4 - Lippincott's) {الأنزيمات الجزء الثالث}

The 3β-hydroxysteroid dehydrogenase/Δ54 isomerase (3β-HSD) isoenzymes are responsible for the oxidation and isomerization of Δ5-3β-hydroxysteroid precursors into Δ4-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3β-HSD activity found in placenta and peripheral tissues, whereas the type II 3β-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3β-HSD gene family strongly suggest that the need for different 3β-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3β-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3β-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3β-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3β-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3β-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

  • I. Introduction

    • A. The role of 3β-hydroxysteroid dehydrogenase activity in steroid formation and degradation

    • B. Subcellular localization

  • II. Human Type I and II 3β-HSD Genes and Pseudogenes

  • III. Structure-Function Relationships

  • IV. Evolution of the 3β-HSD Gene Family

    • A. The rat 3β-HSD gene family

    • B. The mouse 3β-HSD gene family

    • C. The hamster 3β-HSD gene family

    • D. Phylogeny of the 3β-HSD gene family

    • E. Enzymatic characteristics of the 3-KSRs (rat liver-specific type III, mouse types IV and V, and hamster type III)

    • F. 17β-HSD activity of rat type I and IV 3β-HSDs

  • V. Transcriptional Regulation of Human Type I and II 3β-HSD

    • A. Gonadal/adrenal isoenzyme—type II 3β-HSD

    • B. Future directions in transcriptional regulation

    • C. Regulation of placenta/peripheral tissue type I 3β-HSD

    • D. Species similarity/divergence in mechanisms

  • VI. Ontogeny, Localization, and Regulation of 3β-HSD Expression

    • A. Adrenal

    • B. Ovary

    • C. Testis

    • D. Placenta

    • E. Liver

    • F. Breast

    • G. Prostate

    • H. Skin

    • I. Brain

    • J. Other expression sites

  • VII. Molecular Genetics of Human 3β-HSD Deficiency

    • A. Clinical features

    • B. Biological diagnosis

    • C. Molecular diagnosis

    • D. Genotype-phenotype relationships

    • E. Structure-function relationships

    • F. Sequence variants in the HSD3B2 gene vs. nonclassical 3β-HSD deficiency

I. Introduction

STEROID HORMONES PLAY a crucial role in the differentiation, development, growth, and physiological function of most vertebrate tissues. The major pathways of steroid hormone synthesis are well established, and the sequence of the responsible steroidogenic enzymes has been elucidated (Refs.1–7 and references therein) (Fig. 1). For example, in the human, after the conversion of cholesterol to pregnenolone (PREG) by the mitochondrial side-chain cleavage system, the adrenal cortex may direct PREG toward one of three different pathways. First, PREG may remain as a C21,17-deoxysteroid and proceed down the pathway to produce the mineralocorticoid, aldosterone. Second, it may undergo 17α-hydroxylation and proceed down the C21,17-hydroxy pathway to form the principal glucocorticoid, cortisol. The third option is that, after 17α-hydroxylation, it may undergo cleavage of the C17–20 bond to become a C19–17-ketosteroid, leading to the formation of androgens and estrogens. As can be seen in Fig. 1, whichever pathway is followed, the subsequent formation of all classes of steroid hormones relies upon the action of the enzyme 3β-hydroxysteroid dehydrogenase/Δ54-isomerase (3β-HSD) (8–10).

Fig. 1.

Schematic representation of the major mammalian steroidogenic pathways. All P450s are cytochrome enzymes. P450c18, This enzyme mediates 11β-hydroxylation and subsequent reactions involved in the biosynthesis of aldosterone; P450c11, 11β-hydroxylase; P450aro, P450 aromatase.

Fig. 1.

Schematic representation of the major mammalian steroidogenic pathways. All P450s are cytochrome enzymes. P450c18, This enzyme mediates 11β-hydroxylation and subsequent reactions involved in the biosynthesis of aldosterone; P450c11, 11β-hydroxylase; P450aro, P450 aromatase.

It is also well recognized that humans and certain other primates are unique among animal species in having adrenals that secrete large amounts of the inactive steroid precursors, dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S). These steroids do not bind to the androgen receptor (11) but exert either estrogenic or androgenic action after their conversion into active estrogens and/or androgens in target tissues (12, 13). Indeed, in postmenopausal women, almost all sex steroids are synthesized from precursors of adrenal origin except for a small contribution from ovarian testosterone (T) and Δ4-androstenedione (Δ4-DIONE), whereas in adult men, approximately half of androgens are made locally in target tissues (12). Thus, the various types of human enzymes catalyzing 3β-HSD, 17β-hydroxysteroid dehydrogenase (17β-HSD)/ketosteroid reductase (KSR), 5α-reductase activities, and the alternative promoter usage of the aromatase gene, because of their tissue- and/or cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active estrogens and androgens (12, 14–16).

A. The role of 3β-hydroxysteroid dehydrogenase activity in steroid formation and degradation

The nicotinamide adenine dinucleotide (NAD)+-dependent membrane-bound enzyme 3β-HSD, was first described in 1951 by Samuels et al. (17). It is located in the endoplasmic reticulum (ER) and mitochondria (18–23), and it catalyzes the sequential 3β-hydroxysteroid dehydrogenation and Δ5 to Δ4-isomerization of the Δ5-steroid precursors PREG, 17α-hydroxypregnenolone (17OH-PREG), DHEA, and androst-5-ene-3β,17β-diol (Δ5-DIOL) into their respective Δ4-ketosteroids, namely progesterone (PROG), 17α-hydroxyprogesterone (17OH-PROG), Δ4-DIONE, and T. Therefore, this bifunctional dimeric enzyme is required for the biosynthesis of all classes of steroid hormones, namely glucocorticoids, mineralocorticoids, PROG, androgens, and estrogens (Fig. 1). In addition, enzymes of the 3β-HSD family also catalyze the formation and/or degradation of 5α-androstanes and 5α-pregnanes, such as dihydrotestosterone (DHT) and dihydroprogesterone (DHP) (8–10). The 3β-HSD isoenzyme therefore controls critical steroidogenic reactions in the adrenal cortex, gonads, placenta, and a variety of peripheral target tissues (24).

Transient expression of human 3β-HSD isoenzymes provided the first direct evidence that the 3β-HSD and Δ54-isomerase activities reside within a single protein (25–27). However, data obtained from affinity alkylation (28) and inhibition experiments (29) that suggested separate 3β-HSD and isomerase sites are also consistent with a bifunctional catalytic site adopting a different conformation for each activity, as suggested by tryptic peptides associated with both catalytic activities localized using affinity radiolabeled steroids (30–32). Additional studies have supported the hypothesis that reduced NAD (NADH), the coenzyme product of the rate-limiting 3β-HSD reaction, induces a conformational change around the bound 3-oxo-Δ5-steroid (the 3β-HSD product and the isomerase substrate) to activate the isomerase step (33). Finally, as revealed by site-directed mutagenesis of the human type I (placental) enzyme, His261 appears to be a critical amino acid residue for 3β-HSD activity, whereas Tyr253 or Tyr254 participate in the isomerase activity (23).

B. Subcellular localization

Many of the enzymes of the steroidogenic pathway are localized to the smooth ER with the notable exceptions of P450scc (P450 cholesterol side-chain cleavage; CYP11A1), P450c11 (CYP11B1), and aldosterone synthase (CYP11B2). 3β-HSD subcellular localization patterns are unique in that they show various degrees of ER and mitochondrial distribution. The relevance of dual localization is unclear, yet it can be hypothesized that substrate accessibility could be limited with higher degrees of mitochondrial expression due to reduced mitochondrial transport. This would be analogous to the inability of high-efficiency catalysis of cholesterol by P450scc in the absence of the protein controlling cholesterol shuttling, steroidogenic acute regulatory protein (StAR) (34). Smooth ER localized 3β-HSD presumably would have a greater access to cytosolic steroid precursors, such as DHEA and Δ5-DIOL.

3β-HSD activity was detected using histochemical techniques as early as 1965 (35, 36). Similar techniques isolated this activity to the smooth ER and mitochondrial cristae (37). Its membrane localization was not known until studies localized 3β-HSD to the microsomal fraction of human adrenal (38) and chorion/amnion fetal membranes (39), suggesting that 3β-HSD is a membrane-associated enzyme. With the development of antibodies against 3β-HSD, the resolution of its localization increased, and it was verified that it is associated with the ER and mitochondria in human placenta (18, 40, 41), bovine adrenocortical cells (20), and rat adrenal tissue (21).

Submitochondrial fractionation studies show that bovine adrenal 3β-HSD is associated with the inner membrane and with a particulate fraction characterized by contact sites between the two membranes. 3β-HSD activity was higher in this fraction than in the inner mitochondrial membrane, suggesting that intermembrane contact sites may facilitate both the access of cholesterol to the inner membrane where P450scc is localized and the rapid conversion of PREG to PROG by 3β-HSD (19). Elegant biochemical studies have confirmed that a significant amount of adrenocortical 3β-HSD is present in the inner mitochondrial membrane (42). Coprecipitation studies have shown that 3β-HSD is in a functional steroidogenic complex with P450scc in the inner mitochondrial membrane (43), which provides the enzyme with immediate substrate metabolized from cholesterol transported across the mitochondrial membrane. Other work has shown that subcellular distribution in bovine and murine adrenal tissues demonstrated a higher degree of microsomal to mitochondrial localization (44, 45). A similar subcellular distribution was also recently reported in rat ovary, as revealed by immunoelectron microscopic localization, whereas in the testis, the 3β-HSD was restricted to the mitochondria (45).

Although the functional significance of differential 3β-HSD subcellular localization is unknown, studies have been performed to determine whether the dynamics of 3β-HSD subcellular localization can be altered by regulation. Because Ca2+ flux mediates K+ and A-II increases in aldosterone production by zona glomerulosa (ZG) (46), it is possible that Ca2+ could affect the mitochondrial to ER ratio of 3β-HSD. However, bovine ZG cells showed that neither Ca2+ nor A-II had any effect on the subcellular distribution of 3β-HSD and P450scc, but it did affect StAR localization (22). Another report showed that microsomal 3β-HSD activity in the ovary was unchanged during mouse estrous, yet mitochondrial 3β-HSD activity increased and doubled during diestrous in the mouse (47). These results suggest that 3β-HSD activity could be preferentially distributed to the mitochondria under certain physiological conditions, but this may not be a general phenomenon.

II. Human Type I and II 3β-HSD Genes and Pseudogenes

During the past decade, the structure of the isoenzymes of the 3β-HSD family has been characterized in the human and several other vertebrate species (Fig. 2). Human type I 3β-HSD cDNA was isolated and characterized by Luu-The et al. (18, 48, 49) after purification of the 3β-HSD enzyme from human placenta, and this sequence was later confirmed by other workers (25, 50). The second human 3β-HSD isoenzyme, chronologically designated as type II, was isolated from a human adrenal cDNA library (27). The type I 3β-HSD gene (HSD3B1) encodes an enzyme of 372 amino acids predominantly expressed in the placenta and peripheral tissues, such as the skin (principally in sebaceous glands), mammary gland, prostate, and several other normal and tumor tissues (27, 51–54). The purified enzyme has a Michaelis constant (Km) of 3.7 μm and maximal velocity (Vmax) of 43 nmol/min·mg for 3β-HSD substrate (DHEA) and a Km of 28 μm and Vmax of 598 nmol/min·mg for the isomerase substrate (5-androstene-3,17-dione) (54). In comparison, the type II gene (HSD3B2), which encodes a protein of 371 amino acids, shares 93.5% identity with the type I and is almost exclusively expressed in the adrenals, the ovary, and testis (27, 54, 55). The purified enzyme has a Km of 47 μm and Vmax of 82 nmol/min·mg for 3β-HSD substrate (DHEA) and a Km of 88 μm and Vmax of 970 nmol/min·mg for the isomerase substrate (5-androstene-3,17-dione). The higher affinity of type I 3β-HSD could facilitate steroid formation from relatively low concentrations of substrates usually present in peripheral tissues. Based on their differential tissue-specific expression pattern, it is not surprising that classical 3β-HSD deficiency, which will be discussed further in Section VII, results from mutations in the HSD3B2 gene, whereas the HSD3B1 gene is normal in affected individuals (56–59).

Fig. 2.

Comparison of the amino acid sequences of members of the 3β-HSD gene family: human types I and II; macaque; bovine; rat types I, II, III, and IV; mouse types I, II, III, IV, V, and VI; hamster types I, II, and III; horse; pig; chicken; rainbow trout; catfish; and eel types I and II. Residues common to the human type I 3β-HSD are represented by a dot. The members of the mammalian 3β-HSD family have been chronologically designated according to their order of elucidation in each species. The numbers indicated above refer to the human type II sequence. The missense mutations associated with 3β-HSD deficiency are shown by an arrow indicating their position in the human type II sequence. [Adapted from Ref.576 .]

Fig. 2.

Comparison of the amino acid sequences of members of the 3β-HSD gene family: human types I and II; macaque; bovine; rat types I, II, III, and IV; mouse types I, II, III, IV, V, and VI; hamster types I, II, and III; horse; pig; chicken; rainbow trout; catfish; and eel types I and II. Residues common to the human type I 3β-HSD are represented by a dot. The members of the mammalian 3β-HSD family have been chronologically designated according to their order of elucidation in each species. The numbers indicated above refer to the human type II sequence. The missense mutations associated with 3β-HSD deficiency are shown by an arrow indicating their position in the human type II sequence. [Adapted from Ref.576 .]

The structure of each of the HSD3B1 and HSD3B2 genes consists of four exons which are included within a DNA fragment of 7.8 kb and which share 77.4, 91.8, 94.5, and 91.0% identity, respectively (26, 60, 61). The genes are assigned to chromosome 1p13.1, 1–2 cM from the centromeric marker D1Z5 (Fig. 3) (62, 63). Our initial data suggested that the HSD3B1 and HSD3B2 genes and three related pseudogenes (64) are included within a 0.29 megabase SacII DNA fragment, suggesting that the human 3β-HSD gene family exists as a tandem cluster of related genes (63) as observed for the mouse β-HSD genes (65). In support of these findings, in addition to the two expressed genes in the human, five pseudogenes have also been recently cloned and physically mapped (66) (Fig. 3). HSD3Bψ1–5 are unprocessed pseudogenes that are closely related to HSD3B1 and HSD3B2 genes, but contain no corresponding open reading frames. Although mRNA is expressed from ψ4 and ψ5 in several tissues, altered splice sites disrupt the reading frames. The two expressed genes, HSD3B1 and HSD3B2, are located in direct repeat, 100 kb apart; however, separation by two pseudogenes, ψ1 and ψ2, prevents them from sharing common promoter elements (66).

Fig. 3.

Chromosomal localization showing the two expressed genes HSD3B1 and HSD3B2, and five pseudogenes, Ψ1–5. The orientation of four genes is shown by the arrow that points toward the stop codon or its homolog. Clones of yeast artificial chromosomes (alphanumeric identification) are shown as a contig. The information regarding the order of the markers was obtained from the Whitehead Institute/MIT Center for Genome Research, Cambridge, Massachusetts (http://www-genome.wi.mit.edu/). Structure of human type I and type II 3β-HSD genes, mRNA species, and the corresponding proteins. Exons are represented by boxes in which hatched lines demarcate the coding regions, whereas open boxes represent the noncoding regions. Introns are represented by black bold lines. [Adapted from Ref.576 .]

Fig. 3.

Chromosomal localization showing the two expressed genes HSD3B1 and HSD3B2, and five pseudogenes, Ψ1–5. The orientation of four genes is shown by the arrow that points toward the stop codon or its homolog. Clones of yeast artificial chromosomes (alphanumeric identification) are shown as a contig. The information regarding the order of the markers was obtained from the Whitehead Institute/MIT Center for Genome Research, Cambridge, Massachusetts (http://www-genome.wi.mit.edu/). Structure of human type I and type II 3β-HSD genes, mRNA species, and the corresponding proteins. Exons are represented by boxes in which hatched lines demarcate the coding regions, whereas open boxes represent the noncoding regions. Introns are represented by black bold lines. [Adapted from Ref.576 .]

III. Structure-Function Relationships

The two-step reaction of the 3β-HSD/isomerase involves the reduction of NAD+ to NADH by the rate-limiting 3β-HSD activity and the requirement of this NADH for the activation of the isomerase on the same enzyme (41, 67). Stopped-flow spectroscopy studies show that NADH activates the isomerase activity by inducing a time-dependant conformational change in the enzyme, suggesting that the 3β-HSD and isomerase domains of the enzyme are linked by a shared coenzyme domain that functions both as the binding site for NAD+ during the 3β-HSD reaction and as the coenzyme domain for the allosteric activation of the isomerase reaction (33).

The 3β-HSD isoenzymes belong to the short-chain alcohol dehydrogenase superfamily, mainly determined by the nucleotide-binding site sequence located at the amino terminus. It consists of a β-strand, α-helix, β-strand in a fold that provides a hydrophobic pocket for the AMP part of the nucleotide factor. The turn between the first β-strand and the α-helix is a glycine-rich segment, Gly-X-X-Gly-X-X-Gly, similar to the common Rossmann fold sequence Gly-X-Gly-X-X-Gly conserved among most NAD(H)-binding enzymes (68). This well-conserved glycine-rich fragment forms a hydrophobic pocket that allows close association of the AMP part of the cofactor. A preliminary study of rat type III enzyme has targeted Asp (36) as the amino acid that may be responsible for the strict NAD+ specificity of the enzyme (8). More recent mutagenesis studies in human type I enzyme demonstrate that the D36A/K37R mutant shifts cofactor preferences of both 3β-HSD and isomerase activities from NAD(H) to NADP(H), thus showing that the two activities utilize a common coenzyme domain (69).

Affinity labeling of purified human type I identified two tryptic peptides, comprising amino acids Asn176 to Arg186 and Gly251 to Lys274 that contain residues involved in the putative substrate-binding domain (30). These studies have shown that the Gly251 to Lys274 peptide was associated with the site of isomerase activity, whereas Tyr253 appears to function as the general proton donor in the isomerase reaction (24). His261 also appears to be a critical residue for the 3β-HSD activity (23). Additional kinetic analyses of D257L and D258L mutants suggest that this region is part of the isomerase substrate domain (69).

In contrast to other short chain dehydrogenases with a single catalytic Y-X-X-X-K motif (5, 70, 71), there are two potential catalytic motifs (Y154-X-X-X-K158 and Y269-X-X-X-K273) in the primary structure of all 3β-HSDs. Human type I and type II only differ at position 156 in this motif, type I having a tyrosine whereas type II has a histidine residue. The H156Y mutant form of the type I enzyme shifts the substrate kinetics for DHEA and PREG to the same Km and Vmax values exhibited by the type II enzyme; thus, H156 in the type I vs. Y156 in type II 3β-HSD accounts for the substantially higher affinity of the type I 3β-HSD activity for these substrates and inhibitor epostane relative to the type II enzyme (72).

Two membrane-binding domains lying between residues 72 and 89 in the NH2-terminal region and between residues 283 and 310 in the COOH-terminal region were identified. Indeed, deletion of the 283–310 region causes the enzyme to localize in the cytosol without affecting its activities (73). The region is therefore a critical membrane domain of 3β-HSD that can be deleted without compromising enzyme function (54, 73). Deletion of residues 72–89 in the NH2-terminal region produces a mutant protein that is distributed among the microsomes, mitochondria, and cytosol (73). Because 28% of the 3β-HSD and isomerase activities remain in the membranes of microsomes and mitochondria, the presence of the 283–310 domain in this mutant allows the protein to retain significant hydrophobicity. However, a majority (72%) of the protein is shifted into the cytosol, so the 72–89 region does contribute to membrane association. The 8-fold loss of both 3β-HSD and isomerase activity that results from the 72–89 deletion underscores the importance of this region to enzyme function (73). The data obtained by Thomas’ group (73) with the human type I enzyme are consistent with one of our previous studies in which the increased polarity of the domain between residues 75 and 91 in the rat type II 3β-HSD/isomerase was responsible for its having much lower activity than the rat type I enzyme (74). Thus, the presence of this highly conserved hydrophobic domain may be crucial to activity in the entire 3β-HSD gene family. The expression of an active soluble 283–310 deletion mutant of the type I enzyme in a baculovirus expression system provides a valuable tool for crystallographic studies that may ultimately determine the tertiary/quaternary structure of the enzyme (73). A three-dimensional ribbon model has been constructed by Thomas’ group (69), based on the homology data for human type I 3β-HSD and UDP-galactose-4-epimerase. This also represents a useful tool for interpreting biochemical data and designing inhibitors (Fig. 4) (69).

Fig. 4.

Ribbon structure of human type I 3β-HSD/isomerase based on homology modeling with key amino acids identified. The primary sequences of 3β-HSD/isomerase (green) and UDP-galactose-4-epimerase (yellow) were aligned using ClustalX. The NAD and DHEA structures are included. The key Asp36 residue is shown hydrogen binding (gray dotted lines) to the 2′, 3′-hydroxyl groups of the adenosyl ribose group of NAD. The catalytic Tyr154 and Lys158 residues for human type I 3β-HSD activity, the catalytic Tyr253 and Asp257 residues for isomerase activity, and the Asp241 residue that bridges the upper isomerase domain with the lower coenzyme domain are also shown. This ribbon model represents the 3β-HSD/isomerase structure in the 3β-HSD conformation. The oxygen atoms are red, nitrogen atoms are blue, carbon atoms are gray, and phosphorus atoms are pink. [Reproduced from J.L. Thomas et al.: J Biol Chem 278:35483–35490, 2003 (69 ), copyright 2003, with permission from The American Society for Biochemistry and Molecular Biology.]

Fig. 4.

Ribbon structure of human type I 3β-HSD/isomerase based on homology modeling with key amino acids identified. The primary sequences of 3β-HSD/isomerase (green) and UDP-galactose-4-epimerase (yellow) were aligned using ClustalX. The NAD and DHEA structures are included. The key Asp36 residue is shown hydrogen binding (gray dotted lines) to the 2′, 3′-hydroxyl groups of the adenosyl ribose group of NAD. The catalytic Tyr154 and Lys158 residues for human type I 3β-HSD activity, the catalytic Tyr253 and Asp257 residues for isomerase activity, and the Asp241 residue that bridges the upper isomerase domain with the lower coenzyme domain are also shown. This ribbon model represents the 3β-HSD/isomerase structure in the 3β-HSD conformation. The oxygen atoms are red, nitrogen atoms are blue, carbon atoms are gray, and phosphorus atoms are pink. [Reproduced from J.L. Thomas et al.: J Biol Chem 278:35483–35490, 2003 (69 ), copyright 2003, with permission from The American Society for Biochemistry and Molecular Biology.]

IV. Evolution of the 3β-HSD Gene Family

Multiple 3β-HSD isoenzymes have been cloned from several other species, further illustrating that the 3β-HSD gene family is conserved in vertebrate species (Fig. 2 and Table 1). The tissue-specific expression of multiple members of the 3β-HSD family was first demonstrated in the rat (75). Other 3β-HSD cDNAs have been cloned using adrenal/gonadal cDNA libraries from six other species, namely the macaque ovary (76), bovine ovary (77), chicken adrenal (78), horse testis (79), rainbow trout ovary (80), and eel ovary (81). It is important to note that in contrast to the human, which is designated as type II, the adrenal/gonadal 3β-HSD isoenzymes in all other vertebrate species have been designated as type I, due to the chronological order in which they were cloned. The only 3β-HSD sequence available from the pig was obtained using a cDNA library from adipose tissue (616).

TABLE 1.

Kinetic parameters and major expression sites of 3β-HSDs from human, rat, mouse, hamster, macaque, bovine, and rainbow trout

Species . Type . Km PREG/DHEA (μm) . Cofactor . Major sites of expression . First cloning (Ref. no.) . 
Human <1 NAD+Placenta, skin and mammary gland 18
II 1 to 4 NAD+Adrenals and gonads 27
Rat <1 NAD+Adrenals and gonads 82
II >10 NAD+Adrenals and gonads 82
III 3-KSR NADPH Male liver 75
IV <1 NAD+Placenta and skin 83
Mouse <1 NAD+Adrenals and gonads 87
II nd nd Kidney and liver 87
III <1 NAD+Liver > kidney 87
IV 3-KSR NADPH Kidney 88
3-KSR NADPH Male liver 91
VI <1 NAD+Placenta and skin 593
Hamster 2 to 5.5 NAD+Adrenals and gonads 96
II 2 to 9 NAD+Kidney and liver 96
III 3-KSR NADPH Male liver 96
Macaque nd nd Adrenals and gonads 76
Bovine >10 NAD+Adrenals and gonads 77
Rainbow trout DHEA > PREG NAD+Ovary 80
Species . Type . Km PREG/DHEA (μm) . Cofactor . Major sites of expression . First cloning (Ref. no.) . 
Human <1 NAD+Placenta, skin and mammary gland 18
II 1 to 4 NAD+Adrenals and gonads 27
Rat <1 NAD+Adrenals and gonads 82
II >10 NAD+Adrenals and gonads 82
III 3-KSR NADPH Male liver 75
IV <1 NAD+Placenta and skin 83
Mouse <1 NAD+Adrenals and gonads 87
II nd nd Kidney and liver 87
III <1 NAD+Liver > kidney 87
IV 3-KSR NADPH Kidney 88
3-KSR NADPH Male liver 91
VI <1 NAD+Placenta and skin 593
Hamster 2 to 5.5 NAD+Adrenals and gonads 96
II 2 to 9 NAD+Kidney and liver 96
III 3-KSR NADPH Male liver 96
Macaque nd nd Adrenals and gonads 76
Bovine >10 NAD+Adrenals and gonads 77
Rainbow trout DHEA > PREG NAD+Ovary 80

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TABLE 1.

Kinetic parameters and major expression sites of 3β-HSDs from human, rat, mouse, hamster, macaque, bovine, and rainbow trout

Species . Type . Km PREG/DHEA (μm) . Cofactor . Major sites of expression . First cloning (Ref. no.) . 
Human <1 NAD+Placenta, skin and mammary gland 18
II 1 to 4 NAD+Adrenals and gonads 27
Rat <1 NAD+Adrenals and gonads 82
II >10 NAD+Adrenals and gonads 82
III 3-KSR NADPH Male liver 75
IV <1 NAD+Placenta and skin 83
Mouse <1 NAD+Adrenals and gonads 87
II nd nd Kidney and liver 87
III <1 NAD+Liver > kidney 87
IV 3-KSR NADPH Kidney 88
3-KSR NADPH Male liver 91
VI <1 NAD+Placenta and skin 593
Hamster 2 to 5.5 NAD+Adrenals and gonads 96
II 2 to 9 NAD+Kidney and liver 96
III 3-KSR NADPH Male liver 96
Macaque nd nd Adrenals and gonads 76
Bovine >10 NAD+Adrenals and gonads 77
Rainbow trout DHEA > PREG NAD+Ovary 80
Species . Type . Km PREG/DHEA (μm) . Cofactor . Major sites of expression . First cloning (Ref. no.) . 
Human <1 NAD+Placenta, skin and mammary gland 18
II 1 to 4 NAD+Adrenals and gonads 27
Rat <1 NAD+Adrenals and gonads 82
II >10 NAD+Adrenals and gonads 82
III 3-KSR NADPH Male liver 75
IV <1 NAD+Placenta and skin 83
Mouse <1 NAD+Adrenals and gonads 87
II nd nd Kidney and liver 87
III <1 NAD+Liver > kidney 87
IV 3-KSR NADPH Kidney 88
3-KSR NADPH Male liver 91
VI <1 NAD+Placenta and skin 593
Hamster 2 to 5.5 NAD+Adrenals and gonads 96
II 2 to 9 NAD+Kidney and liver 96
III 3-KSR NADPH Male liver 96
Macaque nd nd Adrenals and gonads 76
Bovine >10 NAD+Adrenals and gonads 77
Rainbow trout DHEA > PREG NAD+Ovary 80

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A. The rat 3β-HSD gene family

The secretion of sex steroids originates exclusively from the gonads in rodents and domestic animals, which is in contrast to humans who in addition to secreting sex steroids from the gonads, secrete the sex precursors DHEA and DHEA-S from the adrenal gland. The structures of four members of the rat 3β-HSD family have been characterized (75, 82, 83). With the exception of type III, all isoenzymes catalyze the transformation of 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding Δ4-3-ketosteroids as well as the interconversion of 3β-hydroxy- and 3-keto-5α-androstane steroids. The various isoenzymes show differences in tissue-specific expression (84) (Fig. 5). The rat type I and II 3β-HSD proteins are expressed in the adrenals, gonads, kidney, placenta, adipose tissue, and uterus and share 93.8% identity. The type III protein shares 80% identity with the type I and II proteins but, in contrast to other types, is a specific 3-KSR. The type III gene is exclusively expressed in male liver, and there is marked sexual dimorphic expression, which results from pituitary hormone-induced gene repression in the female rat liver (75, 85). The rat type IV protein shares 90.9, 87.9, and 78.8% identity with that of types I, II, and III proteins, respectively, and is the prominent mRNA species detectable in the placenta and the skin (83). In this respect, it is therefore possible that the rat type IV and the human type I proteins have conserved cis-acting elements in their promoter regions, involved in tissue-specific transcriptional control common to skin and placenta (8). The activities of rat types I and IV are similar (83), whereas there is much lower enzyme activity for the type II compared with the type I, which could be due to a change in four amino acid residues located in a putative membrane-spanning domain, between residues 75 and 91 as described in the previous section (74). Furthermore, types I and IV possess a 17β-HSD activity specific to 5α-androstane-17β-ol steroids, thus suggesting a key role in controlling the bioavailibility of the active androgen DHT (84, 86).

Fig. 5.

Enzymatic 17β-HSD and 3β-HSD activities of rat types I and IV 3β-HSD expressed in intact cells. Reaction 1 corresponds to the androgenic 17β-HSD activity measured in cells expressing the rat type I and IV 3β-HSD isoenzymes. Reaction 2 corresponds to the 3β-HSD activity present in cells expressing rat type I or IV 3β-HSD enzymes. Reaction 3 corresponds to 3α-HSD present in some cells. Reaction 4 corresponds to the 3-KSR activity present in liver cells expressing the rat 3-KRS (type III) enzyme. The hatched arrows indicate predominant reactions expected to use primarily NAD+ as cofactor, whereas the black arrows indicate the predominant reactions expected to use primarily NADPH as cofactor. [Adapted from Ref.84 .]

Fig. 5.

Enzymatic 17β-HSD and 3β-HSD activities of rat types I and IV 3β-HSD expressed in intact cells. Reaction 1 corresponds to the androgenic 17β-HSD activity measured in cells expressing the rat type I and IV 3β-HSD isoenzymes. Reaction 2 corresponds to the 3β-HSD activity present in cells expressing rat type I or IV 3β-HSD enzymes. Reaction 3 corresponds to 3α-HSD present in some cells. Reaction 4 corresponds to the 3-KSR activity present in liver cells expressing the rat 3-KRS (type III) enzyme. The hatched arrows indicate predominant reactions expected to use primarily NAD+ as cofactor, whereas the black arrows indicate the predominant reactions expected to use primarily NADPH as cofactor. [Adapted from Ref.84 .]

B. The mouse 3β-HSD gene family

To date, six distinct cDNAs encoding murine members of the 3β-HSD family have been cloned (87–92), all of which are highly homologous and encode a protein of 372 amino acids. The murine family of 3β-HSD enzymes has been extensively reviewed in the literature (9, 93) and references therein. The genes encoding the different isoenzymes are found closely linked on mouse chromosome 3 (65). Hybridization by Southern blot analysis of restriction enzyme-digested yeast artificial chromosome DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Additional analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment (9, 94). The different forms are expressed in a tissue-specific and developmentally specific manner and fall into two functionally distinct classes of enzymes (92). 3β-HSD types I and III, and most probably type II, function as dehydrogenase/isomerases, and are essential for the biosynthesis of active steroid hormones, whereas 3β-HSD type IV and type V, analogous to rat type III, function as 3-KSRs and are therefore involved in the inactivation of active steroid hormones (88, 91). 3β-HSD I in the adult mouse is expressed in the gonads and the adrenal gland (87), whereas 3β-HSD II and III are expressed in the liver and kidney (87), with much greater expression of type III in the liver than in the kidney. The major site of expression of 3β-HSD IV is in the proximal tubules of the kidney in both the male and female mice (89), with minor expression in the testis (91). The type V isoenzyme appears to be expressed only in the liver of the male mouse, with expression starting during the latter half of pubertal development (91, 95). The type VI isoenzyme functions as a NAD+-dependent 3β-HSD and is the earliest isoform to be expressed during the first half of pregnancy in cells of embryonic origin and in uterine tissue (92). In the adult mouse, 3β-HSD type VI appears to be the only isoenzyme expressed in the skin and is also expressed in Leydig cells of the testis, although to a lesser extent than type I 3β-HSD.

It is hypothesized that mouse 3β-HSD type VI cDNA is orthologous to human 3β-HSD type I cDNA, which has been shown to be the only isoenzyme expressed in the placenta and the skin. The demonstration that the type VI isoenzyme in the mouse functions as a 3β-HSD and is the predominant isoenzyme expressed during the first half of pregnancy in uterine tissue and embryonic cells, suggests that this isoenzyme may be involved in the local production of PROG, which is required for the successful implantation of the blastocyst and/or maintenance of pregnancy (92).

C. The hamster 3β-HSD gene family

The hamster is a rodent species, but in contrast to the rat and mouse, in which the principal corticosteroid is corticosterone, the principal corticosteroid in the hamster is cortisol. A study on the regulation of adrenal steroidogenic enzymes suggested that the hamster could be a good model for studying human steroidogenesis. With this in mind, three isoenzymes of 3β-HSD were characterized in the hamster (96, 97). The type I isoenzyme was isolated from an adrenal cDNA library and was identified as being a low Km 3β-HSD (Km: PREG, 5.5 μm; DHEA, 2.4 μm). A separate isozyme, designated type II was isolated from the kidney and was also found to be a low Km 3β-HSD (Km: PREG, 8.8 μm; DHEA, 2.9 μm). Two cDNAs were isolated from the liver, one which was identical to the type II sequence found in the kidney, and a distinct cDNA encoding an isoform designated as type III, which does not possess any steroid dehydrogenase activity but functions as a 3-KSR. There is sexual dimorphic expression of this liver-specific type III 3β-HSD in the hamster, as seen for the rat liver-specific type III KSR. As is the case for both the rat and mouse, a high affinity 3β-HSD is expressed in the adrenal and gonad of the hamster, consistent with the steroidogenic role of these tissues (96).

D. Phylogeny of the 3β-HSD gene family

McBride et al. (66) indicated no evidence for the presence of other members of the human 3β-HSD family within the physical contig of 0.5 Mb by Southern blot analysis, thus suggesting that in humans there is no comparable liver-specific 3-KSR sharing a high percentage of identity with other members of the HSD3B cluster. Such a conclusion is also well supported by phylogenetic analysis of the mammalian 3β-HSD gene family. Unexpectedly, the phylogenetic tree strongly suggests that independent gene duplications occurred in different species (66, 91), (V. Laudet, personal communication). As illustrated in Fig. 6, our recent analysis shows a first complex of three genes from primates and suggests that an ancestral gene duplicated specifically in the primate lineage to give rise to human types I and II, whereas the macaque gene is the homolog of human type II. It is very likely that an ortholog of the human type I exists in the macaque genome, but yet remains to be identified. The second complex clusters together the single 3β-HSD species characterized in bovine, pig, and horse. The third complex clusters together three clear classes of rodent 3β-HSD genes; firstly, the rat type I, II, and IV as well as the mouse type I, II, III, and VI; secondly, the mouse type IV and V and rat type III, the specific 3-KSRs; and thirdly the hamster type I, II, and III. Because the hamster type III is a liver-specific 3-KSR (97), it is surprising that it is not included in the second class of rodent genes. These findings strongly suggest that the 3β-HSD genes were independently duplicated or triplicated three times in the lineage of the rat, the mouse, and the hamster. It is difficult to understand why the duplication failed to occur earlier in mammalian evolution if there are physiological needs and/or advantages for the presence of multiple isoenzymes. These data may indicate that the need for different 3β-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages.

Fig. 6.

Unrooted phylogenetic tree constructed by the Neighbor-Joining method using 1000 bootstrap replicates. Multiple nucleotide alignments of 3β-HSDs from different species were obtained using PILEUP (Wisconsin GCG package), and the phylogenetic analysis was performed by PAUPsearch, which provides a GCG interface to the tree-searching options in the PAUP program, version 4.0.0d55 (Phylogenetic Analysis Using Parsimony) (590 ). [Adapted from Ref.576 .]

Fig. 6.

Unrooted phylogenetic tree constructed by the Neighbor-Joining method using 1000 bootstrap replicates. Multiple nucleotide alignments of 3β-HSDs from different species were obtained using PILEUP (Wisconsin GCG package), and the phylogenetic analysis was performed by PAUPsearch, which provides a GCG interface to the tree-searching options in the PAUP program, version 4.0.0d55 (Phylogenetic Analysis Using Parsimony) (590 ). [Adapted from Ref.576 .]

It is also of interest to note that although the N-terminal amino acid sequences of the pig hepatic 3β-hydroxy-Δ5-C27-steroid dehydrogenase and the vertebrate 3β-HSD enzymes show some similarities, substrate specificities differ. Although vertebrate 3β-HSD/Δ54 isomerase enzymes are active on C19/C21 steroids, porcine hepatic 3β-hydroxy-Δ5-C27-steroid dehydrogenase is active on C27 steroids such as 7α-hydroxycholesterol, 7α-25-dihydroxycholesterol, 7α-27-dihydrocholesterol, and 3β-7α-dihydroxy-5-cholestenoic acid, and participates in bile acid biosynthesis (98, 99) (Fig. 7). Furthermore, genetic studies of a kindred affected with 3β-hydroxy-Δ5-C27-steroid dehydrogenase deficiency, which is associated with hepatic failure in childhood, showed no genetic linkage to the HSD3B cluster (100). In fact, such hepatic and extrahepatic activity was practically unaffected by trilostane, a well-known C19/C21 3β-HSD inhibitor (99). Gene structure of HSD3B7, as well as positioning of disease-associated mutations on corresponding nucleic and amino acid sequences, is represented in Fig. 8. Moreover, it has recently been suggested that the alcohol dehydrogenase γγ isoenzyme is the sole 3β-HSD using bile acids as a substrate in human liver cytosol (101). Also, it has been demonstrated that the X-linked dominant male-lethal phenotype gene mutated in bare patches and striated mice encodes a novel 3β-HSD (102). This gene encodes an NADPH enzyme, which is likely to be involved in cholesterol biosynthesis and shares only 30% identity with other mammalian 3β-HSD enzymes, thus supporting the phylogenetic divergence between the C19/C21 3β-HSD/Δ54 isomerase and the other enzymes involved in bile acid metabolism and/or biosynthesis of cholesterol.

Fig. 7.

Major bile acid biosynthesis pathways. Two major bile acid biosynthesis pathways are shown. Only major enzymes and intermediates are shown.

Fig. 7.

Major bile acid biosynthesis pathways. Two major bile acid biosynthesis pathways are shown. Only major enzymes and intermediates are shown.

Fig. 8.

Top, Schematic representation of HSD3B7 gene, mRNA, and corresponding protein. Exons are represented by hatched boxes indicating coding region, whereas open boxes represent noncoding regions. Asterisk represents an alternative noncoding exon. Introns are represented by black bold lines. Mutations causing progressive intrahepatic cholestasis are identified on the gene. The nucleotide numbers indicating the positions of individual mutations refer to C27 3β-HSD cDNA (GenBank accession no. AF277719). Bottom, Alignment of amino acid sequences of human type I and type VII 3β-HSD. Residues common to both types are identified by black areas, whereas similar residues are identified by gray areas. Positions of mutations causing progressive intrahepatic cholestasis are identified by an arrow in reference to amino acid sequences on the primary structure. [Adapted from Ref.591 .]

Fig. 8.

Top, Schematic representation of HSD3B7 gene, mRNA, and corresponding protein. Exons are represented by hatched boxes indicating coding region, whereas open boxes represent noncoding regions. Asterisk represents an alternative noncoding exon. Introns are represented by black bold lines. Mutations causing progressive intrahepatic cholestasis are identified on the gene. The nucleotide numbers indicating the positions of individual mutations refer to C27 3β-HSD cDNA (GenBank accession no. AF277719). Bottom, Alignment of amino acid sequences of human type I and type VII 3β-HSD. Residues common to both types are identified by black areas, whereas similar residues are identified by gray areas. Positions of mutations causing progressive intrahepatic cholestasis are identified by an arrow in reference to amino acid sequences on the primary structure. [Adapted from Ref.591 .]

E. Enzymatic characteristics of the 3-KSRs (rat liver-specific type III, mouse types IV and V, and hamster type III)

As mentioned briefly above, the rat type III protein (75) does not display oxidative activity for the classical substrates PREG, DHEA, Δ5-DIOL, and 3β-DIOL, but instead is a specific 3-KSR responsible for the conversion of 3-keto saturated steroids, such as DHT and DHP, into inactive steroids using NADH phosphate (NADPH) as cofactor instead of NADH (86). In addition to using 5α-androstane steroids such as DHT and androstanedione as substrates, the expressed rat 3-KSR also catalyzes the 3β-reduction of DHP into 5α-pregnane-3β,20β-diol (84). The Km and Vmax values of the expressed 3-KSR protein using DHP as substrate and NADPH as cofactor were calculated to be 0.24 μm and 0.83 nmol/min·mg protein, respectively. In comparison, the Km value of the expressed type I 3β-HSD isoenzyme, also using DHP as the substrate in the presence of NADH as cofactor, was 0.55 μm, whereas the calculated Vmax value was 0.18 nmol/min·mg protein (84).

Examination of the 3β-HSD isoenzymes shows a typical βαβ dinucleotide-binding fold with Asp (36) located in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2′-hydroxyl moiety of the adenosine ribose of all known NAD-dependent dehydrogenases (Fig. 2). Using site-directed mutagenesis, it has been shown that the presence of a Tyr residue instead of an Asp residue at position 36 in the typical βαβ dinucleotide-binding fold of the cofactor binding domain of rat type III is responsible for the difference in cofactor specificity of the rat 3-KSR (type III) protein, but this alteration is not sufficient to explain the low activity of the enzyme with Δ5-3β-hydroxysteroid substrates (103). The physiological importance of this peculiar member of the rat 3β-HSD family is well supported by the finding that mouse types IV and V and hamster type III also possess this specific 3-KSR activity (88, 91,

Sours: https://academic.oup.com/edrv/article/26/4/525/2355189

Enzymes hsd

3-beta-hydroxysteroid dehydrogenase deficiency

Description

3-beta (β)-hydroxysteroid dehydrogenase (HSD) deficiency is an inherited disorder that affects hormone-producing glands including the gonads (ovaries in females and testes in males) and the adrenal glands. The gonads direct sexual development before birth and during puberty. The adrenal glands, which are located on top of the kidneys, regulate the production of certain hormones and control salt levels in the body. People with 3β-HSD deficiency lack many of the hormones that are made in these glands. 3β-HSD deficiency is one of a group of disorders known as congenital adrenal hyperplasias that impair hormone production and disrupt sexual development and maturation.

There are three types of 3β-HSD deficiency: the salt-wasting, non-salt-wasting, and non-classic types. In the salt-wasting type, hormone production is extremely low. Individuals with this type lose large amounts of sodium in their urine, which can be life-threatening. Individuals affected with the salt-wasting type are usually diagnosed soon after birth due to complications related to a lack of salt reabsorption, including dehydration, poor feeding, and vomiting. People with the non-salt-wasting type of 3β-HSD deficiency produce enough hormone to allow sodium reabsorption in the kidneys. Individuals with the non-classic type have the mildest symptoms and do not experience salt wasting.

In males with any type of 3β-HSD deficiency, problems with male sex hormones lead to abnormalities of the external genitalia. These abnormalities range from having the opening of the urethra on the underside of the penis (hypospadias) to having external genitalia that do not look clearly male or female (ambiguous genitalia). The severity of the genital abnormality does not consistently depend on the type of the condition. Because of the hormone dysfunction in the testes, males with 3β-HSD deficiency are frequently unable to have biological children (infertile).

Females with 3β-HSD deficiency may have slight abnormalities of the external genitalia at birth. Females affected with the non-salt-wasting or non-classic types are typically not diagnosed until mid-childhood or puberty, when they may experience irregular menstruation, premature pubic hair growth, and excessive body hair growth (hirsutism). Females with 3β-HSD deficiency have difficulty conceiving a child (impaired fertility).

Frequency

The exact prevalence of 3β-HSD deficiency is unknown. At least 60 affected individuals have been reported.

Causes

Mutations in the HSD3B2 gene cause 3β-HSD deficiency. The HSD3B2 gene provides instructions for making the 3β-HSD enzyme. This enzyme is found in the gonads and adrenal glands. The 3β-HSD enzyme is involved in the production of many hormones, including cortisol, aldosterone, androgens, and estrogen. Cortisol has numerous functions such as maintaining energy and blood sugar levels, protecting the body from stress, and suppressing inflammation. Aldosterone is sometimes called the salt-retaining hormone because it regulates the amount of salt retained by the kidney. The retention of salt affects fluid levels and blood pressure. Androgens and estrogen are essential for normal sexual development and reproduction.

3β-HSD deficiency is caused by a deficiency (shortage) of the 3β-HSD enzyme. The amount of functional 3β-HSD enzyme determines whether a person will have the salt-wasting or non-salt-wasting type of the disorder. Individuals with the salt-wasting type have HSD3B2 gene mutations that result in the production of very little or no enzyme. People with the non-salt-wasting type of this condition have HSD3B2 gene mutations that allow the production of some functional enzyme, although in reduced amounts.

Inheritance

This condition is inherited in an autosomal recessive pattern, which means both copies of the gene in each cell have mutations. The parents of an individual with an autosomal recessive condition each carry one copy of the mutated gene, but they typically do not show signs and symptoms of the condition.

Other Names for This Condition

  • 3 beta-HSD deficiency
  • 3 beta-ol dehydrogenase deficiency
  • 3-beta–hydroxysteroid dehydrogenase deficiency
  • 3b-hydroxysteroid dehydrogenase deficiency
  • 3β-HSD deficiency
  • 3β-HSD deficiency congenital adrenal hyperplasia
  • 3β-hydroxysteroid dehydrogenase deficiency
  • Type II 3β-hydroxysteroid dehydrogenase deficiency

Additional Information & Resources

Genetic Testing Information

Genetic and Rare Diseases Information Center

Patient Support and Advocacy Resources

Research Studies from ClinicalTrials.gov

Catalog of Genes and Diseases from OMIM

Scientific Articles on PubMed

References

  • Lutfallah C, Wang W, Mason JI, Chang YT, Haider A, Rich B, Castro-Magana M, Copeland KC, David R, Pang S. Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency. J Clin Endocrinol Metab. 2002 Jun;87(6):2611-22. Citation on PubMed
  • Pan Y, Zhong S, Hu RM, Gong W. Mutation of 3β-hydroxysteroid dehydrogenase (3β-HSD) at the 3'-untranslated region is associated with adrenocortical insufficiency. Mol Med Rep. 2012 Dec;6(6):1305-8. doi: 10.3892/mmr.2012.1107. Epub 2012 Sep 28. Citation on PubMed
  • Pang S, Carbunaru G, Haider A, Copeland KC, Chang YT, Lutfallah C, Mason JI. Carriers for type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) deficiency can only be identified by HSD3B2 genotype study and not by hormone test. Clin Endocrinol (Oxf). 2003 Mar;58(3):323-31. Citation on PubMed
  • Pang S, Wang W, Rich B, David R, Chang YT, Carbunaru G, Myers SE, Howie AF, Smillie KJ, Mason JI. A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2002 Jun;87(6):2556-63. Citation on PubMed
  • Pang S. Congenital adrenal hyperplasia owing to 3 beta-hydroxysteroid dehydrogenase deficiency. Endocrinol Metab Clin North Am. 2001 Mar;30(1):81-99, vi-vii. Review. Citation on PubMed
  • Simard J, Moisan AM, Morel Y. Congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase deficiency. Semin Reprod Med. 2002 Aug;20(3):255-76. Review. Citation on PubMed
  • Takasawa K, Ono M, Hijikata A, Matsubara Y, Katsumata N, Takagi M, Morio T, Ohara O, Kashimada K, Mizutani S. Two novel HSD3B2 missense mutations with diverse residual enzymatic activities for Δ5-steroids. Clin Endocrinol (Oxf). 2014 Jun;80(6):782-9. doi: 10.1111/cen.12394. Epub 2014 Jan 16. Citation on PubMed
  • Welzel M, Wüstemann N, Simic-Schleicher G, Dörr HG, Schulze E, Shaikh G, Clayton P, Grötzinger J, Holterhus PM, Riepe FG. Carboxyl-terminal mutations in 3beta-hydroxysteroid dehydrogenase type II cause severe salt-wasting congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2008 Apr;93(4):1418-25. doi: 10.1210/jc.2007-1874. Epub 2008 Feb 5. Citation on PubMed
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How Do I Lower DHEA Via 3 Beta HSD

11β-Hydroxysteroid Dehydrogenase and Its Role in the Syndrome of Apparent Mineralocorticoid Excess

Abstract

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal tubules and cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine. Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may result in hypokalemia, suppressed plasma renin activity, and hypertension. The syndrome of apparent mineralocorticoid excess(AME) is an inherited form of hypertension in which 11β-hydroxysteroid dehydrogenase (11β-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. Because mineralocorticoid receptors themselves have similar affinities for cortisol and aldosterone, it is hypothesized that the deficiency allows these receptors to be occupied by cortisol, which normally circulates at levels far higher than those of aldosterone. We cloned cDNA and genes encoding two isozymes of 11β-HSD. The liver (L) or type 1 isozyme has relatively low affinity for steroids, is expressed at high levels in the liver but poorly in the kidney, and is not defective in AME. The kidney (K) or type 2 isozyme has high steroid affinity and is expressed at high levels in the kidney and placenta. Mutations in the gene for the latter isozyme have been detected in all kindreds with AME. Moreover, the in vitro enzymatic activity conferred by each mutation is strongly correlated with the ratio of cortisol to cortisone metabolites in the urine [tetrahydrocortisone (THF) + allo-THF]/THE. This suggests that the biochemical phenotype of AME is largely determined by genotype.

Main

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal tubules and cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine (Fig. 1) (reviewed inRef. 1). Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may result in hypokalemia, suppressed plasma renin activity, and hypertension. Such conditions often have a genetic basis. Glucocorticoid-suppressible hyperaldosteronism and congenital adrenal hyperplasia due to 11β-hydroxylase deficiency are examples of autosomal dominant and recessive forms of mineralocorticoid excess, respectively(2). This article reviews studies of another such syndrome, AME, that provide valuable insights into normal and abnormal physiology of mineralocorticoid action.

Schematic of mineralocorticoid action. Top, a normal mineralocorticoid target cell in a renal cortical collecting duct. Aldosterone occupies nuclear receptors (MR) that bind to hormone response elements, increasing transcription of genes and directly or indirectly increasing activities of apical sodium (Na) channels and the basolateral sodium-potassium (Na/K) ATPase. This increases resorption of sodium from and excretion of potassium into the tubular lumen. Cortisol, which circulates at higher levels than aldosterone, cannot occupy the receptor because it is oxidized to cortisone by 11β-HSD.Bottom, a cell from a patient with the syndrome of apparent mineralocorticoid excess. Because 11β-HSD is absent, cortisol inappropriately occupies mineralocorticoid receptors, leading to increased gene transcription, increased activity of sodium channels and the Na/K ATPase, increased resorption of sodium and excretion of potassium, and hypertension.

Full size image

Clinical features of the syndrome of apparent mineralocorticoid excess. AME is an inherited syndrome in which children present with hypertension, hypokalemia, and low plasma renin activity. Other clinical features include moderate intrauterine growth retardation and postnatal failure to thrive. Consequences of the often severe hypokalemia include nephrocalcinosis, nephrogenic diabetes insipidus, and rhabdomyolysis. Complications of hypertension have included cerebrovascular accidents, and several patients have died during infancy or adolescence. Several affected sibling pairs have been reported, but parents have usually been asymptomatic, suggesting that AME is a genetic disorder with an autosomal recessive mode of inheritance.

A low salt diet or blockade of mineralocorticoid receptors with spironolactone ameliorate the hypertension, whereas ACTH and hydrocortisone exacerbate it. Levels of all known mineralocorticoids are low(3, 4). These findings suggest that cortisol(i.e., hydrocortisone) acts as a stronger mineralocorticoid than is normally the case. Indeed, patients with AME have abnormal cortisol metabolism. Cortisol half-life in plasma is prolonged from approximately 80 min to 120-190 min(3). Very low levels of cortisone metabolites are excreted in the urine as compared with cortisol metabolites, indicating a marked deficiency in 11β-HSD, the enzyme catalyzing the conversion of cortisol to cortisone (Fig. 2). This is most often measured as an increase in the sum of the urinary concentrations of THF and allo-THF, divided by the concentration of THE [(THF + allo-THF)/THE]. However, 11-reduction is unimpaired; labeled cortisone administered to patients is excreted entirely as cortisol and other 11β-reduced metabolites(5). There is also an increase in the ratio of 5α- to 5β-reduced cortisol metabolites, allo-THF/THF(6), suggesting that 5β reduction is also impaired. The (THF + allo-THF)/THE is usually much higher than the allo-THF/THF ratio, and the two ratios are linearly related, implying that the primary defect in this disorder is indeed one of 11β-dehydrogenation. This has been assayed directly by administering 11α-[3H]cortisol to subjects and measuring the appearance of tritiated water.

Positions at which enzymes metabolize cortisol.

Full size image

Similar but milder abnormalities occur with licorice intoxication(7). The active component of licorice, glycyrrhetinic acid, inhibits 11β-HSD in isolated rat kidney microsomes(8). Thus, it appears that licorice intoxication is a reversible pharmacologic counterpart to the inherited syndrome of apparent mineralocorticoid excess.

WHY DOES 11β-HSD DEFICIENCY CAUSE HYPERTENSION?

Aldosterone acts through transcriptional effects mediated by a specific nuclear receptor referred to as the mineralocorticoid or “type 1 steroid” receptor. These receptors are expressed at high levels in renal distal tubules and cortical collecting ducts but also in other mineralocorticoid target tissues, including salivary glands and the colon. The mineralocorticoid receptor has a high degree of sequence identity with the glucocorticoid or “type 2” receptor(9), and it has very similar in vitro binding affinities for aldosterone and for glucocorticoids such as corticosterone and cortisol(9, 10).

It has been proposed(7, 11, 12) that oxidation by 11β-HSD of cortisol or corticosterone to cortisone or 11-dehydrocorticosterone, respectively, represents the physiologic mechanism conferring specificity for aldosterone upon the mineralocorticoid receptor (Fig. 1). Although cortisol and corticosterone bind the receptor well in vitro, cortisone and 11-dehydrocorticosterone are poor agonists for this receptor. Aldosterone is a poor substrate for 11β-HSD, because, in solution, its 11-hydroxyl group is normally in a hemiacetal conformation with the 18-aldehyde group. Thus, in AME or licorice intoxication, 11β-HSD deficiency permits cortisol to occupy the mineralocorticoid receptor. Because cortisol normally circulates at levels 100-1000 times those of aldosterone, this leads to signs of mineralocorticoid excess even when aldosterone secretion is suppressed. To confirm this hypothesis, we cloned cDNA and genes for two isozymes of 11β-HSD and tested their involvement in AME.

CLONING OF cDNA-ENCODING ISOZYMES OF 11B-HSD

There are two distinct isozymes of 11β-HSD (Table 1). The first, termed the liver (L) or type 1 isozyme, was originally isolated from rat liver microsomes(13). It is a glycoprotein with a molecular mass of 34 kD that requires NADP+ as a cofactor. A cDNA clone encoding this enzyme was isolated using an antiserum to the purified rat protein(14). The fulllength cDNA is 1.4 kb long including an open reading frame of 876 bp, predicting a protein of 292 amino acids. Although the enzyme purified from rat liver functions only as a dehydrogenase, the recombinant enzyme expressed from cloned cDNA exhibits both 11β-dehydrogenase and the reverse oxoreductase activity (conversion of 11-dehydrocorticosterone to corticosterone) when expressed in mammalian cells(14). At physiologic pH in cell lysates, the kinetic constants for dehydrogenation and reduction (Km of 1.1 and 1.4 μM, respectively) are almost identical(15). These findings imply that this isozyme actually catalyzes a fully reversible reaction and that reductase activity is destroyed during purification from the liver. The corresponding human gene for this isozyme, HSD11L(HSD11B1) is located on chromosome 1, and contains 6 exons with a total length of over 9 kb(16)(Fig. 3). No mutations were identified in this gene in patients with AME(17).

Full size table

Genes encoding 11β-HSD isozymes. Numbered black boxes represent exons, and those exons that encode domains of functional importance are indicated. Highly conserved predicted amino acid sequences are shown in single-letter code; identical residues within these regions are shaded.

Full size image

Other lines of evidence also suggest that this isozyme does not play a significant role in conferring ligand specificity on the mineralocorticoid receptor. This isozyme is expressed at highest levels in the liver, which does not respond to mineralocorticoids, and although it is expressed at high levels in the rat kidney(14), it is expressed at much lower levels in human(16) and sheep(18) kidneys. Even in rat kidney, immunoreactivity to the protein is observed primarily in proximal tubules and not in distal tubules and collecting ducts, the sites of mineralocorticoid action(19).

Accordingly, a second isozyme was sought in mineralocorticoid target tissues. Evidence for such an isozyme was obtained from biochemical studies of isolated rabbit kidney cortical collecting duct cells(20). Activity of 11β-HSD in the microsomal fraction was almost exclusively NAD+ dependent and had a Km for corticosterone of 26 nM. There was almost no reduction of 11-dehydrocorticosterone to corticosterone, suggesting that, unlike the liver(L) isozyme, the kidney (K) or type 2 isozyme catalyzed only dehydrogenation. The enzyme in the human placenta had similar characteristics(21); it was NAD+-dependent and had Km values for corticosterone and cortisol of 14 and 55 nM. Partial purification using AMP affinity chromatography suggested that this isozyme had a molecular weight of 40,000. Thus far, the K isozyme has not been purified to homogeneity in active form from any source. However, a homogenous preparation was recently obtained by a combination of affinity chromatography, affinity labeling, and preparative two-dimensional electrophoresis(22).

Cloning of cDNA encoding the K isozyme of 11β-HSD was rendered more difficult by the unavailability of purified enzyme that could be used to produce an antiserum or to obtain amino acid sequence data. However, because sheep and human kidneys predominantly expressed this type of enzyme, it was feasible to clone the corresponding cDNA by expression screening strategies in which pools of clones were assayed for their ability to confer NAD+-dependent 11β-HSD activity on Xenopus oocytes or cultured mammalian cells. Positive pools were divided into smaller pools and rescreened until a single positive clone was identified. Both sheep(23) and human(24) cDNA encoding this isoform were isolated in this manner. The recombinant K isozyme has properties that are virtually identical to the activity found in mineralocorticoid target tissues. The recombinant enzyme functions exclusively as a dehydrogenase; no reductase activity is detectable with either NADH or NADPH as a cofactor(23, 24). It has an almost exclusive preference for NAD+ as a cofactor and a very high affinity for glucocorticoids. Corticosterone is the preferred substrate, with first order rate constants 10 times higher than those for cortisol, even in mammalian species in which cortisol is the predominant glucocorticoid. Reported Km values for corticosterone are 0.7-10.1 nM and for cortisol, 14-47 nM.

The protein is predicted to contain 404 (sheep) or 405 (human) amino acid residues with a total Mr of 41,000 [the published sheep sequence(23) contains a frameshift error near the 3′ end of the coding sequence]. The human and sheep predicted peptide sequences are 83% identical. A search of sequence databases reveals sequence similarity to members of the short chain alcohol dehydrogenase superfamily. The 11β-HSD K isozyme is most similar (37% sequence identity) to the type 2 (placental, NAD+-dependent, microsomal) isozyme of 17β-HSD(25). It is only 21% identical to the L isozyme of 11β-HSD. The relatively high degree of similarity between the 11β-HSD K isozyme and placental 17β-HSD (comparable to the similarity between cytochrome P450 gene family members) suggests that these two enzymes may be in the same gene family within the short chain dehydrogenase superfamily.

Regions of sequence similarity between the two isozymes (Fig. 3) include part of the putative binding site for the nucleotide cofactor (residues 85-95 in the 11β-HSD K isozyme) and absolutely conserved tyrosine and lysine residues (Tyr-232 and Lys-236 in this enzyme) that function in catalysis(26, 27). The region immediately to the N-terminal side of the catalytic residues forms part of a putative steroid binding pocket in the short chain dehydrogenase that has been analyzed by x-ray crystallography, 3α,20β-HSD(28). This region is notably well conserved (10/18 identical residues) between the two isozymes of 11β-HSD, consistent with a role in binding the substrate.

The corresponding gene, termed HSD11K or HSD11B2, contains five exons spaced over approximately 6.2 kb(29)(Fig. 3). The putative binding site for the NAD+ cofactor (including the core sequence, G XXXG XG) is split between exons 1 and 2, whereas the putative catalytic residues, Tyr-232 and Lys-236, are encoded by exon 4. This structure is different from that of the HSD11L gene encoding the liver (type 1) isozyme of 11β-HSD(16), indicating that the two isozymes belong to different gene families.

HSD11K is expressed in placenta and mineralocorticoid target tissues, particularly the kidney, whereas it is not detected in the liver. Whereas human fetal and adult tissues contain transcripts of 1.9-2.0 kb(24, 29), fetal tissues also express transcripts of 5 and 7 kb(29). These may represent utilization of alternative polyadenylation sites or partially processed transcripts.

DETECTION OF MUTATIONS IN HSD11K IN PATIENTS WITH AME

We identified seven different mutations the HSD11K gene in eight kindreds with AME (Fig. 4). These mutations all affect enzymatic activity or pre-mRNA splicing, thus confirming in its entirety the hypothesis that 11β-HSD protects the mineralocorticoid receptor from high concentrations of cortisol(30). Seven other kindreds have been studied and an additional four mutations detected by others(31–33). Only one patient has been a compound heterozygote for two different mutations, whereas all other patients have carried homozygous mutations. This suggests that the prevalence of AME mutations in the general population is low, so that the disease is found mostly in limited populations in which inbreeding is relatively high. Six kindreds are of Native American origin. Three from Minnesota or Canada carry the same mutation (L250S,L251P), consistent with a founder effect, but the others are each homozygous for a different mutation. The reason for the relatively high prevalence of this very rare disease among Native Americans is not immediately apparent.

Locations of mutations causing apparent mineralocorticoid excess. Intron 1 is not drawn to scale. The predicted percent of wild type enzymatic activity associated with each mutation is listed.

Full size image

Of the mutations identified thus far, two shift the reading frame of translation, a third deletes three amino acids including a crucial catalytic residue (Tyr-232), and one is a nonsense mutation. These mutations are all presumed to completely destroy enzymatic activity. One mutation in the third intron leads to skipping of the fourth exon during processing of pre-mRNA(30). As the fourth exon encodes the catalytic site, the resulting enzyme is again presumably inactive. The other six mutations have been introduced into cDNA and expressed in cultured cells to determine their effects. One (L250P,L251S) is completely inactive and one (R337Δ3nt) has only a trace of activity. The others are all partially active in cultured cells with one, R337C, having greater than 50% of normal activity(34). Only R337C is partially active in lysed cells[although one group reported this mutation to be inactive in cell lysates(35), they did not use appropriate conditions to maximize enzyme stability(36)]. We believe that comparisons of activity are best made using the apparent first order rate constant,Vmax/Km, which predicts reaction velocity at low substrate concentrations. Valid comparisons in whole cells require controls (Western blots or determinations of mRNA levels) for transfection efficiency. However, determinations of apparent Km in whole cells need to be interpreted cautiously, particularly when high concentrations of substrate are used, because many substrates including steroids are subject to active transport into or out of cells(37). Such mechanisms, which have their own kinetics, can confound kinetic measurements of enzymes.

Both the wild type enzyme and most mutants are concentrated in the nucleus as determined by Western blots of cell fractions. All six mutants that we examined are expressed in decreased amounts suggesting that most of the mutations adversely affect protein stability once cells are lysed(34).

Although the number of patients with AME is small, sufficient data now exist to demonstrate a statistically significant correlation between degree of enzymatic impairment and biochemical severity as measured by the precursor:product ratio (THF + allo-THF)/THE(34). This correlation is most obvious for the partially active mutants. We assume that R337C is the only significant mutation in the patients who carry it, even though only one exon of the gene was sequenced(31). If so, a 50% impairment of enzymatic activity is apparently sufficient to compromise metabolism of cortisol in the kidney, suggesting that there is very little excess capacity to metabolize cortisol in this organ. This seems to raise a paradox, because AME is a recessive disorder and heterozygous carriers, who would be expected to have 50% of normal activity, are asymptomatic. Altered stability or kinetic properties of the R337C mutant may be important, including alterations in enzyme inhibition by end product(i.e., cortisone or corticosterone) or by other circulating steroids.

Because of the small numbers of patients, and the possible confounding effects of prior antihypertensive therapy, it is difficult to correlate biochemical severity with measures of clinical severity, although anecdotal reports suggest that mutations that do not destroy activity may be associated with milder disease(30, 31). With the elucidation of the molecular genetic basis of this disorder, ascertainment of additional cases may permit these questions to be answered.

Abbreviations

apparent mineralocorticoid excess

hydroxysteroid dehydrogenase

tetrahydrocortisol

tetrahydrocortisone

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Author information

Author notes
  1. Perrin C White: Recipient of the Society for Pediatric Research 1996 E. Mead Johnsn Award for Research on Pediatrics and presented at the 1996 Annual Meeting of the Pediatric Academic Societies, Washington, DC.

Affiliations

  1. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, 75235-9063, Texas

    Perrin C White, Tomoatsu Mune, Fraser M Rogerson, Kathleen M Kayes & Anil K Agarwal

Corresponding author

Correspondence to Perrin C White.

Additional information

Supported by National Institutes of Health Grant DK42169.

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White, P., Mune, T., Rogerson, F. et al. 11β-Hydroxysteroid Dehydrogenase and Its Role in the Syndrome of Apparent Mineralocorticoid Excess. Pediatr Res41, 25–29 (1997). https://doi.org/10.1203/00006450-199701000-00004

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Mitochondrial 3 beta-hydroxysteroid dehydrogenase (HSD) is essential for the synthesis of progesterone by corpora lutea: An hypothesis

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Abstract

In mouse ovaries, the enzyme 3 beta-hydroxysteroid dehydrogenase (HSD) is distributed between microsomes and mitochondria. Throughout the follicular phase of the estrous cycle, the HSD activity in microsomes is predominant; whereas, after LH stimulation, HSD activity during the luteal phase is highest in the mitochondria. The current study examined whether or not LH stimulation always results in an increase in mitochondrial HSD activity. This was accomplished by measuring the HSD activity in microsomal and mitochondrial fractions from ovaries of pregnant mice. These animals have two peaks of LH during gestation, and one peak of LH after parturition. It was found that mitochondrial HSD activity was highest after each peak of LH. It is proposed that mitochondrial HSD is essential for the synthesis of high levels of progesterone. The increase in HSD activity in mitochondria after LH stimulation occurs because: 1) LH initiates the simultaneous synthesis of HSD and the cholesterol side-chain cleavage enzyme (P450scc); and, 2) HSD and P450scc bind together to form a complex, which becomes inserted into the inner membrane of the mitochondria. High levels of progesterone are synthesized by mitochondrial HSD because: 1) the requisite NAD+ cofactor for progesterone synthesis is provided directly by the mitochondria, rather than indirectly via the rate limiting malate-aspartate shuttle; and, 2) the end-product inhibition of P450scc by pregnenolone is eliminated because pregnenolone is converted to progesterone.

Background

With the exception of 3β-hydroxysteroid dehydrogenase (HSD), the enzymes involved in the conversion of cholesterol to steroid hormones are located in either the mitochondria or the endoplasmic reticulum. HSD is unique in that it is located in both subcellular organelles. In either location, HSD converts pregnenolone and dehydroepiandosterone (DHEA) to progesterone and androstenedione, respectively, using NAD+ as cofactor. The reason for two separate sites for this enzyme is not known.

Establishing the existence of two separate locations for HSD has been a lengthy process. In 1956, Beyer and Samuels reported that the microsomal (endoplasmic reticulum) and mitochondrial fractions from the homogenate of bovine adrenal cortex contained HSD activity [1]. However, the HSD activity found in the mitochondrial fraction was attributed to microsomal contamination and the result of the homogenizion process. While this study established the legitimacy of microsomal HSD, it tended to preclude further research on mitochondrial HSD. For mitochondrial HSD to be considered a distinct and separate entity, additional research over a number of years would be required. Starting in 1965, investigators began to report a dual location for HSD in ovaries [2–4], testes [5, 6], human term placenta [7–10], and rat adrenal cortex [11–14]. In toto, these studies suggested that mitochondrial HSD was indeed a separate entity. Other investigators, however, still considered mitochondrial HSD activity to be due to microsomal contamination [15, 16], and the result of a redistribution artifact [17].

In 1979, we reported the results of an intracellular enzyme distribution study of HSD, cytochrome c oxidase (mitochondrial marker), and steroid 21 hydroxylase (microsomal marker) in rat adrenal cortex [18]. We found that exhaustively washed mitochondria retained 26 % of total HSD activity. In retrospect, this percentage appears to be on the low side. For example, when the specific activity of microsomal HSD is determined, and its contribution to the HSD activity in the remaining cell fractions (nuclear/unbroken cell, mitochondrial, and mitochondrial wash fractions) ascertained, then a maximum of 60 % of total homogenate HSD activity can be attributed to microsomal HSD. This indicates that mitochondrial HSD constitutes 40 % of total homogenate HSD activity, rather than 26 % as we initially reported. In rhesus monkey placenta, HSD activity is equally distributed between mitochondria and microsomes [19], and in bovine adrenal cortex, mitochondrial HSD comprises 30 % of total HSD activity [20, 21].

Our study also found that mitochondrial HSD utilizes matrix space NAD+ as cofactor, indicating that the enzyme is located in the inner mitochondrial membrane [18]. This location for mitochondrial HSD has been established in bovine adrenal cortex [20, 21], and in rat testis [5]. The combined techniques of immuno-cytochemistry and electron microscopy have identified immune reactive HSD in mitochondria of human ovary [22], and in the mitochondria of rat ovary, testis, and adrenal cortex [23]. Mitochondrial HSD has now been isolated from bovine adrenal cortex [21, 24], and from human term placenta [25, 26], and purified to homogeneity. It is now known that microsomal HSD and mitochondrial HSD are identical proteins [25–28]. The reason for two locations for the same enzyme has yet to be determined.

In a study of the intracellular distribution of mitochondrial HSD and microsomal HSD in mouse ovaries over the course of the estrous cycle [29], we reported that during diestrus (luteal phase), the specific activity of mitochondrial HSD was 80 % higher than that of microsomal HSD. This is in sharp contrast to the other three stages of estrus where microsomal HSD had the highest specific activity. In a study in which the cDNA of human placental HSD was transfected into Sf9 cells, the resultant HSD enzyme was distributed between mitochondria and microsomes [27]. In the luteal cells of mouse ovary, the distribution of the enzyme is skewed in favor of the mitochondria. During the luteinization process, luteal cells express high levels of mRNA for the cholesterol side-chain cleavage enzyme (P450scc) and for HSD [30, 31]. The simultaneous synthesis of these two enzymes is very likely the reason for the increase in mitochondrial HSD activity, as will be discussed later.

The results of our previous study [29] tentatively suggested that the increase in mitochondrial HSD activity in mouse ovary during diestrus is due to LH. The pregnant mouse has two peaks of LH during gestation, and one peak of LH after parturition [32]. After each peak of LH, the levels of circulating progesterone increase [33]. The present study examined the distribution of HSD activity in pregnant mouse ovaries to determine whether or not mitochondrial HSD activity also increased after each peak of LH. We found that it did, which led to our proposed explanation for the reason for two separate locations for HSD in corpora lutea.

Methods

Animals

Female mice of the (C3H/HeJ × 129/J)F1 (C31) hybrid were used in the study. Parental stocks were purchased from Jackson Laboratories, Bar Harbor, ME. The mice were housed in an animal room kept at 24°C, with controlled lighting of 14L:10D (lights on at 0600 hr and off at 2000 hr). Purina lab chow (Ralston-Purina, St. Louis, MO) and water were provided ad libitum. The C31 offspring were weaned at 23–28 days of age. Female siblings were housed two per cage. For pregnancy experiments, C31 females were mated with C31 males. Only those with regular estrous cycles (4–5 days) were used for the study.

Determining Stage of the Estrous Cycle and mating

In order to promote a regular estrous cycle in the C31 females, cage shavings were taken from cages containing C31 males, and placed in cages containing the females. Also, male cages surrounded female cages. Vaginal smears were taken daily, usually in the afternoon. The smears were spread on a glass slide in a drop of physiological saline, stained using hematoxylin and eosin Y, and the stage of estrous determined by the method of Rugh [34].

In all experiments, each study group consisted of six females that were between 80 and 190 days of age. Two of the study groups were comprised of animals that were in diestrus and proestrus. The remaining groups all consisted of pregnant animals. These groups were formed as follows: females that were in either proestrus or estrus were mated late in the afternoon or about 2 hr prior to lights off. At lights on the following morning, and for subsequent days if necessary, all females were inspected for the presence of vaginal plugs. The day of vaginal plug was considered as day 0 of pregnancy. Over the gestation period, pregnant females were sacrificed on days 5, 10, 15, and 20. An additional group of females was sacrificed on day 5 postpartum.

Tissue Collection and Processing

Animals were lightly etherized, weighed, and then decapitated. The ovaries were removed, trimmed of fat, weighed in pairs, and placed on an ice-filled petri dish in a few drops of homogenizing buffer (0.3 % BSA, 1 mM EDTA, 0.25 M Sucrose, and 30 mM Tris-HCL [pH 7.4], 4°C), as per Chapman and Sauer [18]. The twelve ovaries were finely minced, then transferred to an ice-cold 5 ml Potter-Elvehjem glass homogenizer. The volume of the sample was brought up to 4 ml with additional homogenization medium, and the ovaries homogenized in the cold room (4°C) with 8 complete strokes of a motorized Teflon pestle. The homogenate was then transferred to a 10 ml centrifuge tube, and the glass homogenizer rinsed with 1 ml of homogenizing medium. The rinse was combined with the homogenate, and a 1 ml sample of the total homogenate removed and saved for later assay.

The uteri were removed from 10 day, 15 day, and 20 day pregnant mice. Trophoblasts were excised from the middle section of each uterine horn. After weighing, the trophoblasts were minced, and processed as described for the ovaries, except that the mince was homogenized in a Thomas glass-glass homogenizer.

Differential Centrifugation

The ovarian homogenate, contained in a 10 ml centrifuge tube, was centrifuged at 700 × g in a Sorvall RC-5 refrigerated centrifuge for 10 min. The supernatant was removed and spun at 10,000 × g for 20 minutes in the same centrifuge. The low-speed pellet, containing nuclei and unbroken cells, was resuspended in 1.5 ml homogenizing buffer. Following the 10,000 × g centrifugation, the resultant mitochondrial pellet was resuspended in 1.5 ml homogenizing buffer. The postmitochondrial supernatant was centrifuged in a Beckman L65 Ultracentrifuge (Beckman Co., Fullerton, CA) for 1 h at 105,000 × g. This yielded cytosol and a pellet of microsomes. Microsomes were resuspended in 1.5 ml homogenizing buffer using a 2 ml Potter-Elvehjem glass homogenizer with Teflon pestle. All fractions, including the total homogenate, were divided into aliquots in 12 × 75-mm borosilicate glass tubes, covered with Parafilm (American Can Co., Greenwich, CT), and frozen at -10°C. Enzymatic analyses were scheduled so that the frozen sub-cellular fractions were thawed only once prior to assay.

Enzymatic Analyses and Other Assays

The total homogenate, low speed pellet (nuclei/unbroken cells), mitochondrial, and microsomal fractions were assayed for HSD activity by measuring the conversion of pregnenolone to progesterone. Duplicate tissue samples of 50 μl and 100 μl were added to 15-ml glass test tubes containing 1 ml of incubation medium (50 mM sucrose, 20 mM KC1, 1 mM EDTA, 30 mM Tris-HCl [pH 7.4], 0.3% BSA), and 0.5 mM NAD+, as per Chapman et al. [29]. The incubates were then placed in a 37°C water bath and allowed to equilibrate for 5 min. The reaction was started by the addition of 100 nmol of pregnenolone in 10 μl of ethanol. After 15 min of incubation, the progesterone product was extracted into 1 ml of spectral grade heptane. The absorbency of progesterone was measured in a Gilford Response spectrophotometer (Gilford Systems, Oberlin, OH). Progesterone has an absorbency peak at 233 nm in heptane and a molar extinction coefficient of 17,000 [18]. In order to access the extraction efficiency of 1 ml of heptane, known concentrations of progesterone standards were run concurrently with the tissue samples. After extraction into heptane, the absorbancy of the standards was compared to their absorbance measured directly in heptane.

Cytochrome c oxidase, an inner mitochondrial membrane marker, was assayed by the procedure of Wharton and Tzalgaloff [35]. Enzymatic activity was determined by the rate of decrease in absorbancy at 550 nm. Protein content was measured by using the method of Bradford [36]. All assays were in duplicate. Replicate data were analyzed for significant differences using ANOVA (Dunnett, and Scheffe' F-test).

Results

The distribution of HSD activity between mitochondria and microsomes undergoes a unique shift in the transition from proestrus to diestrus. At proestrus, for example, the highest HSD activity is in the microsomes. At diestrus, in contrast, mitochondrial HSD activity is almost double that of microsomal HSD [29]. The present study re-examined this phenomenon; and, as Figure 1 shows, the activity of mitochondria HSD increases significantly at diestrus. The activity of the mitochondrial inner membrane enzyme, cytochrome c oxidase, also increases at diestrus. Total ovarian protein, in contrast, decreases.

Protein content, cytochrome c oxidase activity, and HSD activity in total homogenates, and in mitochondrial and microsomal fractions from ovaries of C31 mice that were in either proestrus or diestrus. Each experimental group consisted of 6 mice. Mitochondrial and microsomal fractions were isolated by differential centrifugation. Results are expressed per individual ovary.

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Figure 2 contains the results of two separate experiments in which HSD activity was measured in mitochondrial and microsomal fractions during pregnancy, and 5 days after parturition. As indicated, the activities of mitochondrial HSD and microsomal HSD both increased over the course of the gestation period. However, the increase in HSD activity in the two organelles was inconsistent. For example, at 15 days and 20 days of gestation, the highest HSD activity was in the microsomal fraction. In contrast, at 5 days and 10 days of gestation, and at 5 days postpartum, mitochondrial HSD activity was greater than that of microsomal HSD. These three time points directly follow the peaks of LH [32]. Cytochrome c oxidase activity also increased during pregnancy. At day 20, cytochrome c oxidase activity was more than double the activity measured at 5 days. Total ovarian protein was inversely correlated with the peaks of LH. For example, at 15 days and 20 days of gestation, each ovary contained 1190 μg protein and 1025 μg protein, respectively. In contrast, at 5 days and 10 days of gestation, and at 5 days postpartum, each ovary contained 890 μg protein, 825 μg protein, and 810 μg protein, respectively. This relationship between LH and total ovarian protein also occurs during the estrous cycle [29]. For example, as shown in Figure 1, each ovary at proestrus contained 1030 μg protein; whereas at diestrus, each ovary contained 838 μg protein.

Protein content, cytochrome c oxidase activity, and HSD activity in total homogenates, and in mitochondrial and microsomal fractions from ovaries of C31 mice that were pregnant for 5 days, 10 days, 15 days, and 20 days, or were 5 days postpartum. Each experimental group consisted of 6 mice. Mitochondrial and microsomal fractions were isolated by differential centrifugation. Results of two separate experiments are expressed per individual ovary. Statistical analyses of the enzymatic activities of cytochrome c oxidase and HSD in total homogenates at each time-point showed a significant difference @ P < .05, when compared to animals in diestrus. In addition, the HSD activities in all mitochondrial and microsomal fractions of pregnant mice were significantly different @ P < .05, when compared to animals in diestrus.

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Figure 3 contains the results of the measurement of HSD activity in mitochondrial and microsomal fractions of trophoblasts from 10 day, 15 day, and 20 day pregnant mice. As indicated, HSD activity was not detected (N.D.) in trophoblasts from 10 day pregnant mice. However, trophoblasts from 15 day and 20 day pregnant mice were found to produce 0.4 nmol progesterone/min/trophoblast and 0.6 nmol progesterone/min/trophoblast, respectively. Note that the highest HSD activity in trophoblasts is in the microsomal fraction.

Protein content, cytochrome c oxidase activity, and HSD activity in total homogenates, and in mitochondrial and microsomal fractions from trophoblasts of C31 mice that were pregnant for 10 days, 15 days, and 20 days. Each experimental group in the 10 day and 20 day pregnant animals consisted of 6 mice. N = 12 for the 15 day pregnant group. Mitochondrial and microsomal fractions were isolated by differential centrifugation. Results are expressed per individual trophoblast. N.D. = Not detected.

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Discussion

The results of this and the previous study [29] leave little doubt that mitochondrial HSD activity increases after LH stimulation. How the increase in mitochondrial HSD activity is achieved and for what purpose, are the topics of this discussion.

When mitochondrial HSD was initially purified from the bovine adrenal cortex, the enzyme was found to have a close association with P450scc [24]. This association was of such a high degree that mitochondrial HSD actually copurified with P450scc. Antibodies against mitochondrial HSD precipitated both HSD and P450scc; and, conversely, antibodies against P450scc precipitated both P450scc and mitochondrial HSD. The degree of association between the two enzymes was measured, and a binding constant (KD) of 0.12 μM was determined. As would be expected, P450scc also bound to purified microsomal HSD [24]. HSD is insoluble and inactive in an aqueous medium, due to a segment of the protein, referred to as the "membrane-spanning domain" [27]. Delete this segment and HSD becomes soluble. With the segment in place, HSD is inserted into the membranes of microsomes and mitochondria [27]. The observation that mitochondrial HSD activity increases after LH stimulation suggests that HSD is preferentially inserted into the mitochondrial membrane. The fact that HSD binds to P450scc is very likely the mechanism for its insertion. This suggests that HSD and P450scc bind together, either during, or directly after their synthesis, since it is unlikely that HSD could bind to P450scc, already in place. The mRNAs for HSD and P450scc are expressed concurrently in luteal cells of the rat [37–40], cow [41, 42], sheep [42–44], horse [45], macaque monkey [46], and human [38, 47–49].

Figure 4 is a representative diagram of the proposed effect of the concurrent synthesis of HSD and P450scc on the intracellular distribution of HSD in luteal cells. Initiated by LH; the expression of HSD mRNA and P450scc mRNA results in the simultaneous synthesis of HSD and P450scc. The two enzymes bind to each other to form a complex, which is then inserted into the inner mitochondrial membrane. Molecules of HSD that do not bind to P450scc are inserted into the membrane of the endoplasmic reticulum.

Representative diagram of the proposed effect of the concurrent synthesis of HSD and cytochrome P450sccon the intracellular distribution of HSD in luteal cells. Initiated by LH; the transcription of HSD mRNA and P450scc mRNA results in the simultaneous production of HSD and P450scc. The two enzymes bind to each other to form a complex, which is then inserted into the inner mitochondrial membrane. Molecules of HSD that do not bind to P450scc are inserted into the membrane of the endoplasmic reticulum.

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In the mouse there are peaks of estradiol-17β at proestrus (100 pg/ml) and metestrus (200 pg/ml) [29]. However, these levels are significantly less than the levels of progesterone produced throughout the luteal phase. During pregnancy the levels of circulating progesterone are even higher. One could argue that the increased levels of circulating progesterone in pregnant mice are due to the HSD activity in trophoblasts. This possibility was addressed in the current study. As shown in Figure 3, homogenates of individual trophoblasts from 15 day and 20 day pregnant mice are capable of producing 0.4 nmol progesterone/min/trophoblast and 0.6 nmol progesterone/min/trophoblast, respectively. This level of HSD activity in a single trophoblast is only 3 % of the HSD activity produced by the paired ovaries. However, large litters would increase the percentage. At day 10 there is no question that the ovaries are the major source of circulating progesterone, which averages 55 ng/ml [33]. This level of progesterone is between 275 fold and 550 fold of the peak levels of estradiol-17β produced during the follicular phase.

In order for luteal cells to synthesize high levels of progesterone, a number of events have to occur. First, higher levels of the two enzymes, P-450scc and HSD, have to be produced. This event is initiated when their respective mRNAs are expressed, as referenced above. Secondly, the increase in steroid synthesis requires an increased supply of cholesterol. This is achieved by removing cholesterol from cholesterol ester stores [50, 51], and by initiating the de novo synthesis of cholesterol [52–54]. The latter event is quite likely in anticipation of fertilization of the ova and the need for a supply of cholesterol beyond diestrus. In the pregnant mouse, luteal cells synthesize high levels of progesterone for three weeks [33]. The de novo synthesis of cholesterol requires a carbon source, as well as ATP and NADPH. In luteal cells the carbon source is acetate; ATP is generated through glycolysis; and NADPH is produced during the oxidative decarboxylation of isocitrate and malate [54]. The enzymes that catalyze the latter reaction are NADP+-linked isocitrate dehydrogenase and NADP+-linked malate dehydrogenase. Both enzymes are in abundance in the cytoplasm of luteal cells [55].

The enzyme, P450scc is capable of producing 53 nmol pregnenolone/min/mg protein [56]. This requires an undiminished supply of NADPH, as well as the aforementioned cholesterol. The NADPH that is produced in the cytoplasm is of no direct use to the P450scc enzyme. However, reducing equivalents from NADPH can be transferred from the cytoplasm to mitochondria via the malate-aspartate shuttle [57, 58]. Unfortunately, the mitochondria in luteal tissue lack an NADP+-linked malate dehydrogenase [59, 60]. As a result, NADPH cannot be generated in the mitochondria by the oxidative decarboxylation of malate. This is in sharp contrast to adrenal cortex tissue, which does have a mitochondrial NADP+-linked malate dehydrogenase [61, 62]. In luteal tissue the mitochondria and cytoplasm both contain an NAD+ -linked malate dehydrogenase, each enzyme having a high specific activity [55, 59]. This indicates that there is ample capacity to transfer reducing equivalents from NADH into the mitochondria. If this transfer were to occur in tissues such as liver and heart, the NADH would be used to produce ATP. In steroidogenic tissues, the NADH can also be used to produce NADPH. For example, in mitochondria of luteal tissue, reducing equivalents are transferred from NADH to NADP+ by an energy-independent pyridine-nucleotide transhydrogenase [56]. Sonicates of luteal mitochondria are reported to catalyze the production of 60 nmol NADPH/min/mg protein from NADH [56]. Mitochondria in adrenal cortex tissue have a similar process, except that the transfer is energy-dependent [63]. The main source of NADPH in the mitochondria of luteal tissue is provided by NADP+-linked isocitrate dehydrogenase [56]. This enzyme is capable of reducing 253 nmol NADP+/min/mg protein [56].

In the synthesis of progesterone, NAD+ is reduced to NADH. In order to maintain a high rate of progesterone synthesis, NADH has to be continually oxidized to NAD+. With microsomal HSD, the oxidation of NADH would have to occur via either the α-glycerol phosphate shuttle or the malate-aspartate shuttle. Both shuttles transfer reducing equivalents from NADH into the mitochondria. However, the α-glycerol phosphate shuttle does not operate in the ovary [64], which leaves the transfer of reducing equivalents to the malate-aspartate shuttle. The ovary already heavily utilizes this shuttle. As discussed earlier, the malate-aspartate shuttle transfers reducing equivalents from NADH into the mitochondria for the P450scc enzyme. In addition, the shuttle is involved in the oxidation of the NADH produced during glycolysis. A high rate of glycolysis during the de novo synthesis of cholesterol, for example, generates high levels of NADH. If the levels of NADH exceed the carrying capacity of the shuttle system, the reducing equivalents are transferred to pyruvate via the enzyme, lactate dehydrogenase. A high level of lactate is a signal that the shuttle system is rate limiting.

The ovary produces appreciable amounts of lactate, even during the early follicular phase [65]. As follicular size increases, lactate levels also increase [66], coinciding with the start of antrum formation and detectable estradiol-17β secretion [66, 67]. After the LH surge, the levels of lactate increase an additional 2.5 fold [66, 68]. In luteal tissue, a high percentage of the glucose taken up is metabolized only as far as pyruvate and lactate [69]. Iodoacetate, an inhibitor of glycolysis, abolishes the effect of LH on lactate accumulation and significantly reduces LH-stimulated progesterone synthesis [68, 70].

The fact that the malate-aspartate shuttle is rate-limiting could be the reason for a mitochondrial location for HSD. However, pregnenolone is an end-product inhibitor of the P450scc reaction [71, 72], and a mitochondrial location for HSD would remove the steroid from its site of inhibition. Progesterone does not inhibit the P450scc reaction [71]. Evidence that mitochondrial HSD is involved in the production of high levels of progesterone is provided by the observation that mitochondria from thecal tissue convert only 6.4 % of total 14C-cholesterol to 14C-progesterone (1.2 %) and 14C-pregnenolone (5.2 %); whereas, in contrast, mitochondria from luteal tissue convert 16.1 % of total 14C-cholesterol to 14C-progesterone (13.5 %) and 14C-pregnenolone (2.6 %) [73].

In human adrenals and gonads, HSD is derived from the same gene and has been classified as type II, relative to the placental enzyme, which is classified as type I [27, 28, 74, 75]. In the adrenals and gonads of the rat [74–76] and mouse [77, 78], the enzyme is also derived from the same gene, which in these rodents is classified as type I. The fact that ovaries and adrenal cortex contain the same HSD enzyme indicates that their response to trophic hormone stimulation would also be the same. One would expect, therefore, that ACTH stimulation would cause an increase in mitochondrial HSD activity in adrenal cortex tissue. ACTH stimulation of male and female rats does cause an increase in HSD mRNA and HSD activity [79]. However, it is not known if ACTH stimulation causes a preferential increase in mitochondrial HSD activity.

Aside from the role that Steroidogenic Acute Regulatory protein (StAR) plays in controlling cholesterol access to mitochondria, the rate-limiting step in steroidogenesis is considered to be P450scc [80]. However, the fact that P450scc and HSD are bound together as a complex [24] suggests that the rate-limiting step, or steps, entails the conversion of cholesterol to progesterone. There is a decided advantage in these two enzymes functioning as a unit. Instead of shuttling steroid intermediates from organelle to organelle, cholesterol can be converted to progesterone in what could be described as a single step. The levels of progesterone can be increased even further if the reactions of both enzymes are coupled together, which appears to be the case. The discovery of the energy-independent NADH/NADP+ transhydrogenase initially led to the assumption that it existed to supply P450scc with NADPH [56]. However, the transhydrogenase is capable of producing less than one/half the NADPH needed to synthesize high levels of pregnenolone. If its function is to act as a principal supplier of NADPH, it is inadequate. However, if its main function is to ensure that HSD has an undiminished supply of NAD+, and is operating at Vmax, it is more than adequate. It is an ideal means of coupling HSD to P450scc. Unfortunately, the exchange of one molecule of NADH to produce one molecule of NADPH is insufficient for the overall conversion of progesterone to cholesterol. This is because the P450scc reaction utilizes 3 molecules of NADPH for the synthesis of one molecule of pregnenolone. The remainder of the NADPH for this reaction would have to be supplied by mitochondrial NADP+-linked isocitrate dehydrogenase [56].

The P450scc/HSD enzyme complex is well regulated. In addition to the end-product inhibition exerted by pregnenolone on the P450scc reaction [71, 72], the HSD reaction is affected by the redox state of cytoplasmic pyridine nucleotides [81]. For example, extramitochondrial NAD+ increases mitochondrial HSD activity by up to 40 %; whereas, extramitochondrial NADH inhibits HSD activity by as much as 70 %. Figure 5 is a representative diagram of the proposed regulation of the conversion of cholesterol to progesterone. As indicated, pregnenolone acts as an end product inhibitor of the P450scc reaction, [······ (-)]; and, in the mitochondrial HSD reaction, extramitochondrial NAD+ operates as an allosteric activator [------(+)] and extramitochondrial NADH operates as an allosteric inhibitor [······ (-)]. With this level of control it is difficult to imagine how pregnenolone could ever leave the luteal cells without being converted to progesterone.

The proposed regulation of progesterone synthesis by pregnenolone and by extramitochondrial NAD+/NADH. In the overall conversion of cholesterol to progesterone, pregnenolone acts as an end product inhibitor in the P450scc reaction, [······ (-)]; and, in the mitochondrial HSD reaction, extramitochondrial NAD+ operates as an allosteric activator [------(+)] and extramitochondrial NADH operates as an allosteric inhibitor [······ (-)].

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Finally; it was noted in the results section that total ovarian protein was inversely correlated with the peaks of LH. A decrease in total ovarian protein during diestrus could be due to ovulation. However, this does not explain the lower protein levels following LH stimulation that occurs during pregnancy and after parturition. A rapid and ongoing synthesis of the two enzymes, P450scc and HSD is critical to the production of high levels of progesterone. This necessitates a ready supply of amino acids. It is speculative of course, but the action of LH could include the initiation of proteolysis of protein stores from luteal tissue, which could explain the lower protein levels.

Conclusion

The ovary has two levels of steroid synthesis. One level occurs during the follicular phase, and a higher level of synthesis occurs throughout the luteal phase. We believe the higher level of synthesis is due to, and the reason for, mitochondrial HSD. To synthesize estradiol-17β during the follicular phase, steroid precursors are shuttled from cell type to cell type and from organelle to organelle. The synthesis of progesterone during the luteal phase involves one cell type and two enzymes. With HSD in the mitochondria, rather than in the microsomes, the shuttle of steroid precursors is unnecessary. It also allows for HSD and P450scc to function together as a unit, a decided advantage for producing high levels of progesterone. This is especially true if the two enzymes are coupled together by the NADH/NADP+ transhydrogenase. A mitochondrial location for HSD also solves the problem inherent with the rate-limiting malate-aspartate shuttle, and it removes the end-product inhibition of pregeneolone by converting it to progesterone. Finally, the fact that HSD and P450scc have a strong binding affinity for each other, and are synthesized simultaneously, tentatively suggests a means by which LH stimulation results in an increase in mitochondrial HSD activity.

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Sours: https://rbej.biomedcentral.com/articles/10.1186/1477-7827-3-11


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