Steroidogenic control of liver metabolism through a nuclear receptor-network.

OBJECTIVE: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown. METHODS AND RESULTS: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles. Both gain- and loss-of-function approaches are used to show that hepatic Cyp17a1 is induced by fasting, catalyzes the production of at least one hormone-ligand (DHEA) for the nuclear receptor PPARα, and is ultimately required for maintaining euglycemia and ketogenesis during nutrient deprivation. The feedback-loop that terminates Cyp17a1-PPARα activity, and re-establishes anabolic liver metabolism during re-feeding is mapped to postprandial bile acid-signaling, involving the receptors FXR, SHP and LRH-1. CONCLUSIONS: Together, these findings represent a novel paradigm of homeostatic control in which nutritional cues feed-forward on to metabolic pathways by influencing extragonadal steroidogenesis.

A potential role for CPY17 as a parameter in differentiation between aldosterone-producing adenoma and nodular hyperplasia in patients with hyperaldosteronism.

OBJECTIVE: To investigate CYP17 mRNA and protein expressions in aldosterone-producing adenoma (APA), nodular hyperplasia (NH) and normal adrenal gland (NAG) and if CPY17 might be used as a potential marker to differentiate between APA and NH in patients with hyperaldosteronism. METHODS: Total RNA and protein were extracted from APA, 12 NH, and 15 NAG tissues. mRNA and protein expressions of CPY17 were examined by real-time polymerase chain reaction (PCR) and Western blot analysis. RESULTS: The relative expression levels of CPY17mRNA to glyceraldehyde 3-phosphate dehydrogenase in the APA, NH, and NAG groups are 0.94 ± 0.09, 2.07 ± 0.10, and 3.94 ± 0.19, respectively, when evaluated by real-time PCR. This result was confirmed by the relative protein expression levels of CPY17 to ß-actin, which are 117 ± 13%, 274 ± 19%, and 478 ± 25%, respectively, when evaluated by Western blot analysis. There was a significant difference in mRNA and protein expression level of CYP17 between any two groups (P < .05). Thus, the sequence of the relative expression level of CPY17 is APA < NH < NAG. CONCLUSION: These results indicate that CPY17 was down-regulated in APA compared with that in NH, suggesting a potential role for CPY17 as a marker in differentiation between APA and NH in patients with hyperaldosteronism. Such a study might be helpful to improve the diagnosis and treatment of primary aldosteronism.

Cytochrome P450 17 (CYP17) is involved in endometrial cancinogenesis through apoptosis and invasion pathways.

Cytochrome P450 17 (CYP17) encodes cytochrome P450c17α, an enzyme with 17α-hydroxylase and 17, 20-lyase activities involved in estradiol biosynthesis. Here we examine the role of CYP17 gene in endometrial carcinogenesis. Immunohistochemistry staining of endometrial carcinoma and corresponding uninvolved tissues showed that CYP17 is upregulated in endometrial cancers (15 of 24, 62.5%). To understand the functional significance of this upregulation, we silenced CYP17 gene by introduction of siRNA into endometrial cancer cell line KLE followed by functional studies. Further, to understand the molecular basis of the role of CYP17, we profiled the expression of key pathway-specific genes and identified several components of the apoptosis and invasion pathways that are potentially regulated by CYP17. Silencing of CYP17 caused decreased cell proliferation and induced apoptosis. Significantly, CYP17 depletion leads to downregulation of anti-apoptotic genes B cell lymphoma 2 (Bcl-2) and telomerase reverse transcriptase (TERT), indicating induced apoptosis. Also, attenuation of CYP17 decreased the cellular invasion ability and regulated expression of several invasion pathway components such as melanoma cell adhesion molecule (MCAM), matrix metallopeptidase 2 and 9 (MMP-2 and MMP-9), and tissue inhibitor of metalloproteinase 3 (TIMP3). In conclusion, this is the first report documenting that upregulation of CYP17 in endometrial cancers play a crucial role in endometrial carcinogenesis by targeting multiple components of apoptosis and invasion pathways. Further studies are required to understand the detailed mechanisms underlying CYP17-mediated regulation of these components.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production.

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Evidence that high variation in antral follicle count during follicular waves is linked to alterations in ovarian androgen production in cattle.

Androgens have an important role in ovarian follicular growth and function, but circulating androgen concentrations are also associated with ovarian dysfunction, cardiovascular disease, and metabolic disorders in women. The extent and causes of the variation in androgen production in individuals, however, are unknown. Because thecal cells of follicles synthesize androstenedione and testosterone, variation in production of these androgens is hypothesized to be directly related to the inherently high variation in number of healthy growing follicles in ovaries of individuals. To test this hypothesis, we determined whether thecal CYP17A1 mRNA (codes for a cytochrome P450 enzyme involved in androgen synthesis), LH-induced thecal androstenedione production, androstenedione concentrations in follicular fluid, and circulating testosterone concentrations were lower in cattle with relatively low versus high number of follicles growing during follicular waves and whether ovariectomy reduced serum testosterone concentrations. Results demonstrated that cattle with a low follicle number had lower (P<0.05) abundance of CYP17A1 mRNA in thecal cells, reduced (P<0.01) capacity of thecal cells to produce androstenedione in response to LH, lower (P<0.01) androstenedione concentrations in ovulatory follicles, and lower (P<0.02) circulating testosterone concentrations during estrous cycles compared with animals with high follicle number. Also, serum testosterone in cattle with low or high follicle number was reduced by 63 and 70%, respectively, following ovariectomy. In conclusion, circulating androgen concentrations are lower in cattle with low versus high number of follicles growing during follicular waves, possibly because of a reduced responsiveness of thecal cells to LH.

Postmenopausal estrogen monotherapy-associated breast cancer risk is modified by CYP17A1_-34_T>C polymorphism.

Long-term hormone therapy (HT) is a recognized risk factor for postmenopausal breast cancer. Elevated steroid hormone levels play a critical role in breast carcinogenesis and this may be contributed by the efficiency of hormone biosynthesis. Within this context, genetic polymorphisms related to steroid hormone biosynthesis may modify HT-associated postmenopausal breast cancer risk. CYP17 is a key player of this pathway and the CYP17A1_-34_T > C polymorphism has been suggested to affect breast cancer risk in women using long-term HT. We genotyped 13 polymorphisms of seven genes of the steroid hormone biosynthesis pathway in 3,149 postmenopausal breast cancer patients and 5,489 age-matched controls from Germany. We observed a significant interaction of CYP17A1_-34_T > C and HT use on breast cancer risk in a co-dominant model (P (interaction) = 0.007). Current users of estrogen monotherapy showed a significantly increased risk for duration of use per 5-year increment when they were carriers of the CYP17A1_-34_TC genotype (OR 1.13, 95% CI: 1.04-1.23 per 5 years of use). We conclude that CYP17A1_-34_T > C may be part of the genetic background to contribute to postmenopausal breast cancer risk in women using estrogen monotherapy.

Steroid 17alpha-hydroxylase deficiency: functional characterization of four mutations (A174E, V178D, R440C, L465P) in the CYP17A1 gene.

CONTEXT: Steroid 17alpha-hydroxylase (CYP17A1, alias P450c17) deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The CYP17A1 enzyme catalyzes two distinct reactions, 17alpha-hydroxylase and 17,20-lyase activities. OBJECTIVE: The aim of the study was to analyze the structural and functional consequences of three novel (A174E, V178D, and L465P) and one previously reported (R440C) CYP17A1 mutation found in three patients clinically and biochemically presenting with 17OHD. PATIENTS AND METHODS: Two patients suffering from 46,XY disordered sex development presented at ages 5.5 and 8.8 yr, respectively, with tall stature and hypertension. Mutation analysis revealed compound heterozygous CYP17A1 mutations (A174E/K388X; V178D/R440C). The third patient (46,XX) presented with primary amenorrhea and hypertension at age 15 yr. She was homozygous for the novel L465P mutation. Functional studies employing a yeast microsomal expression system compared wild-type and mutant CYP17A1 both with regard to 17alpha-hydroxylase and 17,20-lyase activity. Mutants were examined in a computational three-dimensional model of the CYP17A1 protein. RESULTS: The activity assays showed that all three mutants retain only 0-7% of both 17alpha-hydroxylase and 17,20-lyase activity relative to CYP17A1 wild-type activity, corresponding to the in vivo situation. Enzyme kinetic studies proved the impairment of both reactions, respectively. Computer-based three-dimensional model analysis of CYP17A1 using CYP2B4 as template showed that three of the mutations had no direct effect on the active center, whereas one affects the heme coordination. CONCLUSION: The functional studies revealed that the described missense mutations result in severe 17OHD. Our data are important to predict the phenotypic expressions and provide important information for patient management and genetic counseling.

Polymorphism T --> C (-34 base pairs) of gene CYP17 promoter in women with polycystic ovary syndrome is associated with increased body weight and insulin resistance: a preliminary study.

The aim of this study was to establish the frequency of gene CYP17 promoter polymorphism in women with polycystic ovary syndrome (PCOS) from a Chilean population and to examine the association of this polymorphism with body weight and estimate of insulin resistance in PCOS patient carriers and noncarriers of the A2 allelic variant. A total of 159 women with clinical and hormonal evidence of PCOS and 93 healthy women (HW) were evaluated. Diagnosis of PCOS was made according to the National Institutes of Health consensus criteria. In PCOS and HW, an oral glucose tolerance test was performed; and serum glucose and insulin were measured before the glucose load and 30, 60, 90, and 120 minutes after. Lipid profile and free fatty acid concentrations were determined in the basal sample. Insulin resistance was evaluated by homeostatic model assessment and insulin sensitivity index composite. A polymerase chain reaction-restriction fragment length polymorphism analysis was performed in all women to determine the A2 allele of the gene CYP17 promoter. The genotype frequency was similar between HW and PCOS women. No differences in anthropometric measurements and metabolic parameters were observed in HW carrier and noncarrier of the A2 variant. In PCOS women, an increase in body mass index, waist circumference, homeostatic model assessment of insulin resistance, and fasting insulin according to the A2 allele dosage was observed (P = .008, P = .016, P = .012, and P = .006, respectively). Polycystic ovary syndrome patient carriers of the A2 allele with a body mass index greater than 29.9 kg/m(2) showed an odds ratio of 9.1 (confidence interval, 3.0-27.4; P < .0001) for developing insulin resistance. These data suggest that the frequency of the A2 allele is similar between PCOS patients and HW; however, the presence of this gene defect in PCOS patients seems to be associated with increase in body weight, abdominal adiposity, and metabolic components.

Phosphorylation of CtBP1 by cAMP-dependent protein kinase modulates induction of CYP17 by stimulating partnering of CtBP1 and 2.

In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/cAMP-dependent protein kinase (PKA) pathway. Carboxyl-terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor-1 in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTH-stimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to steroidogenic factor-1 and the coactivator GCN5 (general control nonderepressed 5) and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD+ ratio stimulate nuclear-cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in CtBP protein partnering and ACTH-dependent CYP17 transcription.

Regulation of the adrenal androgen biosynthesis.

The human adrenal reticularis produces the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S). As opposed to the cortisol and aldosterone little is known regarding the mechanisms that regulate the production of the adrenal androgens. Several recent studies have shown that type II 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5 (CYB5), and steroid sulfotransferase (SULT2A1) play an important role in the regulation of adrenal androgen production. Specifically, adrenal production of DHEA-S is correlated with reticularis expression of SULT2A1 and CYB5. In contrast, HSD3B2 has an inverse correlation with adrenal androgen production likely due to its unique ability to remove precursors from the pathway leading to DHEA. Therefore, its expression is limited to the adrenal glomerulosa/fasciculata but not in reticularis. The differential expression of these three proteins appears to be critical for reticularis function. In this review, we focus on studies that have begun to define the mechanisms regulating the transcription of these genes. Understanding the mechanisms controlling differential expression of these proteins should provide novel information about the human adrenal reticularis and its production of DHEA and DHEA-S.

Abnormal morphology of spermatozoa in cytochrome P450 17alpha-hydroxylase/17, 20-lyase (CYP17) deficient mice.

Cytochrome P450 17alpha-hydroxylase/17, 20-lyase (CYP17) is crucial for cortisol and sex steroid biosynthesis. In a previous study we examined CYP17 function by generating mice with a targeted CYP17 deletion. We found that in addition to its role in steroid biosynthesis, CYP17 is present in germ cells. In the present study we examined the effect of CYP17 on sperm morphology. Disorganization of the sperm midpiece, small sperm mitochondria with reduced inner membranes and matrix, and irregular sperm shape were found to be associated with the CYP17 gene deletion. Treating the mice carrying the CYP17 deletion with testosterone did not alleviate the observed sperm phenotypes, suggesting that CYP17 acts in a testosterone-independent manner. These results suggest that CYP17, in addition to its role in androgen formation, is critical for proper mitochondrial architecture and sperm morphology and thus for sperm function and normal fertility.

Neurosteroids and the songbird model system.

The brain is now widely recognized as having the capacity to make steroids, neurosteroidogenesis. Although many functions are known for steroids that might be made in the brain, the evolution of and natural biological functions for these neurosteroids are not fully understood. In songbirds, neurosteroids may function in the development of neural circuits controlling song and may also participate in the activation of some steroid-dependent behaviors during the non-breeding season. In addition to neuroanatomical and behavioral evidence, we have physiological, molecular, and biochemical evidence for the expression and activity of steroidogenic enzymes in the brains of developing and adult songbirds. We review the evidence published so far for songbird neurosteroidogenesis and discuss why we believe songbird species are excellent models for the study of brain steroid synthesis and action.

CYP17 genotype modifies the association between lignan supply and premenopausal breast cancer risk in humans.

Cytochrome P450c17alpha (CYP17) has been associated with alterations in steroid hormone levels and premenopausal breast cancer risk and could modify the association between phytoestrogen intake and breast cancer risk. We examined plasma concentrations of enterolactone and genistein, estimated dietary phytoestrogen intake, CYP17 5'-UTR MspA1 genetic polymorphism, and breast cancer risk in 267 premenopausal breast cancer patients and 573 age-matched population controls from Germany. Multivariate logistic regression was used to estimate breast cancer risk associated with quartiles of phytoestrogen intake by genotype and to investigate gene-nutrient interactions. Premenopausal breast cancer risk was not significantly associated with the CYP17 A2 genotype. We observed a significant modifying effect of CYP17 genotype on plasma enterolactone-associated breast cancer risk (P for interaction < 0.01). Plasma enterolactone was significantly inversely related to breast cancer risk only in A2A2 carriers, showing odds ratios and 95% CI of 0.02 (0.00-0.41) and 0.01 (0.00-0.21) for the third and fourth quartiles vs. the lowest quartile, respectively. This inverse association was also found for the calculated enterolignan production as well as matairesinol intake. Compared with A1A1 carriers with the lowest enterolactone supply, the risk reduction associated with a high enterolactone supply resulted in a comparably decreased breast cancer risk for all genotypes. For genistein, no clear indication for a differential effect by CYP17 genotype was obtained. Our results suggest that CYP17 genotype modifies the protective effect of lignans on premenopausal breast cancer risk. Women homozygous for A2 allele benefit most from high plasma enterolactone concentrations and a high consumption of dietary precursors.

Identification of a single nucleotide polymorphism in porcine testis cytochrome P450-c17 (CYP17) and its effect on steroidogenesis.

Raising uncastrated male pigs could have significant economic benefits for pig production. Uncastrated male pigs can accumulate high levels of 16-androstene steroids, however, resulting in boar taint, which is highly objectionable to consumers. Cytochrome P450-c17 (CYP17) interacts with cytochrome b5 in the biosynthesis of the 16-androstene steroids and the sex steroids from pregnenolone. Amino acid substitutions in CYP17 could therefore affect the ability of this enzyme to catalyze the reactions leading to the production of androstenone and the sex steroids. In this study, we established a sensitive and flexible single-stranded conformational polymorphism technique capable of detecting a single nucleotide polymorphism. We then used this method to identify a substitution from T to A at nucleotide 1317 of CYP17, which caused a change in the amino acid sequence from Leu(439) to His(439). This mutation, however, did not alter the enzyme activity of CYP17 in the biosynthesis of androstenone or sex steroids. Other polymorphisms previously suggested for CYP17, which are vital for the functional interaction of CYP17 with CYB5 in human, were not observed. This study suggests that the synthesis of androstenone in pig testis is not directly affected by any polymorphisms in the coding region of the porcine CYP17 gene.

Haploinsufficiency of cytochrome P450 17alpha-hydroxylase/17,20 lyase (CYP17) causes infertility in male mice.

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is critical in determining cortisol and sex steroid biosynthesis. To investigate how CYP17 functions in vivo, we generated mice with a targeted deletion of CYP17. Although in chimeric mice Leydig cell CYP17 mRNA and intratesticular and circulating testosterone levels were dramatically reduced (80%), the remaining testosterone was sufficient to support spermatogenesis as evidenced by the generation of phenotypical black C57BL/6 mice. However, male chimeras consistently failed to generate heterozygous CYP17 mice and after five matings chimeric mice stopped mating indicating a change in sexual behavior. These results suggested that CYP17 deletion caused a primary phenotype (infertility), probably not due to the anticipated androgen imbalance and a secondary phenotype (change in sexual behavior) due to the androgen imbalance. Surprisingly, CYP17 mRNA was found in mature sperm, and serial analysis of gene expression identified CYP17 mRNA in other testicular germ cells. CYP17 mRNA levels were directly related to percent chimerism. Moreover, more than 50% of the sperm from high-percentage chimeric mice were morphologically abnormal, and half of them failed the swim test. Furthermore, 60% of swimming abnormal sperm was devoid of CYP17. These results suggest that CYP17, in addition to its role in steroidogenesis and androgen formation, is present in germ cells where it is essential for sperm function, and deletion of one allele prevents genetic transmission of mutant and wild-type alleles causing infertility followed by change in sexual behavior due to androgen imbalance.

Cytochrome P450 17alpha hydroxylase/17,20 lyase (CYP17) function in cholesterol biosynthesis: identification of squalene monooxygenase (epoxidase) activity associated with CYP17 in Leydig cells.

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is a microsomal enzyme catalyzing two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the biosynthesis of adrenal and gonadal steroids. CYP17 is a potent oxidant, it is present in liver and nonsteroidogenic tissues, and it has been suggested to have catalytic properties distinct to its function in steroid metabolism. To identify CYP17 functions distinct of its 17alpha-hydroxylase/17,20-lyase activity, we used MA-10 mouse tumor Leydig cells known to be defective in 17alpha-hydroxylase/17,20-lyase activity. A CYP17 knocked down MA-10 clone (MA-10(CYP17KD)) was generated by homologous recombination and its steroidogenic capacity was compared with wild-type cells (MA-10(wt)). Although no differences in cell morphology and proliferation rates were observed between these cells, the human chorionic gonadotropin-induced progesterone formation and de novo synthesis of steroids were dramatically reduced in MA-10(CYP17KD) cells; their steroidogenic ability could be rescued in part by transfecting CYP17 DNA into the cells. Knocking down CYP17 mRNA by RNA interference yielded similar results. However, no significant difference was observed in the steroidogenic ability of cells treated with 22R-hydroxycholesterol, which suggested a defect in cholesterol biosynthesis. Incubation of MA-10(CYP17KD) cells with (14)C-labeled squalene resulted in the formation of reduced amounts of radiolabeled cholesterol compared with MA-10(wt) cells. In addition, treatment of MA-10(CYP17KD) cells with various cholesterol substrates indicated that unlike squalene, addition of squalene epoxide, lanosterol, zymosterol, and desmosterol could rescue the hormone-induced progesterone formation. Further in vitro studies demonstrated that expression of mouse CYP17 in bacteria resulted in the expression of squalene monooxygenase activity. In conclusion, these studies suggest that CYP17, in addition to its 17alpha-hydroxylase/17,20-lyase activity, critical in androgen formation, also expresses a secondary activity, squalene monooxygenase (epoxidase), of a well-established enzyme involved in cholesterol biosynthesis, which may become critical under certain conditions.

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5.

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.

Functional characterization of mutant CYP17 genes isolated from a 17 alpha-hydroxylase/17,20-lyase-deficient patient.

CYP17 has a dual enzymatic activity that is necessary for steroid hormone biosynthesis. It catalyzes the 17 alpha-hydroxylation of progesterone or pregnenolone and also removes an acetyl moiety of hydroxy-progesterone or hydroxypregnenolone by its 17,20-lyase activity to produce androstenedione or dehydroepiandrosterone (DHEA), respectively. We previously isolated a compound heterozygous mutant of CYP17 from a Korean female patient: 1-base deletion and 1-base transversion mutation at 1 allele and 3-base deletion mutation at the other allele. Here we tested the functional activities of these 2 mutant CYP17 alleles using a transfection analysis in COS-1 cells with radiolabeled substrates and thin layer chromatography. Both mutant CYP17 genes lost not only 17 alpha-hydroxylation activity, but also 17,20-lyase activity in this assay system. This nonfunctional nature of 2 mutant CYP17 genes explains the clinical manifestation of a patient who had 17 alpha-hydroxylase deficiency.