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Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Steroid hormone biosynthesis in the adrenal cortex is controlled by adrenocorticotropin (ACTH), which increases intracellular cAMP, resulting in the activation of cAMP-dependent protein kinase(PKA) and subsequent increase in steroidogenic gene transcription. We have found that a dual-specificity phosphatase is essential for conveying ACTH/cAMP-stimulated transcription of several steroidogenic genes in the human adrenal cortex. In the present study, the role of mitogen-activated protein kinase phosphatase-1 (MKP-1), a nuclear dual-specificity phosphatase, in the transcriptional activation of human CYP17 (hCYP17) in H295R human adrenocortical cells is established. Stimulation of H295R cells with dibutyryl-cAMP (Bt(2)cAMP) induces MKP-1 mRNA and protein expression within 30 min of exposure. In transient-transfection studies, transcriptional activity of an hCYP17 promoter-reporter construct was increased by Bt(2)cAMP and by overexpression of PKA or MKP-1. Furthermore, PKA phosphorylated an MKP-1-glutathione S-transferase fusion protein in in vitro assays and Bt(2)cAMP increased (32)P associated with MKP-1 that was immunoprecipitated from H295R cells. Finally, silencing MKP-1 expression using antisense oligonucleotides attenuated cAMP-stimulated hCYP17 expression, whereas silencing of ERK1/2 increased hCYP17 expression. These findings demonstrate integral roles for MKP-1 and ERK1/2 via regulation of the phosphorylation state of steroidogenic factor-1 (SF-1) in mediating ACTH/cAMP-dependent transcription of hCYP17, thereby maintaining the balance between transcriptional activation and repression.

cAMP-dependent transcription of steroid hydroxylase genes involves activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream target proteins. Although the requirement for the activation of PKA is well established, none of the transcription factors required for steroid hydroxylase gene transcription have been found to be PKA phosphoproteins. In this study we examined the role of changes in phosphorylation state on the expression and transcriptional activity of the human CYP17 gene (hCYP17). Using inhibitors of serine/threonine phosphatase activity (okadaic acid) and phosphotyrosine phosphatase activity (peroxyvanadate), we can inhibit the cAMP-inducible binding of the steroidogenic factor-1 (SF-1), p54(nrb)/NonO, and polypyrimidine tract-binding protein-associated splicing factor (PSF) complex required for regulation of transcription to the promoter of hCYP17. Further, both okadaic acid and peroxyvanadate attenuate cAMP-stimulated increases in endogenous hCYP17 mRNA expression and in hCYP17 promoter-reporter construct luciferase activity. In vivo phosphorylation and immunoprecipitation of SF-1 show a cAMP-stimulated decrease in (32)P-labeled SF-1. Our findings demonstrate that activation of protein phosphatase(s) is essential for cAMP-dependent transcription of hCYP17 in H295R cells and suggest a role for PKA in phosphatase activation, which leads to dephosphorylation of SF-1 and increased gene transcription.

The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

The microsomal 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes the 17alpha-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many species and the 17,20-lyase activity required for the production of androgens in all species. Utilizing the technique of random chimeragenesis we have attempted to map regions of primary sequence that contribute to the species-specific biochemical differences between rat and human P450c17. We have previously reported significant differences between rat and human P450c17 in their activities, stability and substrate-dependent coupling efficiencies even though they share 68% amino acid identity. Identification of the regions of primary sequence that contribute to each of these properties would be helpful in understanding the structure/function relationships in this enzyme. A single plasmid containing the cDNAs encoding both enzymes in a tandem orientation was constructed. This plasmid was linearized at unique restriction sites and used to transform Escherichia coli. A three-step screening protocol identified five chimeras with a uniform distribution of 5' rat and 3' human sequence. All chimeric proteins yield the characteristic reduced-CO difference spectra, indicating proper folding. The chimeras exhibit a range of stability and activities that are not consistent with the degree of parental primary sequence. A chimera containing 301 N-terminal rat P450c17 amino acids and lacking the rat P450c17 phenylalanine 343, had the highest lyase activity. Generation of these functional rat/human chimeras suggests that the tertiary structures of rat and human P450c17 are sufficiently conserved to allow proper folding of chimeric enzymes. However, the properties of these chimeras did not permit identification of a region of primary sequence that contributes to a species-specific property of rat and human P450c17. Stability of these chimeras and insight into the presence of secondary structural elements is discussed., (Copyright 2000 Academic Press.)

Microsomal 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes both the 17alpha-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many animals, and the 17, 20-lyase activity required for the production of androgens in all animals. In rodents such as rat, which utilize corticosterone as the major glucocorticoid, P450c17 is expressed predominantly in the gonads, and is absent in the adrenal. In other species including humans, P450c17 is expressed in both adrenal and gonads and participates in both glucocorticoid and androgen production. Rat and human forms of P450c17 are 69% identical at the amino acid level. Based on the differences in physiological roles between P450c17 in these two species, it could be predicted that major differences would be observed in their hydroxylase activities. Contrary to this hypothesis, using partially purified, recombinant human and rat P450c17, we found that the most significant differences lie in their lyase activities. Lyase activities demonstrate that the rat enzyme favors Delta4 (progesterone) substrates while the human enzyme favors Delta5 (pregnenolone) substrates. This substrate preference is also observed in the ability of steroids to decrease uncoupled H2O2 production and to increase stability during turnover. Cytochrome b5, a microsomal electron-transfer protein, enhances lyase activities of rat and human P450c17. However, the most dramatic stimulatory effect is on the human HO-PROG lyase activity. This enhancement of activities is not associated with electron transfer. These differences in biochemical properties between the two forms of P450c17 indicate that human P450c17 has evolved as an enzyme system that limits androgen production to the gonads where a favorable b5:P450c17 ratio exists. Even though orthologous forms of P450c17 are capable of catalyzing the same enzymatic activities, specific physiological requirements of each species ensure biochemical differences between these enzymes.

Bovine CYP17 is regulated at the transcriptional level by ACTH acting through the second messenger cAMP in adrenal fasciculata and reticularis cells. Promoter analysis has previously identified two regions, proximal and distal, within the CYP17 promoter important in the cAMP dependent transcriptional regulation of this gene. The proximal (-80 to -40) cAMP responsive sequence (CRS2) has been identified as a binding site for Steroidogenic Factor-1 (SF-1)/Ad4BP. The distal region (-243 to -100) is also important for the cAMP transcriptional response as revealed by deletion analysis. Within this distal region from -243 to -225, an independent cAMP responsive sequence referred to as CRS1 has been described. The transcription factors binding CRS1 have been identified as homeodomain transcription factors belonging to an atypical class of homeodomain proteins referred to as TALE. Two families of homeodomain proteins which bind CRS1 are the Pbx and Meis1 families. Proteins from neither of these families can bind CRS1 individually; however, members of the Pbx family interact with members of the Meis1 family to cooperatively bind this element. CRS1 was the first identified cis-acting target element for members of both the Pbx and Meis1 family. Unlike SF-1, these proteins are not expressed in a steroidogenic tissue-specific manner but rather, appear ubiquitous. A current model for the function of these proteins in CYP17 regulation is that they may enhance the cAMP response through the downstream SF-1 binding site.

The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein upon purification using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs extradenticle from Drosophila melanogaster and ceh-20 from Caenorhabditis elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permit these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence.

Pbx1 is a homeodomain transcription factor involved in cAMP-dependent transcriptional regulation of the bovine CYP17 gene. In this study, we have investigated the involvement of Pbx1 in the transcriptional regulation of the human CYP17 gene. Although a sequence identical to previously determined Pbx-binding sites is not present in the promoter region of the human CYP17 gene, three putative Pbx-binding sites are identified by sequence similarity analysis. Coexpression of Pbx1 and a catalytic subunit of protein kinase A (PKA) greatly enhances reporter gene transcription via the 5'-flanking region of the human CYP17 gene. Upon gel shift analysis utilizing nuclear extracts from human adrenal H295R cells, one of the three putative Pbx1-binding sites, -250/-241 bp, shows the typical intense doublet observed with other Pbx-binding sites. 5'-Deletion analyses of the reporter construct containing this Pbx-binding site showed approximately sixfold induction by coexpression of Pbx1 and PKA compared to the basal transcription, suggesting that Pbx1 binds the -250/-241 bp sequence and participates in cAMP-dependent regulation of the human CYP17 gene.

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum., (Copyright 1997 Academic Press.)

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.

We have investigated the developmental pattern of expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein x min) at a level comparable to that in the testis (188 pmol/mg protein x min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein x min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein x min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 expression is not sexually dimorphic. Sequencing confirmed a complete identity in the coding region between hepatic and gonadal mRNAs. Hepatic P450c17 mRNA, however, was 150-200 bases longer than the gonadal counterparts. No significant expression of mRNAs encoding P450scc and P450arom was observed in liver of either sex at any age. In stomach and duodenum, enzyme activity was much lower (maxima at 25 and 14 pmol/mg protein x min, respectively) than that in liver, but persisted from the time of weaning onward. It is suggested that the hepatic peak in P450c17 activity may serve to convert circulating progestogens into androgens for gonadal aromatization during Sertoli and granulosa cell proliferation.

Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acute lymphoblastic leukemia. Previously we reported a cAMP-regulatory sequence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase cytochrome P450 (P450c17) to be the first endogenous Pbx1 binding site and that overexpression of Pbx1 in mouse adrenal Y1 tumor cells enhances cAMP-dependent transcription mediated by this element. Here we report further characterization of Pbx1 binding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated transcription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/G)GA(T/C)A which represents the 5' portion of CRS1. An artificial Pbx-binding sequence (PRS), previously determined by random PCR analysis, is similar to the Pbx1-binding sequence in CRS1 and by both gel shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhancing CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and nonsteroidogenic (HepG2 and S194) cells when coexpressed with the catalytic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of participating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors.

Corticosterone is the major circulating glucocorticoid in adult mice and rats, and this is explained, in part, by the absence of 17 alpha-hydroxylase cytochrome P450 (P450c17) in adrenal glands of these rodents. During embryonic development, however, we discovered transient expression of P450c17 in a subset of adrenocortical cells in the fetal mouse adrenal. This differs from cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, which are expressed continuously in most fetal adrenocortical cells from onset of expression at embryonic days 11-12 (E11-12) until term. Adrenal P450c17 transcripts are detectable in situ at E12.5 and increase in abundance from E12.5 to E14.5. Transcripts are then lost between E16.5 and term (E18.5) and are undetectable in situ in adrenal glands of adult mice. These results are consistent with the presence of pregnenolone 17 alpha-hydroxylase activity in adrenal homogenates of fetal but not adult mice. By using polymerase chain reaction, we determined that murine fetal (E14.5-15.5) adrenal glands contain messenger RNAs (mRNAs) encoding all of the steroid hydroxylases required to produce cortisol and corticosterone but little aldosterone synthase mRNA. Adrenal glands from adult mice contain mRNAs encoding steroid hydroxylases required to produce corticosterone and aldosterone but not cortisol (little P450c17 mRNA). The spatial and temporal expression patterns of P450c17 and aldosterone synthase mRNA, which differ from those of cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, suggest that multiple factors must be required to program cell type- and species-specific expression of these steroid hydroxylases during embryonic development.

Expression of bovine 17 alpha-hydroxylase cytochrome P450 (P450c17) in insect Sf9 cells using baculovirus is the fourth heterologous expression system developed for studying this enzyme. The enzyme in Sf9 cell membranes has all the expected activities and closely resembles that from the other systems: COS1 cells, yeast, and Escherichia coli. One curious feature of baculovirus expression of this hemoprotein is that the immunodetectable P450c17 is present in an insoluble pellet when produced in the absence of added hemin and in a 10,000 g supernatant when expressed in the presence of added hemin. When comparing the level of expression of bovine P450c17 in these different expression systems, E. coli produces the largest quantity per liter culture, while baculovirus produces the most molecules of P450c17 per cell.

Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

A clone bearing the rat CYP17 gene has been isolated and the sequence determined for 417 nucleotides 5' to the translation start site. The sequence shows one difference, a G-C inversion, with that published by Zhang et al. (1992). A transcription initiation site has been located at position -41 relative to the translation start site; this confirms the location published by Nason et al. (1992) with the caveat of the G-C inversion, so that the transcription start site becomes G instead of C. Overall percent similarities of this sequence with those of bovine, human, pig and mouse show values of 58, 64, 65, and 82, respectively. Similar comparisons done for the cAMP-responsive sequences, CRS-1 and CRS-2 (Lund et al., 1990), show 42, 55, 47 and 95% similarities for CRS-1, and 56, 77, 71 and 90% for CRS-2. These comparisons suggest that the highly conserved rat and mouse gene sequences corresponding to the bovine CRS-1 and CRS-2 may be important for either negative regulation of this gene in the adrenal or positive regulation in the gonads of these two species.

Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone and pregnenolone are metabolized to 17 alpha-hydroxysteroids by a cytochrome P450-dependent 17 alpha-hydroxylase (P450c17). The same enzyme can also catalyze the removal of the side-chain of these 17 alpha-hydroxylated steroids to yield androstenedione and dehydroepiandrosterone, respectively. We investigated the metabolism of progesterone by monkey kidney tumor (COS 1) cells transfected with a plasmid vector containing the cDNA encoding the complete amino acid sequence for human cytochrome P450c17. Transfected COS 1 cells converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone, but no detectable androstenedione was produced. However, pregnenolone was converted to 17 alpha-hydroxypregnenolone and, ultimately, dehydroepiandrosterone. No 16 alpha-hydroxypregnenolone was produced. The kinetics of progesterone metabolism by the enzyme expressed in COS 1 cells indicated that both 17 alpha- and 16 alpha-hydroxylated products were products were produced from a common active site. Microsomes prepared from fetal adrenal and adult testis converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone. No detectable androstenedione was produced by these preparations. Antibodies raised against porcine cytochrome P450c17 inhibited the 17 alpha- and 16 alpha-hydroxylation of progesterone to the same extent when using fetal adrenal microsomes, whereas no inhibition of 21-hydroxylation of progesterone was observed. Similar results were obtained with the imidazole antimycotic agent ketoconazole, which is a preferential cytochrome P450c17 inhibitor. From these results we conclude that human cytochrome P450c17 exhibits marked progesterone 16 alpha-hydroxylase activity in addition to its 17 alpha-hydroxylase function when expressed not only in a heterologous cell expression system but also, importantly, in human steroidogenic cells. Furthermore, the human enzyme has extremely low C-17,20-lyase activity toward progesterone, 17 alpha-hydroxyprogesterone, and 16 alpha-hydroxyprogesterone and fails to convert these to corresponding C19 steroids.

A truncated form of bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P450c17) lacking its amino-terminal hydrophobic signal anchor sequence (delta 2-17) was expressed in Escherichia coli to give more than 600 nmol P450 per liter using XL1-blue as the host. The expression level of the truncated P450c17 showed variation among different host strains. When intact E. coli cells were used for analysis, the truncated P450c17 showed the typical cytochrome P450 CO-difference spectrum and type I substrate binding spectra for pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, and 17 alpha-hydroxyprogesterone. The apparent Ks values for these steroids are comparable to those obtained under the same conditions with wild type P450c17. The truncated P450c17 is primarily associated with the membrane fraction from E. coli and remained with this fraction after treatment with 0.1 M Na2CO3. The 17 alpha-hydroxylase activity of the truncated form was observed using intact E. coli. Using isolated membrane fractions, the truncated P450c17 also showed 17,20-lyase activity upon reconstitution with rat liver microsomal NADPH-cytochrome P450 reductase. These results indicate that P450c17 can be associated with E. coli membranes not only via the amino-terminal hydrophobic region as expected for the wild type, nontruncated form, but also through other sequences within the polypeptide chain and that integration of the amino-terminal region into the membrane is not essential for the folding pathway leading to functional P450c17 in E. coli. This is in contrast to mammalian cells where the same truncated P450c17 is found to be inactive and the folding pathway leading to functional P450c17 appears to require a signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.

Maintenance of optimal steriodogenic capacity in the adrenal cortex requires the action of the peptide hormone ACTH. Upon binding to its cell surface receptor ACTH activates adenylate cyclase leading to elevated levels of intracellular cAMP which in turn enhances transcription of the genes encoding the enzymes involved in the conversion of cholesterol to the steroid hormones. By deletion analysis of their upstream regions, the genes encoding the steroid hydroxylases P450c17, P450c21 and P450scc (CYP17, CYP21B and CYP11A, respectively) were found to contain unique cAMP-responsive sequences (CRSs). These sequences are unique in the sense that they have not previously been described to be associated with other genes whose transcription is regulated by cAMP. Furthermore they appear to bind unique nuclear proteins or transcription factors not previously associated with cAMP-dependent transcription. This review summarizes the relatedness of these CRSs in the bovine CYP17 and CYP11A genes and the human CYP12B gene and provides an up-to-date summary of the properties of their nuclear DNA-binding proteins., (Copyright © 1992. Published by Elsevier Ltd.)

17α-Hydroxylase deficiency is characterized by a defect in either or both of 17α-hydroxylase and 17,20-lyase activities, based on the fact that a single polypeptide P450c17 can catalyze both reactions. The clinical manifestations of 17α-hydroxylase/17,20-lyase deficiency seem to be more heterogeneous than expected, varying from the classical type to less symptomatic forms as also observed in 21-hydroxylase deficiency. We have sequenced all eight exons of the CYP17 (P450c17) gene in DNA from several patients, reconstructed the mutations in a human P450c17 cDNA and expressed the mutant P450c17 in COS 1 cells to characterize the kinetic properties of 17α-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17α-hydroxylase deficiency have turned out to be complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular basis generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case clinically reported as isolated 17,20-lyase deficiency, the molecular basis was found to be partial combined deficiency of both activities, somewhat discordant with the patient's clinical profile. Based on the results obtained so far we can predict that those 17α-hydroxylase deficient individuals having a homozygous stop codon in the CYP17 gene positioned at the amino terminal side of the P450c17 heme-binding cysteine (442) will all have the same phenotype. However those individuals having homozygous missense mutations or those who are compound heterozygotes having a missense mutation in at least one CYP17 allele will display their own unique phenotype which clinically will be subtly different from all others., (Copyright © 1992. Published by Elsevier Ltd.)

The gene for human P450(17 alpha) (CYP17) was previously mapped to chromosome 10 through analysis of somatic cell hybrids. Using a modified procedure of fluorescence in situ hybridization, this gene has now been visualized on simultaneously banded chromosomes and localized to a specific subband of chromosome 10 at q24.3. This precise assignment may facilitate the understanding of the molecular basis of 17 alpha-hydroxylase/17,20-lyase deficiency and the evolution of the CYP superfamily of genes.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both the 17 alpha-hydroxylase/17,20-lyase activities. We have, for the first time, elucidated the molecular basis of the deficiency in a male pseudohermaphrodite with ambiguous external genitalia resulting from partial combined deficiency of both activities. The patient is found to be a compound heterozygote, carrying two different inherited mutant alleles in the cytochrome P45017 alpha (CYP17) gene. One allele, from his mother, contains a stop codon (TGA) in place of arginine (CGA) at amino acid position 239 in exon 4. Because this occurs at the N-terminal side of the heme binding sequence, the putative resultant truncated protein is nonfunctional. The second allele, from his father, contains a missense mutation encoding the substitution of proline (CCA) by threonine (ACA) at position 342 in exon 6. Reconstruction of this mutation by site-directed mutagenesis into human P45017 alpha cDNA followed by expression in COS 1 cells leads to the same amount of immunodetectable P45017 alpha protein as found with expression of the normal P45017 alpha cDNA, although both the 17 alpha-hydroxylase and 17,20-lyase activities are found to be reduced to 40-45% of those of the normal enzyme. The presence of ambiguous external genitalia in this 46 XY individual indicates that greater than 20% of the total normal 17,20-lyase activity is required for complete virilization in the male.

To examine the developmental expression and regulation of P450SCC and P450(17 alpha) in the rat placenta, trophoblast and decidual tissue were removed by dissection from conceptuses obtained from rats on selected days of pregnancy. Total cellular and poly(A)+ RNA and microsomal and mitochondrial fractions were isolated and analyzed for the presence of P450(17) alpha and P450SCC messenger RNA (mRNA) and protein by Northern and Western blot analysis. P450(17) alpha and P450SCC mRNA were detected in the trophoblast but not in the decidual tissue. Western blot studies demonstrated that the immunoreactive P450(17) alpha in the rat placenta is a 79-kilodalton protein, having a slower mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than has been reported for other tissue. Antiserum preabsorbed with pure P450(17) alpha was unable to detect this protein, and immunoprecipitation indicated that it is associated with enzyme activity. Development studies have revealed that the two steroidogenic enzymes are differentially expressed during the progression of pregnancy. Whereas P450SCC mRNA and protein increase abruptly between days 10-12 of pregnancy, decline thereafter, and remain low, those of P450(17) alpha increase slowly and progressively, peaking on day 18 and declining just before parturition. It is the changes in P450(17) alpha and not that of P450SCC which appear to be intimately linked to the previously reported changes in placental production of androgen. To examine whether P450(17) alpha and/or P450SCC became expressed from midpregnancy because of the rapid decline in LH that occurs at this stage, pregnant rats were treated with low but sustained levels of human CG (hCG) in order to prevent the drop in LH activity. hCG treatment caused a remarkable down regulation in the expression of both P450SCC and P450(17) alpha message and protein. In summary, the results of this investigation have established, for the first time, the presence of messages for both P450(17) alpha and P450SCC in the trophoblast tissue forming the rat placenta. The results have revealed that these two enzymes are differentially expressed during the progression of pregnancy and that the expression of their genes is down-regulated by LH/hCG.

Optimal steroidogenic capacity in the adrenal cortex is regulated by ACTH via cAMP and involves transcription of the genes encoding the adrenocortical steroid hydroxylases. The microsomal steroid hydroxylases, P45017 alpha and P450C21, are encoded by CYP17 and CYP21, respectively. These genes are thought to have arisen from a common progenitor gene and are coordinately regulated by ACTH. The cAMP responsive sequences (CRS) located in the 5'-flanking regions of these genes are distinct from one another and from known consensus sequences imparting cAMP responsiveness in other genes. The CYP21 CRS binds a putative adrenal-specific nuclear protein. In contrast, the CYP17 CRSI binds a ubiquitous protein that is apparently active only in steroidogenic cells. Thus the ACTH-dependent transcription of these two genes, which have a common evolutionary origin and are coordinately expressed in the adrenal cortex, involves distinct biochemical mechanisms.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

An in vitro transcription system has been developed to study the transcriptional control of steroid hydroxylase gene expression in steroidogenic tissues using upstream sequences of these genes in rabbit beta-globin promoter/reporter constructs. Analysis of deletion mutants of the upstream region of the bovine CYP17 (p45017 alpha) gene in whole cell extracts prepared from mouse Y1 adrenocortical cells revealed that sequences activating transcription in vitro are identical to those previously identified in vivo, namely the novel cAMP-responsive sequences crsI (-243 to -225 base pairs) and crsII (-80 to -40 base pairs). These sequences do not contain the known cAMP response elements, CRE or AP-2. In similar extracts from nonsteroidogenic mouse SVT2 cells, crsI was unable to activate transcription. Partially purified protein factor(s) binding to crsI were prepared from Y1 extracts by crsI-specific DNA affinity chromatography. This fraction activated transcription in vitro in SVT2 extracts only when crsI was present in the template. This in vitro transcription system will permit further investigation of crsI and its cognate factors and of the transcriptional regulation of the other steroid hydroxylase genes.

Cholesterol side chain cleavage cytochrome P450 (P450SCC) and 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha) are key enzymes involved in steroid hormone biosynthesis. The 5'-flanking regions of the corresponding bovine genes have recently been characterized in the adrenal-derived Y1 cell line and specific DNA sequences, required for basal and cAMP-dependent gene expression, were identified. In order to investigate the molecular mechanisms controlling P450SCC and P450(17) alpha gene expression throughout the ovarian cycle, we devised an electroporation protocol to transfect bovine luteal cells in primary culture with specific chimeric DNA constructs. Transfection of such cells with reporter gene constructs containing 186 base pairs (bp) or more of the CYP11A (P450SCC) 5'-regulatory region resulted in cAMP-responsive reporter gene expression. Reporter gene constructs containing 101 bp or less of this regulatory region were expressed neither in the presence nor in the absence of forskolin. Thus, expression of the CYP11A gene in bovine luteal cells in primary culture appears to be controlled by a cis-acting element located between -186 to -101 bp. Transfection of bovine luteal cells in primary culture with reporter gene constructs containing increasing deletions of the bovine CYP17 (P450(17) alpha) 5'-regulatory region clearly demonstrated that none of the sequences were capable of promoting significant reporter gene expression, neither in the presence nor absence of forskolin. Since no 17 alpha-hydroxylase activity and no specific mRNA encoding P450(17) alpha have been detected in the bovine corpus luteum, the failure of expression of the P450(17) alpha reporter gene constructs mimics that of the endogenous gene. These results demonstrate that the regulation of CYP11A gene expression in bovine luteal cells is carried out by the same cis-acting element (-183/-101) which serves this role in the adrenal Y1 cell line. On the other hand, expression of the CYP17 gene in bovine luteal cells appears to be completely repressed, due to the absence of specific positive transcription factor(s) or the presence of negative regulatory factor(s). Thus, transfection experiments using bovine luteal cells in primary culture represent a first step towards the elucidation of molecular mechanisms underlying the regulation of cytochrome P450 gene expression throughout the ovarian cycle.

The endoplasmic reticulum is a major site of localization for eukaryotic cytochrome P-450 mixed-function oxidase complexes. Previous studies have shown that the microsomal forms of P-450 insert into the membrane via their hydrophobic amino terminus through the signal recognition particle-dependent pathway. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the endoplasmic reticulum of COS 1 cells to evaluate the functional role of its hydrophobic amino-terminal sequence and membrane insertion. An NH2-terminal truncated protein, P450 delta 2-17, which lacked amino acids 2-17 was expressed in COS 1 cells, subcellular fractions were isolated, and P450 delta 2-17 was localized by immunoblot analysis. Compared to the full-length P45017 alpha, the NH2-terminal truncation resulted in a 2.5-fold decrease in P45017 alpha protein recovered with the microsomal fraction, 50% of which was an integral membrane protein as defined by resistance to Na2CO3 extraction. Despite correct membrane localization, P450 delta 2-17 was not a functional enzyme in COS 1 cells. A CO difference spectrum of microsomes containing P450 delta 2-17 did not give a typical 450 nm absorbance. We conclude that the hydrophobic amino terminus is required for the expression of a functionally competent P45017 alpha in COS 1 cells and suggest that the insertion of the amino terminus into the membrane is necessary for the folding of this protein into its correct structural form.

In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both of the 17 alpha-hydroxylase/17,20-lyase activities. We have elucidated the molecular basis of the combined deficiency of these activities in a Japanese female who is genotypically male and the child of a consanguineous marriage. The complete exonic sequence of the patient's CYP17 (P45017 alpha) gene revealed a seven-basepair duplication (GCGCACA) in exon 2 which leads to a frame shift and, subsequently, a premature stop codon. Because this stop codon occurs N-terminal to the heme-binding sequence, the presence of this mutation leads to the absence of a functional P45017 alpha-protein in adrenal cortex and testis. This, in turn, leads to an absence of sex steroids and excessive secretion of steroids with mineralocorticoid activity and, consequently, female external genitalia and hypertension in this 46XY patient.

Regions within the 5'-flanking sequence of the bovine CYP17 (P-450(17)alpha) gene which are required for cAMP-dependent regulation of transcription have been localized by transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells. Two sequences have been found which individually confer cAMP responsiveness to reporter genes; they are located at -243/-225 and -80/-40 base pairs (bp). Obvious sequence homology between these two regions is not apparent. Gel shift competition analysis indicates that nuclear protein(s) binding to the -243/-225-bp region can be competed for by the addition of a double-stranded oligonucleotide containing a consensus cAMP-responsive element (CRE) from the human chorionic gonadotropin alpha gene, whereas addition of this CRE does not abolish protein-DNA complexes formed with fragments containing the -80/-40-bp sequence. Gel shift and Southwestern analysis indicate that the -243/-225-bp region of the P-450(17)alpha gene and the CRE both bind a 47-kDa protein and that the CRE binds additional proteins (43 and 68 kDa) not apparently recognized by the -243/-225-bp sequence. Thus cAMP-dependent regulation of the bovine P-450(17)alpha gene appears to involve two independent cis-regulatory regions, neither of which contains a consensus CRE. Based on protein binding analysis, one of these regions (that including -80/-40 bp) is distinct from the consensus CRE while the other (that containing -243/-225 bp) may be related to the consensus CRE.

The promoter/regulatory region of the bovine CYP11A (P-450scc) gene was cloned from a bovine genomic library. One major start site of transcription was identified by primer extension analysis with a minor start site four nucleotides further upstream. A putative TATA box is located at position -31, and at position -68 resides a putative binding site for the transcription factor Sp1. Transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells was used to locate regions within the P-450scc 5'-flanking sequences that are important for basal and cAMP-dependent transcription of the reporter genes. While cAMP-dependent accumulation of mRNA derived from expression of the endogenous bovine P-450scc gene can be inhibited by protein synthesis inhibitors, transcription of reporter gene constructs containing the promoter/regulatory region of the P-450scc gene is not affected by cycloheximide following transient transfection of Y1 cells or primary bovine adrenocortical cells. Basal expression of these constructs as well as cAMP responsiveness is reduced upon deletion of sequences between -186 and -101, further deletion to -50 leading to loss of virtually all the remaining cAMP responsiveness. The sequence between -183 and -83 alone will direct both basal and cAMP-enhanced transcription when fused to a heterologous promoter and is equally active in either the correct or reverse orientation. No homology to the consensus cAMP-responsive element (CRE) or AP-2 binding site is found in this region whereas an activator protein 1-like sequence is found at position -116. It is concluded that the cAMP responsiveness of P-450scc gene expression is mediated by sequences different from canonical consensus regulatory elements. Whether or not there are sequences conferring cAMP responsiveness which are common both to P-450scc and the other steroidogenic P-450 genes remains to be established.

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.

The complete exonic and partial intronic sequence of the bovine CYP17 (P45017 alpha) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when approximately 500 bp of the 5'-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.

Cortisol production requires the activity of only 17 alpha-hydroxylase, whereas the formation of sex steroids requires both 17 alpha-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha), catalyzes both activities. By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450(17) alpha, it has now been established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17 alpha-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17 alpha-hydroxypregnenolone is the substrate than when 17 alpha-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450(17) alpha must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.

Steroid 17 alpha-hydroxylase (cytochrome P-450 17 alpha) catalyzes both 17 alpha-hydroxylation of pregnenolone and progesterone and 17,20-lysis of 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone. In the course of undertaking detailed investigation of the structure-function relationships which exist within this enzyme we have begun to elucidate the molecular basis of human deficiencies in either or both of these activities. Consequently we have determined the exonic structure of the human P-450 17 alpha gene as well as the sequences at the exon/intron boundaries and at the site of initiation of transcription. A single gene in the human genome encodes this protein, being the sole member of a unique gene family (P450XVII) within the P-450 supergene family. A protocol for exonic sequencing of the P-450 17 alpha gene has been established which permits structural analysis of the gene from patients having 17 alpha-hydroxylase and/or 17,20-lyase deficiency. This procedure has been applied to the mutant gene from one individual having combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A four-base duplication is found in exon 8 producing a protein with an altered C-terminal amino acid sequence which results in loss of both enzymatic activities.

Bovine adrenocortical cells in primary culture were used to examine the trophic effect of ACTH on the induction of the 17 alpha-hydroxylase and C-17,20-lyase activities. The addition of exogenous pregnenolone to bovine adrenal microsomes showed the appearance of 17 alpha-hydroxy-pregnenolone before the formation of dehydroepiandrosterone. The same sequence of activities was evident in postmitochondrial supernate from bovine adrenocortical cells cultured 36 h in the presence of 1 microM ACTH but not in postmitochondrial supernate from control cells. In another study, bovine adrenocortical cells were cultured for 36 h after which 30 microM 17 alpha-hydroxypregnenolone was added to the medium and the incubation continued 1 h; there was a 4-fold increase in androgen content in the media from ACTH-treated cells over controls. Measurement of the 17 alpha-hydroxylase and C-17,20-lyase reactions in postmitochondrial supernate from cells cultured 0-72 h in the presence of ACTH or 1 mM dibutyryl cAMP showed concomitant increases in the two activities and both activities were inhibited by the same compounds known to inhibit 17 alpha-hydroxylase activity. These observations support the concept of the co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in response to ACTH; results in keeping with previous studies indicating that the two activities are catalyzed by a single gene product, the polypeptide chain P-45017a.

Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17 alpha-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17 alpha-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17 alpha-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17 alpha-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17 alpha-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17 alpha-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17 alpha-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17 alpha-hydroxylase activity is due, at least in part, to an elevation of cytochrome P-450(17)alpha synthesis.

Among the multifactorial aspects of regulation of steroid hydroxylase gene expression, it is the developmental process which leads to imprinting of expression of particular steroid hydroxylases in specific cell types. We have begun to investigate the ontogeny of steroidogenesis in fetal bovine tissues. Expression of most steroid hydroxylases and related enzymes is detectable in adrenals of the smallest fetuses studied and continues throughout fetal life. Like the other steroid hydroxylases, P-450(17)alpha is detectable in the earliest fetal adrenals studied. However, following an increase in expression, P-450(17)alpha disappears from the fetal adrenal by 100 days gestational age and remains absent until about 230 days gestational age. The absence of P-450(17)alpha is correlated with the absence of cortisol in the fetal adrenal and the absence of ACTH in fetal plasma. Thus expression of P-450(17)alpha in bovine fetal adrenal appears to be strictly dependent on cAMP while expression of other steroid hydroxylases appears to involve both cAMP-dependent and cAMP-independent mechanisms. Furthermore, P-450(17)alpha is expressed in fetal testis at gestational times when it is absent in fetal adrenal. We have begun to examine binding of nuclear proteins from adrenals of various gestational ages to the 5'-flanking region of the bovine P-450(17)alpha gene. Preliminary evidence indicates the presence of a protein in nuclei of fetal adrenals not expressing P-450(17)alpha that is not present in fetal adrenals of other gestational ages.

The effects of adrenocorticotropin (ACTH) on 17 alpha-hydroxylase activity and cytochrome P-450(17 alpha) synthesis have been studied utilizing bovine adrenocortical cells in primary monolayer culture. A 20-fold stimulation of the conversion of pregnenolone to 17 alpha-hydroxypregnenolone was observed in postmitochondrial supernatant fractions from cells treated with ACTH as compared to controls. This increase in 17 alpha-hydroxylase activity was found to be due to a change in the Vmax and not a change in the Km(app). By immunoisolation of newly synthesized protein from an RNA-directed cell-free translation system we found that the level of P-450(17 alpha) was many-fold greater when RNA from ACTH-stimulated cells was used, as compared to RNA from control cells. A similar pattern was obtained when the rate of P-450(17 alpha) synthesis was analyzed by immunoisolation from radiolabeled cellular protein from ACTH-stimulated cells. When the total amount of P-450(17 alpha) was measured by immunoblotting we found that the levels of enzyme present correlated with the 17 alpha-hydroxylase activity. In addition, we found that these ACTH-mediated effects could be mimicked by treatment of cells with analogs of cyclic AMP. These results indicate that the activity of P-450(17 alpha) is regulated primarily by cyclic AMP-mediated changes in synthesis, probably at the transcriptional level, which in turn has a profound effect on the pattern of steroid secretion. Thus, we believe cytochrome P-450(17 alpha) to be a key regulatory enzyme in the steroidogenic pathway.

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

The developmental expression of adrenocortical steroid hydroxylases was studied in bovine fetuses from 40 to 280 days gestational age. The expression of P-450(17 alpha) is first detected at a gestational age of 50 days and reaches a maximum at 60-70 days. The expression of P-450(17 alpha) then declines and is nondetectable at a gestational age of 100 days. P-450(17 alpha) is not expressed again until about 240 days, i.e. shortly before birth (approximately 280 days). P-450scc, P-450c21, P-450(11 beta) and adrenodoxin were present in fetal adrenals throughout gestation. This "on-off-on" pattern of P-450(17 alpha) expression during fetal development was associated with a corresponding episodic production of cortisol. Immunoreactive corticotropin (ACTH) levels in fetal plasma were elevated in small fetuses (corresponding to less than or equal to 100 days) and in near-term fetuses (corresponding to greater than 250 days) compared with those in mid-gestation fetuses. In primary culture, adrenal cells from mid-gestation fetuses contained no detectable P-450(17 alpha) but rapidly responded to ACTH with an increase in P-450(17 alpha) protein and mRNA. The tissue specificity of the developmental patterns is emphasized by the fact that both P-450(17 alpha) and P-450scc were detectable throughout the development of the fetal testes, whereas only P-450scc was detectable in fetal bovine ovary prior to 200 days. Thus, in fetal bovine adrenal it appears that ACTH is the major regulatory factor effecting the intermittent presence of P-450(17 alpha), whereas the presence of the other steroid hydroxylases is either regulated by additional factors or shows a much different sensitivity to ACTH.