Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.

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 pathway for the metabolism of vitamin D3: unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1)

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.     

Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.     

Hormonal and developmental regulation of adrenodoxin messenger ribonucleic acid in steroidogenic tissues

Adrenodoxin is an iron-sulfur protein found in the mitochondria of steroidogenic tissues. It participates in steroidogenesis as an electron transport intermediate for mitochondrial cytochromes P450, including P450scc, the cholesterol side-chain cleavage enzyme. Using a human adrenodoxin cDNA probe recently cloned in our laboratory, we examined the distribution and hormonal regulation of adrenodoxin mRNA in a variety of steroidogenic tissues. Adrenodoxin mRNA was found in all steroidogenic tissues examined. In human fetal testes, adrenodoxin mRNA was more abundant in early gestation, diminishing toward midterm in a pattern closely similar to that we reported previously for P450scc. Unlike P450scc, however, significant amounts of adrenodoxin mRNA were detected in human fetal ovaries, with no discernible gestation-dependent change. The abundance of adrenodoxin mRNA was increased in cultured human granulosa cells by treatment with hCG, FSH, cAMP, and cholera toxin. In human fetal adrenal cells, ACTH and cAMP stimulated accumulation of adrenodoxin mRNA, while in cultured human fetal testicular cells and cultured fetal rhesus monkey ovarian cells, both hCG and cAMP stimulated accumulation of adrenodoxin mRNA. In all of these systems, the accumulation of adrenodoxin mRNA closely paralleled the response of P450scc. These data suggest that the genes for these functionally related but structurally unrelated proteins are regulated in a coordinate manner.     

Compound heterozygous mutations in the cholesterol side-chain cleavage enzyme gene (CYP11A) cause congenital adrenal insufficiency in humans

Cholesterol side-chain cleavage enzyme (P450scc) catalyzes the conversion of cholesterol to pregnenolone in mitochondria, which is the first step in the biosynthesis of all steroid hormones. Until now, no homozygous or compound heterozygous mutations in CYP11A have been described in humans. Here we describe novel compound heterozygous mutations in CYP11A in a patient with congenital adrenal insufficiency born to healthy parents. One mutation, a maternally inherited R353W mutation, resulted in markedly reduced P450scc activity by the single amino acid substitution, indicating that Arg(353) is a crucial amino acid residue for P450scc activity. The other mutation, a de novo A189V mutation in the paternal allele, did not affect the P450scc activity by the single amino acid substitution and turned out to be a splicing mutation, which created a novel alternative splice-donor site. It resulted in a deletion of 61 nucleotides in the open reading frame and thus partially inactivated CYP11A. These experimental data are consistent with the clinical findings indicating that the patient had partially preserved ability to synthesize adrenal steroid hormones. This is the first report of the compound heterozygote for the CYP11A mutations with congenital adrenal insufficiency and the phenotypically normal heterozygote in humans.     

Current developments in the molecular genetics of the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is most likely a complex trait with an oligogenic basis. In this article, we present evidence from molecular genetic studies for involvement of the steroid synthesis gene CYP11a (coding for P450 cholesterol side-chain cleavage) in the aetiology of hyperandrogenism. Variation in the regulatory region of the insulin gene also appears to contribute to the development of PCOS.     

Differential capacity for cholesterol transport and processing in large and small rat luteal cells.

The aim of this investigation was to determine whether a specific luteal subpopulation is responsible for the hypertrophic development of the corpus luteum at midpregnancy in the rat and to determine whether there was an underlying cellular basis for the differential production of steroids by the luteal cell subtypes. To examine this, we have dispersed and separated rat luteal steroidogenic cell populations into small (< 20 microns) and large (> 30 microns) cell types by elutriation. Luteal cells were examined at early (day 3) and midpregnancy (day 14) for differences in protein content and for differential expression of proteins required for steroid production. Specific proteins examined include the P450side chain cleavage enzyme (P450scc), adrenodoxin and adrenodoxin reductase, proteins required for cholesterol conversion to progestagens in the corpus luteum, and sterol carrier protein-2 (SCP2), a protein thought to be involved in intracellular cholesterol transport. The cytochrome P450(17)alpha hydroxylase (P450(17)alpha), a key enzyme responsible for androgen biosynthesis was also examined in the isolated luteal cells. The large luteal cell population displayed an increase in total cellular protein content while the small cell type did not change with luteal development. In addition, the large luteal cells expressed proteins unique to or elevated in that cell type. Analysis by two-dimensional polyacrylamide gel electrophoresis revealed that the large cell-specific proteins had molecular masses of 23 K and 32 K and that a 14 kilodalton (kDa) protein was elevated in the large cell type relative to the small cells. The small luteal cell on day 3 of pregnancy expressed a 36 kDa protein which was barely detectable in the large cell. Immunocytochemical and Western analysis indicated that the large luteal cells contain 5.3-fold more SCP2 (P < 0.05) and 5.6-fold more P450scc (P < 0.001) relative to the small cell type. Immunocytochemical staining of adrenodoxin and adrenodoxin reductase indicate these proteins were elevated in the large cell as well. Human CG administration stimulated P450(17)alpha expression mainly in the large luteal cell population. The results of this investigation indicate, for the first time, that the large luteal cell of the rat, in contrast to the small cell type, undergoes a dramatic increase in protein content with luteal development, and that with this increase in cell size there is a concomitant increase in the large cell capacity to produce steroids. This occurs as a direct result of the enhanced expression of SCP2, P450scc, adrenodoxin and adrenodoxin reductase, proteins specifically required to transport and process cholesterol for steroid production in the large luteal cell.     

Lanosterol 14alpha-demethylase and MAS sterols in mammalian gametogenesis

The lanosterol 14alpha-demethylase protein complex is composed of a cytochrome P450 enzyme CYP51 and its redox partner NADPH cytochrome P450 reductase. The complex participates in cholesterol biosynthesis and produces folicular fluid meiosis activating sterol (FF-MAS) from lanosterol. FF-MAS is metabolized further by sterol Delta14-reductase to testis-meiosis activating sterol (T-MAS). Additional enzymatic steps are needed before cholesterol is produced. Using the anti-human CYP51 antibody we have studied CYP51 protein expression by confocal microscopy in male and female mouse gonads. Leydig cells and acrosomes of spermatids express the highest levels of the CYP51 protein. CYP51 protein is also detected in primary mouse oocytes of non-treated mice and in some granulosa cells. While regulatory mechanisms responsible for FF-MAS accumulation in the ovary are not yet established, two mechanisms contributing to production the of T-MAS in the testis have been found. Potential in vivo roles of FF-MAS and T-MAS in fertilization are discussed.     

Structure and mechanism of the complex between cytochrome P4503A4 and ritonavir

Ritonavir is a HIV protease inhibitor routinely prescribed to HIV patients that also potently inactivates cytochrome P4503A4 (CYP3A4), the major human drug-metabolizing enzyme. By inhibiting CYP3A4, ritonavir increases plasma concentrations of other anti-HIV drugs oxidized by CYP3A4 thereby improving clinical efficacy. Despite the importance and wide use of ritonavir in anti-HIV therapy, the precise mechanism of CYP3A4 inhibition remains unclear. The available data are inconsistent and suggest that ritonavir acts as a mechanism-based, competitive or mixed competitive-noncompetitive CYP3A4 inactivator. To resolve this controversy and gain functional and structural insights into the mechanism of CYP3A4 inhibition, we investigated the ritonavir binding reaction by kinetic and equilibrium analysis, elucidated how the drug affects redox properties of the hemoprotein, and determined the 2.0 ? X-ray structure of the CYP3A4-ritonavir complex. Our results show that ritonavir is a type II ligand that perfectly fits into the CYP3A4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen, which decreases the redox potential of the protein and precludes its reduction with the redox partner, cytochrome P450 reductase.     

Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system

A fusion construct for the human cholesterol side-chain cleavage enzyme system termed F2 (H(2)N-P450scc-adrenodoxin reductase-adrenodoxin-COOH), was stably expressed in nonsteroidogenic COS-1 cells. Multiple clones were obtained and analyzed, identifying the clone COS-F2-130 as the most active in converting 22R-hydroxycholesterol (22R-OH-C) to pregnenolone. The F2 fusion construct was properly transcribed and translated in COS-F2-130 cells, indicating that these cells did not proteolytically cleave the F2 protein. Steroid analyses show that the COS-F2-130 cells do not convert appreciable quantities of pregnenolone to other steroids. Isolated COS-F2-130 mitochondria showed enhanced steroidogenesis when incubated with biosynthetic N-62 StAR protein in vitro. The cells were easily transfectable with StAR expression vectors, showing that COS-F2-130 cells exhibited both StAR-independent and StAR-dependent activity. Transient expression of either full-length or N-62 StAR stimulated steroidogenesis to approximately 45% of the maximal steroidogenic capacity, as indicated by incubation with 22R-OH-C. Single, double, and triple transfections of individual vectors expressing P450scc, adrenodoxin reductase, and adrenodoxin demonstrated that the P450 moiety of the F2 fusion protein could only receive electrons from the covalently linked adrenodoxin moiety, but that free adrenodoxin reductase could foster activity of the fusion enzyme. COS-F2-130 cells provide a useful system for studying steroidogenesis, as these are the only cells described to date that convert cholesterol to pregnenolone but lack downstream enzymes that catalyze other steroidogenic reactions.     

The influence of the amino acid substitution I98K on the catalytic activity of baboon cytochrome P450 side-chain cleavage (CYP11A1)

Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and "rate-limiting" step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS-1 cells with 25-hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 microM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.     

Regulation of mRNAs encoding the steroidogenic acute regulatory protein and cholesterol side-chain cleavage enzyme in the elasmobranch interrenal gland

The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes.     

Zone-specific regulation of two messenger RNAs for P450c11 in the adrenals of pregnant and nonpregnant rats.

Adrenal mitochondria possess two steroidogenic cytochrome P450s. P450c11 converts deoxycorticosterone to corticosterone and aldosterone, and P450scc converts cholesterol to pregnenolone. These P450s receive electrons from NADPH via adrenodoxin reductase and adrenodoxin. A single bovine P450c11 protein has 11-hydroxylase, 18-hydroxylase, and 18-oxidase activities, but this series of enzymatic steps may be mediated by more than one enzyme in rats. Enzymatic assays of purified rat mitochondrial proteins have suggested that one enzyme found in all zones of the adrenal cortex has both 11- and 18-hydroxylase activities, whereas another enzyme, found exclusively in the zona glomerulosa, catalyzes 18-hydroxylation and 18-oxidation of corticosterone. We studied the number and zonal distribution of P450c11 mRNA species in the rat adrenal and how these mRNAs are regulated in the adrenals of normal and pregnant rats. Rats synthesize two similar, but distinct, P450c11 mRNAs. One, P450c11A, is found in both the zona glomerulosa and fasciculata/reticularis, whereas the second, P450c11B, is found only in the zona glomerulosa. The abundance of neither P450c11A mRNA nor P450c11B mRNA is affected by a high-salt diet. However, when rats receive a low-salt diet, P450c11A mRNA decreases and P450c11B mRNA increases. Dexamethasone decreases the amount of P450c11A mRNA without affecting P450c11B mRNA. The combination of a high-salt diet and dexamethasone decreases the amount of both mRNAs further to almost undetectable amounts. Rats given a low-salt diet and dexamethasone have a dramatic increase in the abundance of P450c11B mRNA. Thus both forms of P450c11 mRNA are regulated independently in the rat adrenal cortex. In situ hybridization studies show that only the P450c11 found in the zona glomerulosa is regulated by salt treatment in vivo, whereas glucocorticoid treatment in vivo regulates P450c11 in all zones. In the adrenals of pregnant rats, P450c11B is regulated in a similar fashion to its regulation in the nonpregnant rat adrenal, despite major differences in sodium retention and intravascular volume in pregnant and nonpregnant rats. In the pregnant rat, a low-salt diet increases the abundance of P450c11B to a greater degree than in the nonpregnant rat. By contrast, dexamethasone does not diminish the abundance of P450c11A mRNA in the pregnant rat but reduces it to an almost undetectable amount in the nonpregnant rat. Thus, the regulation of glucocorticoid and mineralocorticoid production in the pregnant and nonpregnant rat occurs by different mechanisms.     

19-Hydroxylase inhibition of adrenal mitochondrial P450 11 beta/18/19-hydroxylase by a suicide inhibitor

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid that is increased in some forms of experimental and human hypertension. The pivotal step in 19-nor-DOC biosynthesis is adrenal P450 19-hydroxylase, but this enzyme has not been clearly distinguished from P450 11 beta/18-hydroxylase. This study attempted to specifically inhibit adrenal 19-hydroxylation of deoxycorticosterone (DOC) using a suicide aromatase inhibitor, 19-acetylenic androstenedione (19-AA). Purified bovine P450 11 beta/18/19-hydroxylase was incubated with excess substrate DOC, adrenodoxin, and adrenodoxin reductase in the presence of increasing doses of the inhibitor, 19AA. 11 beta-, 18-, and 19-hydroxylation were measured by quantification of corticosterone, 18-OH-DOC, and 19-OH-DOC respectively. Measurements of these products demonstrated that 11 beta- and 18-hydroxylation was not inhibited whereas 19-hydroxylation was inhibited as manifested by decreased 19-OH-DOC formation (p less than .05). The IC50 of 19-AA was approximately 10(-12) M. The specific inhibition of 19-hydroxylation suggests that the 19-hydroxylase may be an enzyme distinct from the P450 11 beta/18-hydroxylase. This further suggests that 19-nor-DOC biosynthesis may be under independent regulation and may be amendable to specific in vivo inhibition.     

Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women

Abstract Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients. In the present study we have screened, using heteroduplex analysis, the genes encoding StAR and SF-1 as well as DAX-1 and CYP11A for mutations in genomic DNA from 19 women presenting with hirsutism and increased serum androgen levels. When variants were found, analysis was extended to a larger group of hyperandrogenic patients and nonaffected women. Two variants were identified in the SF-1 gene. A G-->C change in exon 6, resulting in an Arg(365)Pro mutation, was found in 1 of 45 patients, but not in controls. Also, a Gly(146)Ala missense mutation, resulting from a G-->C change in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected individuals. We identified a C-->T base pair change at position -33 of the StAR gene. Three of 48 patients and 3 of 43 controls presented this variant. No mutations were found in coding regions of the StAR gene. Analysis of CYP11A-coding regions identified a G-->A change in exon 3, resulting in a Val(179)Ile missense mutation. This mutation was found in 1 of 29 patients studied and was not present in 50 controls. Finally, analysis of DAX-1 showed no variant in any of the women studied. In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.     

Construction of gene therapy vectors targeting adrenocortical cells: enhancement of activity and specificity with agents modulating the cyclic adenosine 3',5'-monophosphate pathway

In preliminary studies we demonstrated that the CYP11B1 (11beta-hydroxylase) promoter could direct specific expression of a suicide gene in adrenocortical cancer cells, providing a potentially specific therapeutic option for adrenocortical cancer. In this present study we describe our attempts to enhance the activity of the CYP11B1 promoter while maintaining its specificity for adrenal cells. Using a putative enhancer element from the cholesterol side-chain cleavage (P450scc) gene, the activity of the CYP11B1 promoter in and its specificity for adrenocortical cells were enhanced. Treatment with 8-bromo-cAMP or forskolin resulted in further enhancement. In stably transfected Y-1 cells, in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the CYP11B1 promoter with the P450scc enhancer element, HSV-TK expression and ganciclovir sensitivity were augmented by treatment with 8-bromo-cAMP, forskolin, and ACTH. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity to adrenocortical cells. We propose that a similar strategy using differentiating agents may be useful in the gene therapy of tumors with unique differentiated properties, including those arising from other endocrine organs.     

Homozygous disruption of P450 side-chain cleavage (CYP11A1) is associated with prematurity, complete 46,XY sex reversal, and severe adrenal failure

Disruption of the P450 side-chain cleavage cytochrome (P450scc) enzyme due to deleterious mutations of the CYP11A1 gene is thought to be incompatible with fetal survival because of impaired progesterone production by the fetoplacental unit. We present a 46,XY patient with a homozygous disruption of CYP11A1. The child was born prematurely with complete sex reversal and severe adrenal insufficiency. Laboratory data showed diminished or absent steroidogenesis in all pathways. Molecular genetic analysis of the CYP11A1 gene revealed a homozygous single nucleotide deletion leading to a premature termination at codon position 288. This mutation will delete highly conserved regions of the P450scc enzyme and thus is predicted to lead to a nonfunctional protein. Both healthy parents were heterozygous for this mutation. Our report demonstrates that severe disruption of P450scc can be compatible with survival in rare instances. Furthermore, defects in this enzyme are inherited in an autosomal-recessive fashion, and heterozygote carriers can be healthy and fertile. The possibility of P450scc-independent pathways of steroid synthesis in addition to the current concept of luteoplacental shift of progesterone synthesis in humans has to be questioned.     

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells.

Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.