Steroidogenic factor 1/adrenal 4 binding protein transforms human bone marrow mesenchymal cells into steroidogenic cells

Steroidogenic factor 1/adrenal 4 binding protein (SF-1/Ad4BP) is an essential nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. Mesenchymal bone marrow cells (BMCs) contain pluripotent progenitor cells, which differentiate into multiple lineages. In a previous study, we reported that adenovirus-mediated forced expression of SF-1 could transform mouse primary long-term cultured BMCs into steroidogenic cells. For future clinical application, trials using human BMCs would be indispensable. In this study, we examined whether SF-1 could transform human BMCs into steroidogenic cells and compared the steroid profile of these cells with that of mouse steroidogenic BMCs. Primary cultured human BMCs infected with adenovirus containing bovine SF-1 cDNA could produce progesterone, corticosterone, cortisol, dehydroepiandrosterone, testosterone, and estradiol. Such a mixed character of adrenal and gonadal steroid production in human BMCs was supported by the expressions of P450scc, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c21, P450c11, P450c17, 17beta-HSD, and P450arom mRNAs. Unlike mouse steroidogenic BMCs, introduction of SF-1 into human BMCs caused dramatic inductions of both ACTH and LH receptors, thus leading to good responsiveness of the cells to ACTH and LH respectively. Importantly, among several factors that are known to be closely associated with adrenal and/or gonadal development, introduction of only SF-1 enabled the human BMCs to express P450scc and to produce cortisol and testosterone, suggesting that SF-1 is truly a master regulator for the production of steroidogenic cells from human BMCs.     

SF-1, DAX-1, and acd: molecular determinants of adrenocortical growth and steroidogenesis

The formation of the adrenal cortex in humans is notable for the presence of two discrete zones, the fetal zone (FZ) which regresses soon after birth and the definitive zone (DZ) which gives rise to the classic steroidogenic zones of the adult cortex. Mice possess an analogous structure to the FZ referred to as the X-zone (XZ) which regresses at puberty in the male and during the first pregnancy in the female. Similar to the human FZ in X-linked Congenital Adrenal Hypoplasia caused by loss of function mutations in DAX-1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita critical region on the X chromosome), the mouse XZ does not regress when DAX-1 is mutated. Only in humans with DAX-1 mutations, however, is the DZ small and hypofunctional. Patients and mice with SF-1 mutations have complete adrenal aplasia with absence of both the DZ and FZ/XZ. Lastly, the phenotype of the Autosomal Recessive Adrenocortical Dysplasia (acd) mouse is strikingly similar to human Miniature Adult Congenital Adrenal Hypoplasia, lacking an XZ/FZ and possessing a dysfunctional DZ. Current work has addressed the regulation of SF-1 and DAX-1 dependent adrenocortical growth and steroidogenesis in vivo utilizing mouse models of simple and combined SF-1 and DAX-1 deficiency. In addition, the model of compensatory adrenal growth in SF-1 haplo-insufficient mice has been applied to evaluate the potential role of SF-1 in adrenocortical proliferation. Additional efforts aim to positionally clone the acd gene, predicated on the hypothesis that it is a critical component of the adrenal developmental cascade.     

Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa

Gonadal differentiation in some species of amphibians is sensitive to steroids. The phenotypic sex of XX/XY-type frogs such as Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles, but little is known about the molecular mechanism of this sex reversal. To elucidate the mechanism of the sex differentiation, we examined the role of P450 aromatase (P450arom), an enzyme that converts testosterone to estrogen, during gonadal differentiation of amphibians. In this study, we first cloned a P450arom cDNA homolog of the frog R. rugosa and analyzed by RT-PCR its expression profile in developing and in female-to-male sex-reversed gonads. P450arom expression was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary in which only immature oocytes were observed. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, when oocytes were growing; and it was no longer seen in adult ovaries. By RT-PCR, we also examined the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it declined rapidly after the injection of testosterone. In contrast, no change in the SF-1 (also known as Ad4BP) expression was observed. Moreover, to identify the type(s) of cells expressing P450arom protein, we performed immunostaining with an antibody against frog P450arom protein. Cells giving positive signals were observed around oocytes in the ovary of frogs 1 month after metamorphosis. They were identified as follicle cells by both light and electron microscopy. The results, taken together, indicate that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa.     

A shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation

Gonadotropins (GTHs; FSH and LH) require two major steroidal mediators, estradiol-17 beta (E(2)) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP) to act as critical hormones to execute oocyte growth and maturation, respectively. A two-cell type model has been proposed, where the theca cells provide the precursor steroids, and the granulosa cells produce the two steroidal mediators under the direct influence of FSH and LH. A distinct shift in steroidogenesis, i.e. from E(2) to 17 alpha,20 beta-DP as well as the steroidogenic enzyme genes from ovarian cytochrome P450 aromatase (oP450arom) to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD), occurs in the granulosa layers of ovarian follicles prior to oocyte maturation. The triggering of the steroidogenic shift by GTHs in granulosa cells occurs through the subjugation of Ad4BP/SF-1 expression in respect of oP450arom, followed by an over-expression of 20 beta-HSD probably through the CREB.     

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.     

Regulation of steroidogenesis and steroidogenic acute regulatory protein in R2C cells by DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1)

In the present study, steroidogenesis in two different Leydig tumor cell lines was compared. One, the MA-10 mouse tumor cell line, produces steroids and the steroidogenic acute regulatory (StAR) protein only when stimulated by trophic hormones and cAMP analogs. The other, the R2C rat tumor cell line, produces steroids and the StAR protein constitutively without stimulation. We observed that high levels of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1), a repressor of steroidogenesis and StAR gene expression, were present in MA-10 cells but not in R2C cells. Based upon this observation, we hypothesized that the absence of DAX-1 might result in constitutive steroidogenesis in R2C cells. To test this hypothesis, DAX-1 was overexpressed in the R2C cells using the Tet-on inducible gene expression system and resulted in a 45% decrease in steroid production, a 35% decrease in StAR protein, and a 39% decrease in cytochrome P450 side chain cleavage expression. Further, using retroviral infection with DAX-1, StAR expression and steroidogenesis were decreased 50-60% and 60% in R2C cells, respectively. These results corroborate previous findings that DAX-1 negatively regulates steroid synthesis through the inhibition of StAR expression and indicate that the absence of DAX-1 in R2C cells is, at least in part, responsible for the constitutive steroidogenesis and StAR expression observed.