Douglas M. Stocco

DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis

Mutations in the DAX-1 gene are responsible for congenital X-linked adrenal hypoplasia, a disease that is associated with hypogonadotropic hypogonadism,. DAX-1 expression is tissue-specific and is finely regulated throughout development, suggesting that it has a role in both adrenal and gonadal function. DAX-1 is an unusual member of the nuclear-receptor superfamily of transcription factors which contains no canonical zinc-finger or any other known DNA-binding motif. Binding sites for DAX-1 are found in the promoters of the dax-1 and StAR (for steroidogenic acute regulatory protein) genes. Here we show that DAX-1 binds DNA and acts as a powerful transcriptional repressor of StAR gene expression, leading to a drastic decrease in steroid production. We provide in vitro and in vivo evidence that DAX-1 binds to DNA hairpin structures. Our results establish DAX-1 as the first member of the nuclear receptor superfamily with novel DNA-binding features and reveal that it has regulatory properties critical to the understanding of its physiological functions.

Phosphorylation of Steroidogenic Acute Regulatory Protein (StAR) Modulates Its Steroidogenic Activity

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR’s action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase.32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitrotranscription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [γ-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis

The rate-limiting, hormone-regulated, enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme system (CSCC), which is located on the matrix side of the inner mitochondrial membrane. However, it has long been observed that hydrophilic cholesterol-like substrates capable of traversing the mitochondrial membranes are cleaved to pregnenolone by the CSCC in the absence of any hormone stimulation. Therefore, the true regulated step in the acute response of steroidogenic cells to hormone stimulation is the delivery of cholesterol to the inner mitochondrial membrane and the CSCC. It has been known for greater than three decades that transfer of cholesterol requires de novo protein synthesis; however, prior to this time the regulatory protein(s) had yet to be identified conclusively. It is the purpose of this commentary to briefly review a number of the candidates that have been proposed as the acute regulatory protein. As such, we have summarized the available information that describes the roles of transcription, translation, and phosphorylation in this regulation, and have also reviewed the supporting cases that have been made for several of the proteins put forth as the acute regulator. We close with a comprehensive description of the Steroidogenic Acute Regulatory protein (StAR) that we and others have identified and characterized as a family of proteins that are synthesized and imported into the mitochondria in response to hormone stimulation, and for which strong evidence exists indicating that it is the long sought acute regulatory protein.

Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives

Steroid hormones are synthesized in the adrenal gland, gonads, placenta and brain and are critical for normal reproductive function and bodily homeostasis. The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroid biosynthesis, i.e. the delivery of cholesterol from the outer to the inner mitochondrial membrane. The expression of the StAR protein is predominantly regulated by cAMP-dependent mechanisms in the adrenal and gonads. Whereas StAR plays an indispensable role in the regulation of steroid biosynthesis, a complete understanding of the regulation of its expression and function in steroidogenesis is not available. It has become clear that the regulation of StAR gene expression is a complex process that involves the interaction of a diversity of hormones and multiple signaling pathways that coordinate the cooperation and interaction of transcriptional machinery, as well as a number of post-transcriptional mechanisms that govern mRNA and protein expression. However, information is lacking on how the StAR gene is regulated in vivo such that it is expressed at appropriate times during development and is confined to the steroidogenic cells. Thus, it is not surprising that the precise mechanism involved in the regulation of StAR gene has not yet been established, which is the key to understanding the regulation of steroidogenesis in the context of both male and female development and function.

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3β-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3β-HS...

Submitochondrial Distribution of Three Key Steroidogenic Proteins (Steroidogenic Acute Regulatory Protein and Cytochrome P450scc and 3β-Hydroxysteroid Dehydrogenase Isomerase Enzymes) upon Stimulation by Intracellular Calcium in Adrenal Glomerulosa Cells

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 ± 3.2 and 151.1 ± 18.1% of controls, respectively) and in IM (to 119 ± 5.1 and 124.5 ± 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 ± 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 ± 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3β-hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.

Intramitochondrial cholesterol transfer.

Cholesterol serves as the initial substrate for all steroid hormones synthesized in the body regardless of the steroidogenic tissue or final steroid produced. The first steroid formed in the steroidogenic pathway is pregnenolone which is formed by the excision of a six carbon unit from cholesterol by the cytochrome P450 side chain cleavage enzyme system which is located in the inner mitochondrial membrane. It has long been known that the regulated biosynthesis of steroids is controlled by a cycloheximide sensitive factor whose function is to transfer cholesterol from the outer to the inner mitochondrial membrane, thus, the identity of this factor is of great importance. A candidate for the regulatory factor is the mitochondrial protein, the steroidogenic acute regulatory (StAR) protein. Cloning and sequencing of the StAR cDNA indicated that it was a novel protein, and transient transfections with the cDNA for the StAR protein resulted in increased steroid production in the absence of stimulation. Mutations in the StAR gene cause the potentially lethal disease congenital lipoid adrenal hyperplasia, a condition in which cholesterol transfer to the cytochrome P450 side chain cleavage enzyme, P450scc, is blocked, filling the cell with cholesterol and cholesterol esters. StAR knockout mice have a phenotype which is essentially identical to the human condition. The cholesterol transferring activity of StAR has been shown to reside in the C-terminal part of the molecule and a protein sharing homology with a region in the C-terminus of StAR has been shown to display cholesterol transferring capacity. Recent evidence has indicated that StAR can act as a sterol transfer protein and it is perhaps this characteristic which allows it to mobilize cholesterol to the inner mitochondrial membrane. However, while it appears that StAR is the acute regulator of steroid biosynthesis via its cholesterol transferring activity, its mechanism of action remains unknown.

Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression

The rate-limiting, committed, and regulatable step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory (StAR) protein. In steroidogenic cells, the StAR protein is regulated by cAMP-dependent mechanisms. However, the StAR promoter lacks a consensus cAMP response-element (CRE), suggesting the involvement of alternate regulatory factor(s) in cAMP responsiveness. These regulatory elements are found to be located in a transcription factor-binding site-rich region (consisting of approximately 150 nucleotides upstream of the transcription start site) of the StAR promoter, and appears to be the most important region in regulating transcription of the StAR gene. The StAR promoter sequences in mouse, rat and human are highly homologous, and in the absence of a canonical CRE, multiple cis-elements have been shown to be instrumental in the regulation of StAR gene expression. Nevertheless, it has become apparent that functional cooperation, interaction, and alteration of different transcription factors are involved in the fine-tuning of the regulatory events associated with StAR gene transcription.

The steroidogenic acute regulatory protein is induced by angiotensin II and K+ in H295R adrenocortical cells.

Adrenal steroid hormone biosynthesis can be activated by the protein kinase A pathway by ACTH, the protein kinase C pathway by angiotensin II (AII), or by increasing intracellular Ca2+ levels by AII or K+. Although their mechanisms of action are not known, each of these pathways is dependent upon the de novo synthesis of a protein that is required for the acute production of steroids. We have recently proposed the steroidogenic acute regulatory (StAR) protein as this required protein, therefore, we examined the effect of different agonists on StARs expression in H295R human adrenocortical carcinoma cells. (Bu)2cAMP, AII, K+, BAYK8644 (a calcium channel agonist) and TPA are all shown to induce StAR. Aldosterone synthesis was stimulated by all the agonists with the exception of TPA, indicating that AII-stimulated steroid production is mediated by increases in intracellular calcium. Thus, these data suggest that regulation of StAR expression may represent a common mechanism for divergent pathways to acutely control adrenal steroidogenesis.

Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis

Background: Translocator protein (TSPO) has been considered a mitochondrial cholesterol transporter critical for steroid hormone production. TSPO knock-out mice were reported to be embryonic lethal. Results: TSPO knock-out mice are viable with no effects on steroidogenesis. Conclusion: TSPO is not essential for steroidogenesis and is not necessary for sustaining life. Significance: This study rectifies a serious inaccuracy in the current understanding that is critical for treating steroid hormone disorders. Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo−/−) mice. Contrary to the early report, Tspo−/− mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo−/− mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo−/− mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo−/− mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.