GENERAL COMMENTARY article Front. Endocrinol., 24 July 2020Sec. Cellular Endocrinology Volume 11 - 2020 | https://doi.org/10.3389/fendo.2020.00472

Abstract This two-part presentation regarding acute regulation of steroid biosynthesis documents discovery of the StAR protein and resolves controversy regarding mitochondrial cholesterol transport. The acute regulation of steroid biosynthesis was known to require de novo synthesis of a regulator protein to mediate the transfer of cholesterol, the substrate for steroids, from the outer to the inner mitochondrial membrane where it was converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme. We discovered a novel protein that was tightly correlated with steroid biosynthesis and had the requisite characteristics for the putative acute regulator of cholesterol transfer for steroid synthesis. Further studies confirmed that StAR protein is an indispensable component in the process of mitochondrial uptake of the cholesterol substrate for steroidogenesis. The translocator protein (TSPO) is a mitochondrial outer membrane protein suggested to import cholesterol to the inner mitochondrial membrane. However, it was demonstrated in vivo in Leydig cell specific TSPO conditional knockout mice that TSPO was not required for testosterone production or fertility. Similarly, global TSPO knockout (TSPO/-) mice were viable and fertile with fecundity equivalent to TSPO floxed (TSPOfl/fl) controls. Adrenal and gonadal steroidogenesis did not differ between TSPOfl/ fl and TSPO-/- mice. In vitro use of different steroidogenic cell line models (MA-10, MLTC, Y-1, H295R and R2C) demonstrated that siRNA-knockdown of TSPO did not affect steroidogenesis. Also, CRISPR/ Cas9-mediated TSPO deletion did not affect MA-10 cell steroidogenesis. These results directly 1) refute the suggestion that TSPO is indispensable for viability and steroid hormone biosynthesis; and, 2) substantiate the primal role of the StAR protein as the rate limiting factor in steroid hormone biosynthesis.

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate-limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years and facts surrounding their rise or decline. Only two have persisted—translocator protein (TSPO) and the steroidogenic acute regulatory protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

The steroidogenic acute regulatory (STAR) protein is an absolute requirement for the transfer of cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone, thus initiating the synthesis of steroid hormones. This transfer of cholesterol is considered the rate-limiting step in the synthesis of steroid hormones in steroidogenic cells. The induction of the STAR protein following treatment of cells with steroidogenic stimuli is characterized by extremely rapid phases of both transcription and translation. Following the cloning and characterization of the Star gene structure in 1994 (1), 1 of the first endeavors in the further study of this gene was to determine the factors that were involved in regulating its rapid expression. Because it was known that STAR expression and steroidogenesis used the cyclic adenosine monophosphate (cAMP) signaling pathway in which protein kinase A (PKA) is activated, the earliest studies concentrated on the search for known PKAactivated transcription factors to determine if they target the promoter region and induce transcription of this gene. The 50–flanking region of the Star gene was shown to contain a number of consensus sequences for the binding of specific protein transcription factors, and thus, several of these factors were studied in greater detail. The use of various length promoter sequences joined to luciferase reporter genes allowed for the rapid determination of the ability of a number of known transcription factors to activate the Star gene as well as their approximate location within the promoter. In general, it was found that full transcriptional potential of the Star gene was contained within the first 151 nucleotides of the Star promoter region. Predictably, 1 of the first transcription factors studied was steroidogenic factor 1 (SF-1). SF-1 is an orphan nuclear receptor that plays a key role in the expression of many cAMPregulated genes that are involved in steroidogenic functions as well as in adrenal and gonadal development (2). The first such functional SF-1 binding motif was determined to be at position2135 in the Star promoter (3). Following that initial investigation, several other functional SF-1 binding sites were also characterized (4–6) and found to be capable of inducing transcription of Star. There followed a great deal of additional activity on the characterization of the Star promoter and, as a result, in addition to SF-1, many other protein factors were shown to be involved in the regulation of Star expression. The regulation of Star was also found to be positive in the cases of factors such as CCAAT/enhancer binding protein (C/EBP) (7, 8); a zinc finger family transcription factor member (SP1) (5); the sterol regulatory element binding protein (9, 10); a member of the GATA family of transcription factors (GATA4) (11, 12); the cyclic adenosine monophosphate response element binding protein (13, 14); members of the activator protein 1 transcription factor family, (fos and jun) (15, 16), and the liver-X receptor–retinoid X receptor/retinoic acid receptor complex (17). There also emerged transcription factors that were able to negatively regulate Star promoter activity, as shown in studies on dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X-chromosome (gene 1) (18, 19), yin yang factor 1 (20), and the transcription factor forkhead box O3 (21). Together, all of these factors were shown to interact directly or in complexes with other proteins with specific

The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summarized, and some recent findings that propose a serious challenge for the role of TSPO in this process are discussed.

Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:Tspo Delta/Delta) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:Tspo Delta/Delta cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:Tspo Delta/Delta and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo(-/-)) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspo(fl/fl)) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism.

Recent reports on Leydig cell-specific Tspo conditional knockout Tspo(c Delta/Delta) mice (1), viable global Tspo knockout (Tspo(-/-)) mice from two independent laboratories (2, 3), and clones of CRISPR/Cas9-mediated Tspo-deleted MA-10 Leydig cells (MA-10Tsp(o Delta/Delta)) (4) established that TSPO is not essential for steroid hormone biosynthesis or viability [reviewed in Ref. (5, 6)]. These reports refuted 25 years of dogma that described TSPO as a mitochondrial cholesterol transport protein, indispensable for steroidogenesis. In response, the research group involved in most of the early studies linking TSPO and steroidogenesis investigated Leydig cell-specific and adrenocortical cell-specific Tspo(c Delta/Delta) mice (7) and presented results that seem to repudiate the recent findings and revive the old model. In this commentary, we would like to point out that interpretations made in the manuscript by Fan et al. (7) are seriously flawed.

Translocator protein (TSPO) is a mitochondrial outer membrane protein of unknown function with high physiological expression in steroidogenic cells. Using TSPO gene-deleted mice, we recently demonstrated that TSPO function is not essential for steroidogenesis. The first link between TSPO and steroidogenesis was established in studies showing modest increases in progesterone production by adrenocortical and Leydig tumor cell lines after treatment with PK11195. To reconcile discrepancies between physiological and pharmacological interpretations of TSPO function, we generated TSPO-knockout MA-10 mouse Leydig tumor cells (MA-10:TspoΔ/Δ) and examined their steroidogenic potential after exposure to either dibutyryl-cAMP or PK11195. Progesterone production in MA-10:TspoΔ/Δ after dibutyryl-cAMP was not different from control MA-10:Tspo+/+ cells, confirming that TSPO function is not essential for steroidogenesis. Interestingly, when treated with increasing concentrations of PK11195, both control MA-10:Tspo+/+ cells and MA-10:TspoΔ/Δ cells responded in a similar dose-dependent manner showing increases in progesterone production. These results show that the pharmacological effect of PK11195 on steroidogenesis is not mediated through TSPO.

The 18-kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial cholesterol import that is essential for steroid hormone production. In contrast to this initial proposition, recent experiments reexamining TSPO function have demonstrated that it is not involved in steroidogenesis. This fundamental change has forced a reexamination of the functional interpretations made for TSPO that broadly impacts both basic and clinical research across multiple fields. In this minireview, we recapitulate the key studies from 25 years of TSPO research and concurrently examine their limitations that perhaps led towards the incorrect association of TSPO and steroid hormone production. Although this shift in understanding raises new questions regarding the molecular function of TSPO, these recent developments are poised to have a significant positive impact for research progress in steroid endocrinology.

Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is an outer mitochondrial membrane protein. TSPO has been shown to cooperate with steroidogenic acute regulatory protein (StAR) and function in the transport of cholesterol into mitochondria. TSPO has also been considered as a structural component of the mitochondrial permeability transition pore (MPTP). However, recent advances have changed these views of TSPO's functions and have prompted a re-evaluation of established concepts. This review summarizes the history of TSPO, key elements of the debate, and functional experiments that have changed our understanding. Moving forward, we examine how this fundamental change impacts our understanding of TSPO and affects the future of TSPO as a therapeutic and diagnostic target.

Retinoids (vitamin A and its derivatives) are critical for a spectrum of developmental and physiological processes, in which steroid hormones also play indispensable roles. The StAR protein predominantly regulates steroid biosynthesis in steroidogenic tissues. We have reported that regulation of retinoid, especially atRA and 9-cis RA, responsive StAR transcription is largely mediated by an LXR-RXR/RAR heterodimeric motif in the mouse StAR promoter. Herein we demonstrate that retinoids are capable of enhancing StAR protein, P-StAR, and steroid production in granulosa, adrenocortical, glial, and epidermal cells. Whereas transient expression of RARα and RXRα enhanced 9-cis RA induced StAR gene transcription, silencing of RXRα with siRNA, decreased StAR and steroid levels. An oligonucleotide probe encompassing an LXR-RXR/RAR motif bound to adrenocortical and epidermal keratinocyte nuclear proteins in EMSAs. ChIP studies revealed association of RARα and RXRα with the StAR proximal promoter. Further studies demonstrated that StAR mRNA levels decreased in diseased and elderly men and women skin tissues and that atRA could restore steroidogenesis in epidermal keratinocytes of aged individuals. These findings provide novel insights into the relevance of retinoid signaling in the up-regulation of steroid biosynthesis in various target tissues, and indicate that retinoid therapy may have important implications in age-related complications and diseases.

To determine if oxysterol levels, produced by macrophages, are elevated in peritoneal fluid of women with endometriosis. Prospective Case-Control. Forty women (age 18-40 years) undergoing scheduled laparoscopy (diagnostic or bilateral tubal ligation) were consented and participated in the study. 25 participants were diagnosed with endometriosis. 15 participants had no evidence of endometriosis (controls). One to 10 mL of peritoneal fluid was aspirated from the posterior cul-de-sac from each participant. The fluid was evaluated to determine the role of oxysterols in endometriosis. In addition, human endometrial stromal cells (HESC) were treated with either synthetic 22-hydroxycholesterol (22-HC), 25-HC and 27-HC or peritoneal fluid samples from study participants to determine levels of steroidogenic acute regulatory (StAR) protein, StAR mRNA and pregnenolone synthesis. Among the 25 study participants with endometriosis, 3 had Stage I (minimal), 16 had Stage II (mild), 2 had Stage III (moderate) and 4 had Stage IV (severe) endometriosis per American Society for Reproductive Medicine scale for classification of endometriosis. Preliminary data indicates that levels of 25-HC and 27-HC increased in a stage-dependent manner in the peritoneal fluid of women with endometriosis. In addition, StAR protein and mRNA expression were strikingly higher in peritoneal fluid from participants with Stage IV endometriosis compared with controls. HESC treated with either peritoneal fluid from study participants or with oxysterols showed increases in StAR protein, StAR mRNA expression and pregnenolone levels. Oxysterols, oxygenated metabolites of cholesterol secreted by macrophages, are elevated stage dependently in peritoneal fluid of women with endometriosis. HESC treated with peritoneal fluid samples showed steroidogenic activity by increased StAR protein, StAR mRNA expression and pregnenolone synthesis. Detection of either 25-HC or 27-HC levels through blood or peritoneal fluid sampling may lead to less invasive and less costly techniques to diagnose endometriosis or to determine its recurrence. Additionally, pharmaceutical advances directed at inhibiting the production of these oxysterols by macrophages may lead to new treatment options for patients with endometriosis.

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.

Non-vesicular intracellular cholesterol transport is an important mechanism for maintaining membrane cholesterol homeostasis. Recent reports of studies directed at soluble cholesterol transport protei

The events that regulate the rapid synthesis of steroid hormones in response to trophic hormone stimulation of the steroidogenic cells have been the ongoing subject of intense interest for several decades. Much of the early work performed in this area determined that the acute regulation of steroid hormone biosynthesis required the rapid, de novo synthesis of a protein(s) whose function appeared to be involved in mediating the delivery of cholesterol, the substrate for all steroid hormones, from the outer mitochondrial membrane to the inner mitochondrial membrane. The existence of a protein that was necessary for intramitochondrial transfer of cholesterol was first postulated by James Ferguson in 1963 (1). This transfer is an absolute requirement for steroid biosynthesis, because the cholesterol side-chain cleavage enzyme system that converts cholesterol to pregnenolone, the first steroid synthesized, resides on the inner side of the inner mitochondrial membrane. As such, the hydrophobic cholesterol substrate is unable to traverse the aqueous intermembrane space and reach the inner mitochondrial membrane through diffusion. The next 3 decades saw a focused interest in determining the identity of this putative regulator protein (2–13). These studies gave rise to a list of the characteristics that the putative protein regulator appeared to possess, including those described above. Later work introduced other putative regulator proteins, the sterol carrier protein 2 (14), the steroidogenesis activator polypeptide (15, 16), the peripheral benzodiazepine receptor (PBR) (17), and the steroidogenic acute regulatory protein (StAR) (18). Although space limitations do not allow for a critical evaluation of the characteristics of each of the candidates that have been put forth as the putative regulator protein over the years, these candidates have been described in an earlier review (19).

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5'-flanking region of the StAR gene demonstrated the importance of the -254/-1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the -254/-1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells.