Pulak R. Manna

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.

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.

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.

Elements involved in the regulation of the StAR gene.

The steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane, the regulated step in steroidogenesis. A most interesting facet of this protein is the manner in which its expression is acutely regulated. In this regard, a number of studies have concentrated on the search for consensus cis regulatory elements within its promoter, and, more importantly, on whether these elements are involved in its expression. This short review will summarize some of the findings that have been reported concerning the nature of how the expression of this gene is regulated.

Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene

Transcriptional regulation of the steroidogenic acute regulatory (StAR) protein gene by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP-response element (CRE; TGACGTCA) and is mediated by several sequence-specific transcription factors. We previously identified three CRE-like sites (within the -151/-1 bp cAMP-responsive region of the mouse StAR gene), of which the CRE2 site overlaps with an activator protein-1 (AP-1) motif (TGACTGA, designated as CRE2/AP-1) that can bind both CRE and AP-1 DNA-binding proteins. The present studies were aimed at exploring the functional crosstalk between CREB (CRE-binding protein) and cFos/cJun (AP-1 family members) on the CRE2/AP-1 element and its role in regulating transcription of the StAR gene. Using MA-10 mouse Leydig tumor cells, we demonstrate that the CRE and AP-1 families of proteins interact with the CRE2/AP-1 sequence. CREB, cFos, and cJun proteins were found to bind to the CRE2/AP-1 motif but not the CRE1 and CRE3 sites. Treatment with the cAMP analog (Bu)(2)cAMP augmented phosphorylation of CREB (Ser(133)), cFos (Thr(325)), and cJun (ser(73)). Chromatin immunoprecipitation studies revealed that the induction of CREB, cFos, and cJun by (Bu)(2)cAMP was correlated with protein-DNA interactions and recruitment of the coactivator CREB-binding protein (CBP) to the StAR promoter. EMSA studies employing CREB and cFos/cJun proteins demonstrated competition between these factors for binding to the CRE2/AP-1 motif. Transfection of cells containing the -151/-1 StAR reporter with CREB and cFos/cJun resulted in trans-repression of the StAR gene, an event tightly associated with CBP, demonstrating that both CREB and Fos/Jun compete with each other for binding with limited amounts of intracellular CBP. Overexpression of adenovirus E1A, which binds and inactivates CBP, markedly suppressed StAR gene expression. Ectopic expression of CBP eliminated the repression of the StAR gene by E1A and potentiated the activity of CREB and cFos/cJun on StAR promoter responsiveness. These findings identify molecular events involved in crosstalk between CREB and cFos/cJun, which confer both gain and loss of function on a single cis-element in fine-tuning of the regulatory events involved in transcription of the StAR gene.

The role of specific mitogen-activated protein kinase signaling cascades in the regulation of steroidogenesis.

Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models.

Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling

The steroidogenic acute regulatory (StAR) protein plays a central role in the regulation of steroid biosynthesis. While steroidogenesis is influenced by many processes, their modes of actions, in a few cases, remain obscure. In this study, we explored the mechanism of action of one such signaling pathway, the extracellular signal-regulated kinase 1/2 (ERK1/2), in regulating StAR expression and steroidogenesis in conjunction with the protein kinase A (PKA) and protein kinase C (PKC) pathways. Using MA-10 mouse Leydig tumor cells, we demonstrate that the activation of PKC and PKA signaling, by phorbol-12-myristate-13-acetate (PMA) and dibutyryl cAMP (dbcAMP)/human chorionic gonadotropin (hCG) respectively, was able to phosphorylate ERK1/2, an event markedly decreased by an upstream kinase inhibitor, U0126. Treatment with PMA enhanced StAR protein expression (associated with a slight increase in progesterone synthesis) but not its phosphorylation (P-StAR), which, in contrast, coordinately increased in response to dbcAMP/hCG. Inhibition of ERK1/2 activity by U0126 decreased PMA-treated StAR expression but increased dbcAMP/hCG-mediated StAR and P-StAR; however, progesterone levels were attenuated. U0126 was found to affect StAR expression and steroidogenesis both at the transcriptional and translational levels. Further studies demonstrated that the effect of U0126 on PMA- and dbcAMP/hCG-mediated StAR expression and steroid synthesis was tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). In fact, both DAX-1 and SR-B1 appear to play important roles in hormone-regulated steroidogenesis. These findings clearly demonstrate that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways and is stimulus specific in mouse Leydig cells.

Biphasic Action of Prolactin in the Regulation of Murine Leydig Tumor Cell Functions1

We investigated in this study the effects of ovine PRL on endocrine functions of cultured murine Leydig tumor cells (mLTC-1). The parameters studied were the activation of signal transduction systems involving cAMP and intracellular free Ca, the expression of Janus kinase 2 (JAK2), expression and function of LH and PRL receptors (R), expression of the steroidogenic acute regulatory (StAR) protein, and stimulation of steroidogenesis. Very similar biphasic doseand timedependent responses of all the parameters studied were found upon PRL stimulation, comprising a fast inhibition within 24 h in response to high PRL doses (

Mitochondrial A-Kinase Anchoring Protein 121 Binds Type II Protein Kinase A and Enhances Steroidogenic Acute Regulatory Protein-Mediated Steroidogenesis in MA-10 Mouse Leydig Tumor Cells

30 mg/liter), and a slow stimulation, between 48–72 h, in response to lower PRL doses (1–10 mg/liter). In addition, extracellular Ca (1.5 mmol/liter) increased the effect of PRL on human CG (hCG)-stimulated StAR messenger RNA expression and progesterone (P) production. Importantly, the biphasic effects of PRL on LHR gene expression and hCG-mediated P production were abolished in the presence of anti-PRL antiserum, demonstrating specificity of PRL action. The PRL effects on StAR expression, and steroid and cAMP production, apparently reflect its effects on LHR function. The relevance of the PRL effects observed in mLTC-1 cells was supported by demonstration of similar PRL responses in hCG-stimulated testosterone production of isolated mouse Leydig cells. Collectively, these findings clearly demonstrate the biphasic regulatory actions of PRL, and clarify some facets of the controversial role of this hormone in Leydig cell function. (Endocrinology 142: 308–318, 2001) T ROLE of PRL in the regulation of gonadal functions remains controversial (1, 2). Numerous studies have elucidated effects of PRL on testis, in particular on Leydig cells, although the exact nature and regulatory pathways involved in these actions are still poorly understood. A confounding factor affecting the results could be the heterologous nature of PRL preparations used, although there are also genuine differences in effects of PRL on Leydig cells of different species. For example, some studies demonstrate that PRL stimulates Leydig cells, and in particular the LHR function, whereas others demonstrate inhibitory actions on the same parameters. Induction of hypoprolactinemia by bromocriptine is known to suppress the LHR levels in rat testes (3–5). PRL treatment has been shown to increase the number of Leydig cells and LHR in hypophysectomized immature rats (6–8), as well as testicular LH-mediated testosterone production (9, 10). However, hyperprolactinemia in men is known to be associated with azoospermia (1, 11), hypogonadism (12), and impaired gonadal function (13), and it induces direct inhibitory effects on Leydig cell steroidogenesis in the rat (14). A reason for such apparently conflicting findings may lie in the fact that the nature of PRL actions on Leydig cells is dependent on their functional state, in addition to being time and dose dependent. PRL is known to induce a biphasic effect on hCG-stimulated steroid production in MA-10 mouse Leydig tumor cells (15, 16). Further aspects of the apparently biphasic effects of PRL on Leydig cell steroidogenesis have yet to be investigated. The steroidogenic acute regulatory (StAR) protein, a novel 30-kDa mitochondrial factor, has recently been purified, cloned, and characterized in MA-10 cells (17). The rate-limiting and regulated step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the mitochondrial inner membrane (18, 19), which has recently been found to be mediated by StAR protein. It has also been demonstrated that StAR protein is intimately associated with the acute regulation of steroidogenesis in steroidogenic cells (20–23). Recently, we demonstrated that human CG (hCG) markedly enhanced the StAR protein and StAR messenger RNA (mRNA) levels, which were consistent with progesterone (P) production in mLTC-1 cells (22). The possible connections of PRL and StAR protein actions are not known in the regulation of steroidogenesis. The PRL action is mediated through its binding to a plasma membrane receptor, a member of the cytokine/PRL/GH receptor family, including those of GH, and a large number of lymphokines and related growth factors (24–26). This receptor family is characterized by a single transmembrane domain and conserved homology in the extracellular domain (25, 26). In the testis, PRL receptors are expressed in Leydig (27–29), Sertoli, and spermatogenic cells (30). Several receptors of this family, including those of PRL, were found to induce tyrosine phosphorylation and activation of receptorassociated tyrosine kinases of the Janus kinase (JAK) family (31, 32). In addition, PRL is known to activate the cytoplasmic signal transducers and activators of transcription (STATs), possibly through direct phosphorylation by the JAK2 tyrosine kinase (33). An important pathway of PRL actions is Received April 5, 2000. Address all correspondence and requests for reprints to: Ilpo T. Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi@utu.fi. * This study was supported by a research grant from the Sigrid Jusélius Foundation. † Equal contributors to this study. 0013-7227/01/

On the role of skin in the regulation of local and systemic steroidogenic activities

03.00/0 Vol. 142, No. 1 Endocrinology Printed in U.S.A. Copyright