Expression and roles of steroidogenic acute regulatory (StAR) protein in ‘non-classical’, extra-adrenal and extra-gonadal cells and tissues

Molecular and Cellular Endocrinology 371 (2013) 47–61

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

Expression and roles of steroidogenic acute regulatory (StAR) protein in ‘non-classical’, extra-adrenal and extra-gonadal cells and tissues Eli Anuka a, Michael Gal b, Douglas M. Stocco c, Joseph Orly a,⇑ a

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel IVF Unit, Department of Obstetrics and Gynecology, Shaare-Zedek Medical Center, The Hebrew University School of Medicine, Jerusalem 91904, Israel c Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA b

a r t i c l e

i n f o

Article history: Available online 13 February 2013 Keywords: StAR expression ‘Non-classical’ steroidogenic cells Non-steroidogenic StAR activity

a b s t r a c t The activity of the steroidogenic acute regulatory (StAR) protein is indispensable and rate limiting for high output synthesis of steroid hormones in the adrenal cortex and the gonads, known as the ‘classical’ steroidogenic organs (StAR is not expressed in the human placenta). In addition, studies of recent years have shown that StAR is also expressed in many tissues that produce steroid hormones for local use, potentially conferring some functional advantage by acting via intracrine, autocrine or paracrine fashion. Others hypothesized that StAR might also function in non-steroidogenic roles in specific tissues. This review highlights the evidence for the presence of StAR in 17 extra-adrenal and extra-gonadal organs, cell types and malignancies. Provided is the physiological context and the rationale for searching for the presence of StAR in such cells. Since in many of the tissues the overall level of StAR is relatively low, we also reviewed the methods used for StAR detection. The gathered information suggests that a comprehensive understanding of StAR activity in ‘non-classical’ tissues will require the use of experimental approaches that are able to analyze StAR presence at single-cell resolution. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Contents 1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. StAR discovery in ‘Classical’ steroidogenic tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. StAR – the rate limiting step in steroidogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. StAR mutations and StAR independent placental steroidogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3. Mechanism of StAR action – still enigmatic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Exploring StAR expression in ‘Non Classical’ tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-classical steroid hormones synthesizing cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2. Sebocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Adipose tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 48 48 48 49 49 49 49 51 51 52 52 52 53 53

Abbreviations: OMM, outer mitochondrial membrane; IMM, inner mitochondrial membranes; TSPO, translocator protein; VDAC, voltage dependent anion channel; CNS, central nervous system; SR-BI, class-B type-I scavenger receptor; PTII, type II pneumonocytes; CCHCR1, Coiled-Coil a-Helical Rod protein 1; NR5A1, nuclear receptor subfamily 5, group A, member 1, SF-1; NR5A2, liver receptor homolog-1; HMG-CoA, hydroxymethylglutaryl-coenzyme (reductase); LXR, liver X receptors; PPAR, peroxisome proliferator-activated receptor; SERM, selective estrogen receptor modulator; COUP-TFII or NR2F2, nuclear receptor subfamily 2, group F, member 2; WT1, Wilm’s tumor tumor suppressor gene1. ⇑ Corresponding author. Tel.: +972 54 427 2948; fax: +972 2 658 6448. E-mail address: [email protected] (J. Orly). 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.02.003

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3.

4.

5. 6.

E. Anuka et al. / Molecular and Cellular Endocrinology 371 (2013) 47–61

2.7. Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-steroidogenic StAR expressing cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. StAR and oxysterol synthesis-potential significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Liver cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Vasculature StAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Vascular endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer and diseased states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Endometrial carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Endometriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Ovarian carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Intestine/colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Adrenal tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. StAR discovery in ‘Classical’ steroidogenic tissues 1.1.1. StAR – the rate limiting step in steroidogenesis The ’’classical’’ steroidogenic tissues expressing StAR include the adrenal cortex and the reproductive organs consisting of the ovary, testis, and the placenta in non-human mammals. The first step in steroidogenesis is the conversion of cholesterol to the first steroid, pregnenolone, formed in the mitochondria of these cells. This conversion occurs via the action of the cholesterol side chain cleavage cytochrome P450 (CYP11A1) that resides in the inner mitochondrial membrane in all steroidogenic cells (Farkash et al., 1986; Hall, 1985; Simpson and Waterman, 1983). Pregnenolone then exits the mitochondria and in steroidogenic tissues of most species, is converted to progesterone by 3b-hydroxysteroid dehydrogenase (HSD3B), which resides in the microsomal compartment; subsequently, pregnenolone is transformed to a variety of active steroid hormones, depending on the specialized cells in each tissue, e.g., glucocorticoids (cortisol in humans or corticosterone in rodents) and mineralcorticoids (aldosterone) in the adrenal cortex, and sex hormones (progesterone, androgens, estrogens) in the gonads (Payne and Hales, 2004). Like most biosynthetic pathways, the steroidogenic pathway has a rate-limiting step, which was believed to be the activation of the CYP11A1 enzyme (Karaboyas and Koritz, 1965). However, it soon became clear that the regulated, and more accurately defined rate-limiting step was the acute delivery of the substrate cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) where CYP11A1 is located (Black et al., 1994). This step turned out to have an absolute requirement for the synthesis of new proteins since puromycin inhibited ACTH induction of corticoid synthesis in cells of the adrenal gland (Ferguson, 1963). Hence, a three decades long search sought putative candidate regulator proteins that are hormone stimulated, acutely synthesised and puromycin/cycloheximide sensitive, as previously reviewed (Stocco and Clark, 1996). A leading candidate among those was described as an ACTH-induced 30 kDa phosphoprotein in rat and mouse adrenocortical cells, and as an LH-induced protein in rat ovary corpus luteum cells and mouse testicular Leydig cells (Alberta et al., 1989). Later studies determined that the 30 kDa mitochondrial protein was processed from a 37 kDa precursor form (Epstein and Orme-Johnson, 1991; Stocco and Sodeman, 1991; Stocco and Ascoli, 1993). The

53 53 54 54 54 54 54 54 55 55 55 55 56 56 56 57 57 57 57 57

protein purification and cloning of the cDNA for the 37 kDa protein precursor was successfully accomplished in 1994 (Clark et al., 1994). Expression of the novel cDNA-derived protein resulted in a significant increase in steroid production in the absence of hormone stimulation, indicating a direct role for the 37–30 kDa proteins in hormone-regulated steroid production. The protein was named the Steroidogenic Acute Regulatory Protein or StAR (Clark et al., 1994) and in the earliest of the studies on StAR, this protein was indeed thought to be confined to the ‘‘classical’’ steroidogenic tissues of the adrenals and gonads. 1.1.2. StAR mutations and StAR independent placental steroidogenesis Shortly following the cloning of the StAR cDNA it was demonstrated that mutations in the StAR gene resulted in congenital lipoid adrenal hyperplasia (lipoid CAH), the etiology of which provided compelling evidence for the essential role of this vital protein in the regulation of steroidogenesis (Lin et al., 1995). Targeted disruption of the StAR gene in mice successfully produced StAR knockout mice that displayed characteristics very similar to human lipoid CAH and further substantiated the necessity for StAR action in stimulated steroid biosynthesis in the adrenal cortex and the gonads (Caron et al., 1997; Miller and Strauss, 1999; Stocco, 2002). In this regards, an exceptional steroidogenic tissue that does not express StAR is the human placenta (Sugawara et al., 1995b). Yet, this organ produces as much as 300 mg progesterone daily in order to maintain uterine muscle tranquility and avoid preterm labor (Tang et al., 2001; Hardy et al., 2006; da Fonseca et al., 2009). Strauss et al. (1996) calculated that per trophoblast cell mass (100 g syncytiotrophoblasts/placeta), such a rate of steroidogenesis is roughly 8–10 times less efficient when compared to the rate of luteal steroidogenesis (1 g tissue producing 25 mg/day progesterone). That suggests that, as expected, the absence of StAR in the human placenta probably accounts for the lower rate of placental steroidogenesis, which is probably required to compensate for the much larger mass of the steroidogenic component of this organ. As for the mechanism of StAR-free steroidogenesis in the human placenta, it has been suggested that a truncated form of MLN64, known as a StAR like late-endosome protein expressed in several human tissues, might substitute, at least in part, for StAR activity in this tissue (Watari et al., 1997; Bose et al., 2000). In this regard, it should be mentioned that StAR independent ‘basal’ rate of steroidogenesis is also possible in steroidogenic cell types, where the OMM translocator protein (18-kDa TSPO, further

E. Anuka et al. / Molecular and Cellular Endocrinology 371 (2013) 47–61

discussed herein) is expressed; it is also tempting to speculate that the TSPO may account for a StAR-like activity as a partial compensatory mechanism for glucocorticoid synthesis in StAR deficient mice that survive without glucocorticoid replacement therapy when denied as of the day of weaning (Caron et al., 1997; Ishii et al., 2002). It should be stressed, however, that only very few of such mice do survive this treatment (Tomohiro Ishii, personal communication). 1.1.3. Mechanism of StAR action – still enigmatic An early model of StAR action postulated that in response to trophic hormone stimulation, StAR is synthesised in the cytosol and during import into the mitochondrial inner compartment, contact sites between the inner and outer membranes were formed and served as a hydrophobic bridge for transfer of cholesterol from the OMM to the IMM (Stocco and Clark, 1996). It then became clear that this model would require substantial modification when it was observed that expression of N-terminal truncations of the StAR protein that removed as many as 62 amino acids and were not imported into the mitochondria, retained cholesterol transfer activity and steroid production (Arakane et al., 1996; Wang et al., 1998). This observation was strengthened when it was found that bacterially produced StAR protein lacking the first 62 N-terminal amino acids did not undergo import, but was able to fully support steroidogenesis when added to isolated mitochondria (Arakane et al., 1998). Furthermore, StAR activity was retained even if N-62 StAR was covalently linked to the outer face of the OMM, suggesting that StAR import into the matrix is not essential for cholesterol mobilization activity of the protein (Bose et al., 2002). In vitro studies by Kallen and colleagues demonstrated that StAR can act as a sterol transfer protein to enhance sterol desorption from one membrane to another (Kallen et al., 1998). They also showed that StAR is directed to the mitochondria via its N-terminus and, presumably utilizing C-terminal sequences, produces alteration in the OMM that results in the transfer of cholesterol from the outer to the inner membrane. In another study by Bose et al., StAR was subjected to limited proteolysis at different pH values and showed that the molecule behaves differently as the pH decreases and forms a molten globule structure at low pH values (Bose et al., 1999), a scenario possibly recapitulating the effect of charged phospholipid heads on StAR pausing at the outer face of the OMM just before import. If the transition to a molten globule occurs, this structural change might lower the energy required to open the StAR structure, possibly exposing a cholesterol channel or, it may prolong the interval with which StAR can reside on the outer membrane, thus allowing increased transfer of cholesterol during this period. Is all that done by StAR alone? While the mechanism of action of StAR is still unknown, there arose a belief that StAR is likely to functionally interact with components such as proteins, lipids and/or other factors of the mitochondrial membranes, which results in cholesterol transfer. The identification of such factors has proven to be quite difficult. For example, studies by Rone et al., have recently suggested that cholesterol transfer is promoted upon association of StAR with a large complex that spans the two mitochondrial membranes and includes the OMM translocator protein (TSPO/PBR) and the voltage-dependent anion channel 1 (VDAC1), along with CYP11A1 and other proteins of the IMM (Rone et al., 2012). Others have suggested that physical and functional interaction of StAR with VDAC requires a prior phosphorylation of StAR by membrane associated protein kinase A, which in turn, depends on StAR protein–protein interactions with the OMM phosphate carrier protein (PCP) (Bose et al., 2008). A detailed account of how physical and/or functional interactions of StAR with the mitochondrial membrane proteins facilitate cholesterol passage to become subjected to CYP11A1 catalysis is yet to be defined.

49

1.2. Exploring StAR expression in ‘Non Classical’ tissues Since StAR discovery 18 years ago, PubMed lists over 1500 papers when queried with ‘‘steroidogenic acute regulatory protein’’. That includes tens of studies appearing in recent years, which demonstrated StAR presence and its potential roles outside the ‘classical’ steroidogenic tissues. The present article reviews a list of ‘nonclassical’ cells and tissues that express StAR, including the brain and eye, heart, lung, pancreas, kidney, skin cells, fat, liver, vascular endothelial cells, intestine, as well as diseased tissues of the gut, prostate, uterine endometrium and endometriosis, ovarian and adrenal cancers. Two or three rationales were behind the studies on StAR function outside the steroidogenic organs: first, the largest group perceived StAR as a prominent marker for a local de novo production of steroid hormones; such were the reasons for searches for StAR in tissues expected to produce glucocorticoids or mineralcorticoids (heart, lung, fat, and intestine and adrenal tumors), while other tissues were expected to synthesize steroid derivatives and sex hormones including the brain and eye, kidney, skin, fat, pancreas and tumors of the prostate, uterus, and the ovary. A different rationale guided the exploration for StAR expression in cells that do not synthesize steroid hormones but, instead, express the mitochondrial cytochrome P450 27-hydroxylase (CYP27A1) that transforms cholesterol to oxysterols. Similarly to CYP11A1, CYP27A1 is an inner mitochondrial membrane resident and a potential acceptor for cholesterol supply provided by StAR (Sugawara et al., 1995a). Within this group, the liver is rich in CYP27A1, hence StAR was considered a candidate protein to mediate bile acid synthesis. The role of StAR was also examined in context of lipid homeostasis known to be regulated by oxysterols produced in extra-hepatic cells bearing high CYP27A1 levels; those include monocytes and macrophage ‘foam cells’, aortic tissue and endothelium. Table 1 provides a summary of StAR expressing cells presented in an ‘at-a-glance’ fashion. The reader can quickly grasp and compare the basic parameters covered in each of the ‘non-classical’ steroidogenic tissues, including the animal/cell models, the methods used for documentation of the StAR gene products, i.e., protein assessed by immuno histo/cyto-chemistry and/or Western analysis, and mRNA determinations by Northern analysis, RT-PCR or qRTPCR. Also mentioned are other steroidogenic enzymes co-expressed with StAR presence and, finally, the suspected functions of StAR in each case.

2. Non-classical steroid hormones synthesizing cells 2.1. Brain The presence of StAR, CYP11A1 and other steroidogenic enzymes responsible for the synthesis of steroids in the CNS (Baulieu, 1998; Mellon and Griffin, 2002) was recently reviewed elsewhere (King and Stocco, 2011; Lavaque et al., 2006). Briefly, active neurosteroids are not necessarily restricted to classical steroid hormones and may include metabolites derived from circulating steroids such as progesterone, 11-deoxycorticosterone or testosterone; other active neurosteroids locally synthesised de novo, includenon-classical hormones such as pregnenolone and DHEA. The synthesis of the latter is catalyzed by CYP11A1, StAR and CYP17A1 expression in both glial cells and neurons of both embryonic, neonate and adult nervous systems (Mensah-Nyagan et al., 1999; Mellon and Griffin, 2002; King et al., 2004; Seol et al., 2009). The functions of the neurosteroids in normal and diseased states (citations reviewed in Mellon and Griffin, 2002) range from non-genomic activation of GABAergic receptors involved in seizure

Cancer and diseased states

Endothel

Macrophages

bEnd.3 cell line; primary heart microvascular cells; mini-vein of connective tissue; intima of aorta; thoracic aorta of hyperlipidemic rats Prostate cancer cell lines: DU145, PC3, LNCaP(C33, C81)

H.M.R

H

bEnd.3

M

H,M

M

Hepatoblastoma cell lines: HepG2 and Reuber H35 Primary macrophages from ApoE KO and C57BL/6J mice models; RAW264.7 cell line THP1.RAW264.7

H,R

H

Liver

Distal tubules, thick ascending limb of Henle’s loop Primary hepatocytes, HepG2 cell line

Primary hepatocytes

R

Kidney

Entire organ

R

H

H

Pancreas

H

Skin

Omental and subcutanous adipose depots

M

Lungs

H

Sebocytes

H.R.M

Heart

Adipose tissue

Keratinocytes

R

Eye

Epithelium, endothelium and neurons Normal and failing human hearts; experimental myocardial infarction in rodents; aortic banding in mouse; cultured rodent cardiomyocytes Fetal lung tissue (E15)

H.R.M

Brain

Species cell type

Table 1 StAR expression in ’non-classical’ cells and tissues.

RT-PCR (30–34 Cs), qRT-PCR

In vivo, primary culture

RT-PCR (28 Cs)

Primary, cell line

Overexpression

qRT-PCR

RT-PCR (30–35 Cs)

Cell line

Cell line, primary culture, In vivo

Cell line

Cell line

Overexpression

qRT-PCR

Cell line

Primary culture

RT-PCR (32 Cs), Northern analysis Overexpression

RT-PCR (33–38 Cs)

In vivo

Primary culture, cell line

RT-PCR (35 Cs)

qRT-PCR

In vivo

In vivo

qcRT-PCR

RT-PCR (25 Cs); qRT-PCR

In vivo, cell line (SZ95)

In vivo, primary culture, cell line

qRT-PCR

–

In vivo

Protein

Co-expressed with

CYP27A1

WB (30 lg mito extract)

–

WB (50 lg)

–

CYPllAl,3bHSDl, CYP17, 17bHSD

ICC, IHC, WB (50 lg)

WB (30 lg)

–

–

–

WB

CYP27A1

CYP11A1 and SFl

CYP11A1.HSD3B2, CYP21B, CYP19, HSD11B1.HSD17B3, HSD17B5, HSD17B7 CYP11A1

CYP11A1.3bHSD, CYP21B, CYP11B1, 11bHSD1 CYP11A1,3bHSD, CYP17, CYP21.CYP11B1 3bHSD

CYP11A1.CYP11B1

SR-BI

WB (60 lg mito extract); IHC

IHC

–

IHC

IHC, ICC

–

–

IHC

CNS cell types, details reviewed elsewhere

mRNA

In vivo

Model

Endogenous androgen synthesis allows tumor growth despite castration

StAR overexpression upregulates ABC transporters (potential increase of cellular cholesterol efflux) Reciprocal to the above, LDL, cholesterol, 25-HC upregulate StAR expression

Inhibition of apoptosis by increasing cholesterol efflux and regulation of pro- and anti-apoptotic genes

Marked augmentation of bile acid synthesis via the ’acidic pathway’ Anti-apoptotic, confers chemotherapy resistance

Claimed bile acid synthesis

Low expression levels, questionable role Pregnenolone synthesis, unclear role

Provost and Tremblay (2005)

Corticosterone synthesis, embryonal lung development, type II pneomocyte maturation Testosterone production correlates with hair loss; aberrant cortisol production in psoriasis and eczema StAR and 3bHSD were higher in androgenic alopecia patient’s bald skin Unclear function of 11deoxycorticosterone

Dillard et al. (2008)

Ning et al. (2006), Suren Castillo et al. (2008)

Ning et al. (2009), Taylor et al. (2010), Bai et al. (2010) Ning et al. (2010)

Ma et al. (2007)

Montero et al. (2008)

Pandak et al. (2002)

Hall et al. (2005)

Dalla Valle et al. (2004), Pagotto et al. (2011)

Morales et al. (2008)

MacKenzie et al. (2008)

Tiala et al. (2007), Hannen et al. (2011), Inoue et al. (2012), Chen et al. (2006) Chen et al. (2006)

Young et al. (2001), Casal et al. (2003), Ohtani et al. (2009)

King and Stocco (2011), Lavaque et al. (2006) Provost et al. (2003)

References

Synthesis of glucocorticoids and mineralocorticoids

Neurosteroidogenesis

Function

50 E. Anuka et al. / Molecular and Cellular Endocrinology 371 (2013) 47–61

2.2. Eye

Abbreviations: H, human; R, rat; M, mouse; Cs, cycles; IHC, immunohistochemistry; ICC, immunocycochemistry; WB, Western blotting; mito, mitochondria.

Non functional adrenal tumors H

In vivo

RT-PCR (30 Cs)

–

CYP11A1,CYP11B1, LRH-1 CYP11A1 – qRT-PCR Colorectal cancer H

In vivo

CYPllAl,3bHSD IHC RT-PCR (45 Cs) In vivo Epithelial ovarian carcinoma H

H

51

disorders (pregnenolone sulfate, allopregnanolone), to myelination (progesterone), neurite outgrowth (DHEA, DHEAS), effects on behavioral patterns of stress (allopregnanolone), anxiety (THDOC or androstanediol), several depression patterns, and memory gain and loss in rodents (pregnenolone, DHEA and DHEAS). Pregnenolone and progesterone also act as endogenous neuroprotectants against acute or chronic neurotoxicity. In sum, the multiplicity of StAR and CYP11A1 expression in many of the brain regions reflects the diversity of the neurosteroids roles in the CNS functions. It is likely that future approaches using cell-specific regulated ablation of endogenous steroidogenesis could be instrumental in characterizing the consequences on given cognitive deficits, developmental or pathological disorders in the brain.

Zenkert et al. (2000)

Sidler et al. (2011)

Abd-Elaziz et al. (2005)

Tsai et al. (2001), Attar et al. (2009) CYPllAl,3bHSD, CYP17, CY Parom WB (25 lg mito extract) In vivo

qcRT-PCR, qRTPCR

CYP11A1,CYP17, 3bHSD H

Endometrial carcinoma cell lines HHUA and HOUA-1 Endometriosis

Cell line

RT-PCR (35 Cs)

WB (15 lg mito extract)

No clear role for androgen production; CYParom is not expressed. High StAR-mediated estradiol synthesis may contribute to survival of the retrograded endometrium StAR as a potential marker for a good prognosis due to progesterone inhibition of cell proliferation Cortisol synthesis may confer tumor immune escape Unclear role of StAR

Sugawara et al. (2004)

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Extending a previously published observation of neurosteroid synthesis in rat retinal cells (Guarneri et al., 1994), a search for ocular StAR was conducted using immunohistochemical approaches that identified a high degree of StAR co-localization with class-B type-I scavenger receptor (SR-BI) in different cells of the rat eye retinal and non-retinal tissues (Provost et al., 2003). SR-BI is a HDL receptor that mediates selective cholesterol uptake and is essential for steroidogenesis in the adrenal and gonads of rodents (Azhar and Reaven, 2002). The documented co-localization of StAR and SR-BI in ocular cells (Provost et al., 2003) could potentially serve for neurosteroid synthesis, but other than a previous observation of CYP11A1 expression in retinal ganglionic cells (Guarneri et al., 1994), no direct evidence has been presented for the concomitant presence of the ocular StAR/SR-BI with CYP11A1 and 3bHSD. 2.3. Heart Attempts to find and characterize key enzymes catalyzing the synthesis of glucocorticoids and mineralcorticoids in the heart (Casal et al., 2003; Gomez-Sanchez and Gomez-Sanchez, 2001; Heymes et al., 2004; Kayes-Wandover and White, 2000; Silvestre et al., 1998; Young et al., 2001) were motivated by the presence of their cognate receptors, widely expressed in this organ (Barnett and Pritchett, 1988; Lazar et al., 1990), and found involved in cardiovascular diseases (Funder, 1997; Gustafsson et al., 2012; Messaoudi et al., 2012; Pitt et al., 2003, 1999). Noteworthy is the detrimental impact of aldosterone interaction with its heart receptors under conditions of heart failure. For example, reduced mortality was observed in post-infarct patients treated with inhibitors of the aldosterone receptors (Hayashi et al., 2003; Messaoudi et al., 2012; Pitt et al., 1999; Zannad et al., 2011). This observation is consistent with the finding that aldosterone receptor knock-down or deletion in genetically manipulated mice, had a positive effect on the recovery of the animals after experimental myocardial infarction (Beggah et al., 2002; Fraccarollo et al., 2011; Milliez et al., 2005). Attention was first focused on the terminal enzymes required for cortisol/corticosterone synthesis and aldosterone production executed by 11b-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), respectively (Kayes-Wandover and White, 2000; Silvestre et al., 1998; Young et al., 2001). Further search for these gene products in the heart was mostly focused on RT-PCR measurements of the transcripts and enzyme activity assays (KayesWandover and White, 2000; Silvestre et al., 1998; Young et al., 2001). As to the question of whether the human heart, a normal or failing one, can synthesize steroids de novo, Young et al. were doubtful (Young et al., 2001) since, despite the apparent expression of StAR and CYP11A1 transcripts, the HSD3B2 mRNA was barely observed (Kayes-Wandover and White, 2000; Young et al., 2001). In

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rodents, frequently used for failing and hypertrophied heart models, myocardial infarction and induced heart hypertrophy generated relative increases of Star transcripts in the mouse and rat heart (Casal et al., 2003; Young et al., 2001). However, Cyp11a1 mRNA was demonstrated in the rat only (Ohtani et al., 2009) and information on Hsd3b expression was not provided. Yet, since the rat heart expresses Cyp11b1 and Cyp11b2 transcripts- it was assumed to be capable of synthesizing glucocorticoids and aldosterone from cholesterol (Heymes et al., 2004; Ohtani et al., 2009; Silvestre et al., 1998). To uncover which cell type might be responsible for putative heart steroidogenesis, two studies utilizing heart tissue cell suspension culture methods were conducted; in both cases isolated cardiomyocytes were shown to be capable of de novo steroidogenesis (Casal et al., 2003; Ohtani et al., 2009). Yet, when the entire array of studies came full circle, the findings from both in vivo and tissue culture experiments led to an understanding that the levels of the steroidogenic transcripts are at best 100–10,000 lower than those found in the cortical cells of the adrenal (Heymes et al., 2004; Kayes-Wandover and White, 2000). As such, the possible contribution of extra-adrenal cholesterol conversion to locally made aldosterone and glucocorticoids in the heart remained an open issue (Funder, 2004; Gomez-Sanchez et al., 2004; Gomez-Sanchez and Gomez-Sanchez, 2001). 2.4. Lung Glucocorticoids are essential for the pre-term maturation of infant lungs that need to convert from fluid-filled organ to respiratory gas exchange (Bolt et al., 2001). Although the origin of the fetal glucocorticoids is probably the fetal adrenal that is the first to become active due to the early maturation of the hypothalamic–pituitary-adrenal axis, circulating maternal glucocorticoids can compensate in the case of fetal adrenal insufficiency, both in humans and mice (Muglia et al., 1995; Venihaki et al., 2000). Glucocorticoid binding to lung receptors is essential for anatomic and functional maturation of the pulmonary alveoli (Cole et al., 1995; Muglia et al., 1995) that undergo thinning of the alveolar epithelium by production of surfactant material in type II pneumonocytes (PTII) (Ballard et al., 1997; McCormick and Mendelson, 1994). Interestingly, in addition to circulating glucocorticoids (GCs), a local expression of StAR and the entire enzyme cascade required for GC synthesis in the mouse fetal lung itself was identified (Provost and Tremblay, 2005). The levels of CYP11A1, StAR, HSD3B, CYP21B, CYP11B1 and HSD11B1 in the lung were about equal to the levels of fetal adrenals, and about 1–4% of the levels of these proteins in adult adrenals. Aldosterone synthase and 11b-HSD2 are not expressed in the prenatal rodent lung, ensuring no downstream corticosterone transformation or metabolism, which allows a pure GC responsive tissue context. Furthermore, consistent with an expected relevance of local GC production in the prenatal lung, high expression of the steroidogenic enzymes in the developing lung transiently peaks on gestational day 15, 2 days before the surge of surfactant synthesis. Which cells in the fetal lung are steroidogenic is unclear at the moment. In the absence of such information, one cannot address the physiological relevance of local lung steroidogenesis by use of conditional knockout approaches. At the time of publication, Provost and Tremblay mentioned possible paracrine and intracrine responses to the lung GC (Provost and Tremblay, 2005). This observation leaves all options possible since the GC receptors are ubiquitously distributed among many cell types of the organ (Adcock et al., 1996; Condon et al., 1998). Interestingly, a recent genetic dissection of mouse models carrying a GC receptor conditional knockout in different lung cell types revealed that ablation of the GC receptor was fatal if it occurred in the mesenchymal, rather than

epithelial or endothelial cells of the lung alveoli (Habermehl et al., 2011). These results are consistent with the long-known assessment that differentiation of the PTII cells depends on multifactorial paracrine communication with the alveolar fibroblasts (Shannon and Hyatt, 2004). In light of the fact that GCs activate essential gene expression in the fibroblasts (Wang et al., 1995), further studies are required to understand whether fibroblast support of lung maturation follows their own response to endogenously synthesised GCs, or takes place due to their activation by GCs made by other cell types. 2.5. Skin 2.5.1. Keratinocytes A considerable number of the studies in skin have sought to determine the impact of the sex hormones on skin physiology. Several of these studies explored the role of estrogen in the physiological functions of aging skin (review ref Verdier-Sevrain et al., 2006), or studied the abnormalities caused by androgenic involvement in either excessive hair growth (hirsutism) in women, or scalp hair loss (alopecia) in men (Finasteride, 2002; Randall, 2008). An intriguing question becomes, to what extent can one expect local skin steroidogenesis to play a role beyond the well-established effects of circulating sex hormones? Inoue et al. have recently correlated the levels of StAR and several other key steroidogenic enzymes, to the levels of sex steroids they measured in 17 human skin specimens. This meticulous examination revealed that the expression level of StAR was positively correlated with skin concentrations of testosterone in the male (Inoue et al., 2012). More specifically, StAR mRNA was predominantly expressed in the epidermis, a finding consistent with immunohistological localization of the protein in the epidermal granular layers and the hair outer root sheath cells of the human male skin. Furthermore, StAR was found to be significantly higher in the vertex area of the scalp, where hair density was lower than in the temporal area. Finally, complementing data were generated to show that testosterone synthesis was achieved in commercially available human keratinocytes when incubated in the presence of cholesterol (Bingham and Shaw, 1973). This suggested that local StAR expression may help create the androgenic environment known to be associated with alopecia in the vertex area of the scalp. Expression patterns similar to that of StAR were also observed for the CYP19A1 transcript in skin samples of both sexes (Inoue et al., 2012), a finding consistent with skin estrogen production previously attributed to skin biotransformation of circulating steroids (Labrie et al., 2000). However, despite this promising finding on StAR expression, the steroidogenic context of de novo hormone synthesis in the skin requires more exploration due to the fact that expression of CYP11A1 and HSD3B were absent in the skin samples that were utilized (Inoue et al., 2012). In agreement with the above information, StAR was also immunohistochemically identified in the basal layer of the scalp, i.e., colocalized in stem and proliferating keratinocytes; the latter pattern was aberrant in psoriatic and atopic dermatitis skin (eczema) (Hannen et al., 2011). Although StAR protein was not shown in primary keratinocytes prepared from face-lift material, pregnenolone processing to cortisol was subsequently demonstrated, leaving the question of de novo skin steroidogenesis still open. In a similar study, Tiala et al. also provided visual evidence for the expression of StAR in basal keratinocytes studied in normal and psoriatic skin (Tiala et al., 2007). Interestingly, these investigators sought the mechanism affecting keratinocytes by StAR binding protein (Sugawara et al., 2003) now named Coiled-Coil a-Helical Rod protein 1 (CCHCR1) located within the major psoriasis susceptibility locus and involved in the etiology of early stages of keratinocyte transformation (Suomela et al., 2009). However, so far, no

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functional linkage has been established between StAR in the keratinocyte, de novo skin steroidogenesis and the pathogenesis of psoriasis or neoplasia.

of the adrenal gland, respectively. Thus, the question becomes whether such limited steroidogenic capacity plays a physiologically meaningful function in fat depots.

2.5.2. Sebocytes Being highly androgen sensitive and accounting for the vast majority of androgen metabolism in the skin, the sebocytes were the obvious target cell type to look for StAR expression and de novo steroidogenesis, in particular in alopecia scalp skin known to overproduce DHT by virtue of high levels of the two 5a-reductase isozymes (Hoffmann, 2003). Chen et al. analyzed the transcript levels of StAR, CYP11A1 HSD3B2 and CYP17A1 in 51 men with androgenic alopecia (AGA) undergoing hair transplantation. StAR and HSD3B, but not the transcripts of the P450s, were higher in the bald skin (Chen et al., 2006). Immunohistochemistry showed moderate expression of StAR protein in the basal layer of the epidermal keratinocytes, outer root sheath of hair follicles and the eccrine sweat ducts but not the glandular cells. Strong StAR staining was also observed in pre-differentiated SZ95 sebocytes that are known to replicate in response to DHT. In summary, as is noted in several of the described studies, the general agreement is that the biochemical significance of high StAR levels in the face of missing CYP11A1 and CYP17A1 expression in the skin is presently not clear (Thiboutot et al., 2003).

2.7. Pancreas

2.6. Adipose tissue The search for de novo steroid hormone synthesis and StAR expression in the adipose tissue was naturally expected because of earlier studies describing the extra-adrenal steroidogenesis in brain and heart tissues (Davies and MacKenzie, 2003). Numerous studies have revealed the endocrine gland characteristics of fat cells, which include the secretion of adipocytokines, as well as the presence of a renin–angiotensin system and sex steroids and glucocorticoid metabolizing enzymes (Kershaw and Flier, 2004; Morton et al., 2004; Turgeon et al., 2006). As for sex steroids, the well-established synthesis of estradiol from circulating androgens is the most discussed endocrine function of adipose tissue (Bulun et al., 2012; Simpson et al., 2002). More relevant for fat de novo steroidogenesis are the glucocorticoids, long-known to affect fat cell differentiation and distribution (Hauner et al., 1987; Lonn et al., 1994). Reciprocally, the association of obesity with hyperaldosteronism (Goodfriend et al., 1998) is probably contributed to, at least in part, by adrenal StAR expression and steroidogenesis regulated by adipocyte secretions (Schinner et al., 2007). Beyond the unquestionable linkage between obesity and the role of fat cell expression of HSD11B1 that increases the local level of cortisol by regenerating it from circulating inactive cortisone (Masuzaki et al., 2001), MacKenzie et al. were the first to perform an extensive study aiming to find whether human adipose tissue can produce cortisol and aldosterone from cholesterol (MacKenzie et al., 2008). This study used tissue biopsies obtained from 8 women undergoing caesarean section, in order to perform carefully normalized real-time RT-PCR assessments of transcript levels in paired omental fat biopsies and the abdominal subcutaneous fat. The results clearly showed that the abdominal fat depots probably cannot produce cortisol and aldosterone by de novo steroidogenesis since the key enzymes CYP17A1, CYP11B1 and CYP11B2 are not expressed in this tissue. Instead, the mRNAs encoding StAR, CYP11A1, HSD3B2 and CYP21B are present in adipose tissue and probably allow for the conversion of cholesterol to 11-deoxycorticosterone synthesis. However, comparison of the transcript quantity in the abdomen fat samples relative to human adrenal RNA (commercially available) revealed that the adipose capacity to produce progesterone from cholesterol is low, with levels of StAR, CYP11A1 and HSD3B2 mRNAs being only 0.6%, 1% and 0.005% those

The search for pancreatic StAR seemed to address a 29 year old finding of high estrogen receptor binding in pancreatic carcinoma tissue (Greenway et al., 1981). Studies of enzyme activities related to androgen-estrogen bio-transformations followed that seminal notion (Fernez-del Castillo et al., 1991; Iqbal et al., 1983; Mendoza-Hernez et al., 1988), and included evidence for de novo pancreatic steroidogenesis; Morales et al. revealed the presence of CYP11A1 mRNA (RT-PCR) and measured pregnenolone synthesis in isolated mitochondria of rat pancreas (Morales et al., 1999). Upon revisiting this project using human pancreas (Morales et al., 2008), the investigators needed 35 PCR cycles to recover relatively equal levels of StAR, CYP11A1, SF-1 and the liver receptor homolog-1 (NR5A1 and NR5A2, respectively) transcripts in male and female samples normalized to cyclophilin; no adrenal control for tissue-specific comparisons was included. Yet, the overall low expression of the pancreatic gene products could be, in part, explained by subsequent immunohistochemical studies showing islet-specific CYP11A1 localization. Successive sections stained with anti-StAR did not yield convincing data on the presence of StAR protein in the pancreas or even positive controls of human ovary, possibly due to use of a less effective antiserum. Also unexplained was the immunohistochemical localization of SF-1 and SF2 that appeared to be cytoplasmic instead of the expected expression in nuclei of the steroidogenic islet cells; SF-1 was strongly stained in the cytosol of the pancreatic interstitial cells, while SF2 was identified in the endothelial cells of the pancreas. 2.8. Kidney It seems possible that a developmental biology ‘curiosity’ could have been a reason for the search for StAR in the kidney (Dalla Valle et al., 2004), as the mammalian kidney shares the same embryonic origin (intermediate mesoderm) with the steroidogenic cells of the gonads and the adrenal cortex. An earlier immuno-histochemical survey documented renal StAR expression in the distal convoluted tubules of the human adult, fetal and carcinomas of the kidney (Pollack et al., 1997). Addressing this issue in a rat model revealed similar results obtained by two separate groups who provided evidence for renal steroid production from cholesterol (Dalla Valle et al., 2004; Pagotto et al., 2011). The latter investigators showed convincingly that the genes encoding Star, Cyp11a1, Tspo and Nr5a1 are indeed expressed at low levels compared to the adrenal and testicular gene products, requiring 33–38 PCR cycles to detect the mRNAs, and 6–20 times more protein required to detect the protein band on Western blot. Supported with immunohistochemical evidence, Della Valle et al. detected the CYP11A1 protein (no StAR data) in the renal cortical distal tubules of neonates and juvenile animals up to puberty (Dalla Valle et al., 2004). Therefore, de novo steroidogenesis was proposed to play a role in development. Pagotto et al., however, identified CYP11A1 and StAR co-expressed in the thick ascending limb of Henle’s loop cells in the outer medulla, and in cortical cells of the distal convoluted tubules (Pagotto et al., 2011). The fact that these enzymes were observed in the adult kidney, suggested that the role of pregnenolone is not necessarily restricted to development. Importantly, expression of 3bHSD needed for the biotransformation of pregnenolone to active steroid hormones is missing in the kidney (Dalla Valle et al., 2004; Pagotto et al., 2011), hence the physiological roles of locally produced pregnenolone and of renal de novo steroidogenesis remain unanswered. The precedence for pregnenolone

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production and function in the CNS (Waters et al., 1997) may suggest a protective role of this steroid in the kidney as well. 3. Non-steroidogenic StAR expressing cells 3.1. StAR and oxysterol synthesis-potential significance One cannot underestimate the potential significance of StAR activity when delivering substrate to an alternative acceptor of cholesterol in the IMM, i.e., CYP27A1. The liver was the first example for this process, in which the ability of StAR to facilitate the translocation of cholesterol to the IMM where it can be utilized for initiation of hepatic bile acid synthesis, was described. Briefly stated, transformation of cholesterol to primary bile acids can proceed by two pathways; in the ‘classical neutral’ pathway, 7ahydroxylation of cholesterol is initiated by the endoplasmic reticulum enzyme cholesterol 7a-hydroxylase CYP7A1, followed by 27-hydroxylation of the sterol intermediate that diffuses into the mitochondria and becomes a substrate for the IMM monooxygenase enzyme complex of CYP27A1 (Vlahcevic and Stravitz, 1999). In the ‘alternative acidic’ pathway of bile acid production, cholesterol first enters the mitochondrion, where it undergoes what is commonly termed as 27-hydroxylation (Fakheri and Javitt, 2012) by CYP27A1, also referred to as sterol 26-hydroxylase (Andersson et al., 1989; Atsuta and Okuda, 1981; Cali and Russell, 1991; Javitt, 1990; Sato et al., 1977). This enzyme is also present in extra-hepatic tissues (Russell, 2000; Schroepfer, 2000) including vascular cell types relevant to the issues of this review. The putative physiological importance of non-steroidogenic StAR, when co-expressed with CYP27A1, is premised strongly on the multiple functions of this enzyme product, i.e., 27-hydroxycholesterol (27-HC). The latter is the most prevalent among the oxysterols that also include 24-hydroxycholesterol (24HC), and 25hydroxycholesterol (25HC). Besides being precursors for bile acid synthesis, the oxysterols function in reverse cholesterol transport to deliver sterols from peripheral tissues to the liver (Russell, 2009). Additionally, oxysterols inhibit cellular cholesterol synthesis by both blocking the signaling mechanism of sterol regulatory element-binding protein (SREBP), known to upregulate transcription of sterol-sensitive genes, as well as by upregulating the degradation rate of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the key enzyme in regulating cholesterol synthesis (Olkkonen and Hynynen, 2009). Oxysterols are also known to be activating ligands for the liver X receptors (LXR), and thereby have additional potential impacts on the transcription of genes controlling cholesterol and lipid metabolism (Janowski et al., 1996). Finally and most surprisingly, recent studies in mouse models have identified 27-hydroxycholesterol as the first endogenous selective estrogen receptor modulator, SERM, that can promote estrogen dependent growth of breast cancer cells and has adverse effects on bone mineralization and estrogen related cardiovascular protection (Umetani and Shaul, 2011). 3.2. Liver Similar to CYP11A1, CYP27A1 requires O2, mitochondrial NADPH, ferrodoxin and ferrodoxin reductase. Accordingly, low but measurable StAR expression was found in human primary hepatocytes when examined by Western blot of 30 lg of isolated mitochondrial protein and by Northern analysis (Hall et al., 2005). Low levels of StAR expression probably also occur in primary rat hepatocytes (Pandak et al., 2002). Over-expression of the protein in such cells was able to markedly increase bile acid production, thus proving the potential synergy between the CYP27A1 and StAR activities (Pandak et al., 2002; Sugawara

et al., 1995). In addition, CYP27A1 over-expression led to increased StAR expression, as did treatment with 27-hydroxycholesterol, which resulted in a 2-fold higher expression of StAR in the liver (Hall et al., 2005; Sugawara et al., 1995). Nevertheless, despite all expectations, it is likely that liver StAR does not play a central role in bile acid synthesis in normal hepatocytes. This notion is understandable considering the fact that under normal physiological conditions, the alternative ‘acidic’ pathway of bile acid biosynthesis is probably only secondary to the predominant ‘neutral’ pathway. Thus, the vast majority of hepatic cholesterol metabolism is initiated first by microsomal CYP7A1 activity, and results in the production of more hydrophilic 7a-hydroxycholesterol that does not require the presence of StAR for entering the mitochondria for further processing by CYP27A1. 3.3. Liver cancer Mitochondria are considered cholesterol-poor organelles containing no more than 3–5% of the plasma membrane content of this sterol (Maxfield and Tabas, 2005). Interestingly, when compared to primary hepatocytes, the levels of mitochondrial cholesterol (Crain et al., 1983; Feo et al., 1975) and StAR protein were found to be higher in transformed human hepatocytes (Hall et al., 2005). These observations led Montero et al. to explore the hypothesis that cholesterol enrichment in the mitochondria may result in less susceptibility to apoptotic cell death and make the cells more resistant to cancer therapy (Montero et al., 2008). In accordance, doxorubicin treatment of cells resulted in more apoptosis upon reduction of endogenous cholesterol synthesis by statins, or partially knocking-down (by 40–50%) the otherwise elevated levels of StAR in human hepatocellular carcinoma cells. Interestingly, StAR reduction by siRNA resulted in a net decrease of mitochondrial cholesterol levels and, therefore, it was not clear whether the observed resistance to chemotherapy resulted from lower delivery of cholesterol to mitochondria from extra-mitochondrial sources, or the impaired mobilization of cholesterol from the OMM to the IMM. An alternative mechanism for the StAR initiated protection from chemotherapy induced apoptosis may result from the interaction of StAR with the VDAC (Bose et al., 2008; Rone et al., 2012, 2009); this OMM protein was previously proposed to be an essential factor in the onset of apoptosis (Keinan et al., 2010). Hence, it may be hypothesized that StAR expression is advantageous for a cancer cell due to impairment of VDAC function in apoptosis. 3.4. Vasculature StAR Whereas StAR/CYP27A1 mediated production of oxysterols in the liver may serve for cholesterol biotransformation in bile acid synthesis, the premise that StAR is also a rate limiting factor for oxysterol synthesis in extra-hepatic cells led to studies of the cell types involved in what is termed ‘cholesterol efflux’ from cells, a process implicated in the reverse cholesterol transport pathway (Escher et al., 2003). Two typically CYP27A1 rich cells that are perceived as pivotal for the prevention of atherosclerosis are macrophages (Bjorkhem et al., 1994) and vascular endothelium (Reiss et al., 1994). 3.4.1. Macrophages A firm interest in the process of atherosclerosis led the Yin group and others to explore the potential role of StAR in macrophages (Bobryshev, 2006; Ning et al., 2010; Taylor et al., 2010). In symmary, these studies, macrophage accumulation of cholesterol originating from the uptake of LDL and cytotoxic oxidized LDL, can turn these cells into foam cells and trigger their apoptotic and necrotic death, a process associated with the progression of atherosclerosis (Bobryshev, 2006; Pennings et al., 2006). Primarily,

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these studies were conducted in cultured cell lines and most of the observations were based on the consequences of StAR over-expression. Thus, over-expression of StAR in THY-1 monocyte-derived macrophage cells prevented their apoptotic death by a mechanism involving increased expression of anti-apoptotic genes (Bcl-2), and concomitant down-regulation of pro-apoptotic gene expression (Bax, caspase-3) (Bai et al., 2010). Although no direct data were presented to explain the impact of StAR on gene transcription, the authors hypothesized that cholesterol is mobilized into the mitochondria by StAR. CYP27A1 then catalyses the formation of 25-hydroxycholesterol, assuming that this oxysterol, like 27hydroxycholesterol, acts as an activating ligand of the LXR transcription factor (Fu et al., 2001; Venkateswaran et al., 2000). This hypothesis was based on a previous study from this group (Ning et al., 2009) that demonstrated that adenovirus mediated StAR over-expression in differentiated THP-1 cells attenuated apoptosis, and was concomitantly linked with characteristic changes in gene products that are implicated in the progression of foam cell pathology. StAR over-expression reduced the levels of cytotoxic intracellular cholesterol, apparently as a result of the increased expression of CYP27A1 and ABC transporters known to support cellular cholesterol efflux. StAR presence also led to a decreased secretion of the pro-inflammatory cytokines TNFa and IL-1b; both phenomena could have resulted from the observed up-regulation of the LXRPPAR transcription factors previously shown to control both cholesterol trafficking and cytokine expression (Chinetti et al., 2006; Van Eck et al., 2005; Wang and Wan, 2008). Consistent with the above pattern, a relatively low level of endogenous StAR expression (mRNA) was observed in human THP-1 cells (Borthwick et al., 2009) and RAW264.7 mouse macrophage cells, whereas the proinflammatory cytokines (TNFa IFNc) down-regulated the Star gene products (Ma et al., 2007). Extending the use of the latter cell line model, Taylor et al. have recently shown that over-expression of StAR enhanced LXR expression (mRNA), which in turn increased apoAI and ABCA1-dependent cholesterol efflux, probably occurring downstream of the conversion of cholesterol to oxysterols by CYP27A1 (Taylor et al., 2010). 3.4.2. Vascular endothelium Low power images of immunohistochemical and cytochemical StAR staining have suggested that the protein is expressed in a variety of endothelial cells in vivo and in-culture, including miniveins of the human connective tissue, intima of human aorta, rat aorta, primary cultured rat heart microvascular endothelial cells and immortalized mouse brain microvascular endothelial cell line, bEnd.3 (Ning et al., 2006; Suren Castillo et al., 2008; Ning et al., 2010). None of the above observations presented morphological evidence for mitochondrial StAR localization. Yet, in the endothelial bEnd.3 cell line, Ning et al. were able to show StAR expression by Western analysis, apparently demonstrating both StAR pre-protein and its mature mitochondrial product. Moreover, in these cells StAR was induced 2-fold (qRT-PCR and Western) by the addition of cholesterol, LDL or 25-hydroxycholesterol to the culture medium (Ning et al., 2006). Recently, these investigators also applied an adenovirus infected StAR over-expression approach to show that, similarly to the response of the THP-1 macrophages, a 160-fold increase of StAR content in the bEnd.3 cells induced the expression of ABC transporters by 2-fold (Ning et al., 2010). Such results were consistent with the possibility that StAR enhanced oxysterol production in the endothelial cells can up-regulate gene products involved in cholesterol efflux. Collectively, the evidence for a putative anti-atherosclerotic activity of StAR in CYP27A1-rich cells is attractive, particularly when explored ex vivo and under over-expression circumstances. However, due to the marginally detected endogenous levels of StAR in the liver, macrophages or the vascular endothelial cells,

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further studies that target StAR ablation in these cells, may be required in order to provide convincing evidence that the involvement of StAR in cholesterol homeostasis is physiologically meaningful in vivo. 4. Cancer and diseased states In addition to the increase in robustness that liver carcinoma cells acquire following StAR expression, it turned out that tumors of ‘non-classical’ tissues may also harness an acquired ability to make steroid hormones locally, in order to gain a survival advantage. 4.1. Prostate The rationale behind the search for StAR and the other genes required for testosterone/DHT synthesis in prostate tumors was straightforward and took into account the unique evolution of this disease. Prostate cancer is the second most frequent cancer-related cause of death among men in the United States (Jemal et al., 2005). Prenatal development and normal function of this gland in the adult depends on the testicular supply of testosterone that is converted to the more active hormone DHT in each of the target cells. Androgens are also essential for initiation of prostate cancer and, therefore, removal of the androgen source by castration is required for endocrine therapy (Griffiths et al., 1997). However, progression of tumor growth was observed to be associated with apparent loss of androgen dependency (Feldman and Feldman, 2001) and the tumors become castration-resistant (Mostaghel et al., 2007). Yet, castration-resistance did not involve the acquisition of new pathways bypassing the need for androgen receptors (AR), or loss of the latter, since receptor blockers cause tumor growth arrest; moreover, some tumors expressed spare receptors and became supersensitive responders able to maintain growth and survival despite the antiandrogen therapy (Chen et al., 2004). Instead, it was shown that genes regulating cholesterol biosynthesis are upregulated in androgen ablation resistant prostate cancer cells (Holzbeierlein et al., 2004), along with genes encoding enzymes capable of forming testosterone from adrenal precursors (El-Alfy et al., 1999; Nakamura et al., 2005; Stanbrough et al., 2006). Taking this into consideration, Khan and colleagues raised the possibility that advancing prostate tumors acquire the capability to synthesize testosterone/DHT in order to support their own receptor-mediated survival even in the absence of testicular derived hormone (Dillard et al., 2008). Using a series of cell lines, including androgen-dependent (C33) and androgen-independent (C81) prostate tumor cells, these investigators showed that the C81 cells express markedly higher transcript levels of StAR and CYP17A1, when compared to the C33 cell line. Both cell types expressed comparable levels of CYP11A1, HSD3B and HSD17B transcripts at levels observed in the H295R human adrenal adrenocortical carcinoma cell line. This proof-of-concept study explains why castration may not achieve androgen deficiency and, therefore, inhibition of steroidogenesis is required for complete abolishment of androgen effects on the tumor growth (Eichholz et al., 2012). 4.2. Endometrial carcinoma Cancer of the endometrium is the most common, yet highly curable gynecologic malignancy in women. A high proportion of endometrial carcinomas are hormone responsive and express estrogen and progesterone receptors (McCarty et al., 1979). Such features are consistent with the normal physiology of the endometrium, whereby follicular estrogen is mitogenic while the post-ovulatory luteal secretion of progesterone arrests cell growth and supports

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differentiation of the implantation-ready endometrium. Therefore, the long-known anti-proliferative activity of progesterone is powerful in combination with chemotherapy cocktails used to cure this cancer (Kelley and Baker, 1961; Mutter et al., 2001). Similar to the experimental paradigm seeking steroidogenic capacities in prostate tumor cells altered to become hormone-independent (Dillard et al., 2008), Sugawara et al. examined HHUA cells expressing both ER and PR, and compared them to HOUA cells, which are undifferentiated endometrial carcinoma cells, expressing neither ER nor PR (Ishiwata et al., 1984; Sugawara et al., 2004). CYP11A1 and StAR were significantly expressed at the levels of transcript and Western blot; transcripts encoding HSD3B and CYP17A1 were also detected. These results suggested that progesterone and aromatizable androgens can be synthesised in these cells, but not estrogens (Sugawara et al., 2004). No clear difference was noted between a de novo steroidogenic capacity of the differentiated and the non-differentiated cell types. Also, the expression and potential role for the steroidogenic genes and their products in endometrial tumors have not been reported so far. Interestingly, a search for StAR, CYP11A1, and HSD3B in the pregnant uterus was successful in rodents, where the latter genes were expressed not only in the mid-gestation trophoblast giant cells (Arensburg et al., 1999; Schiff et al., 1993; Sher et al., 2007; Yivgi-Ohana et al., 2009). 4.3. Endometriosis Endometriosis is a common and long-known gynecological disease (Sampson, 1927). In this disease endometrial cells appear in the abdomen due to retrograde menstruation via the fallopian tubes and result in the ectopic flourishing of the cells outside the uterine cavity, most commonly on the peritoneum (Olive and Schwartz, 1993) or on the extraovarian wall (Attar et al., 2009). Also interesting is the second possibility for the etiology of endometriosis, known as coelomic (peritoneal) metaplasia, that is based on the assumption that the tumor arises from undifferentiated cells in the peritoneal cavity (Mutter et al., 2001). Endometriosis affects roughly 10% of women of reproductive age. Not so surprising is the fact that growth of the ectopic endometrial cells is estrogen dependent and, to this end, these cells can provide their own supply of the hormone by expressing aromatase (Bulun et al., 2000; Kitawaki et al., 1999). Several studies aimed to find whether this tissue independently provides its own needs by synthesizing estradiol from cholesterol, or depends on circulating androgens to be locally transformed to estradiol by the endometriotic aromatase activity. In this regard, Tsai and colleagues questioned if endometriotic stroma cells can synthesize progesterone from cholesterol (Tsai et al., 2001). Indeed, StAR gene products were markedly induced in the stroma of the endometriotic lesions from early and advanced stages of endometriosis that were also comparable to normal endometrium samples taken from diseasefree women. Progesterone synthesis and StAR expression were induced by PGE2 treatment. In contrast to StAR, the transcripts of CYP11A1 and HSD3B were present, but remained at basal levels in all three tissues, suggesting that StAR fulfills the expectations of being the sole rate-limiting factor in this tissue model. Then, based on the known enrichment of estradiol levels in the endometriotic cells (Noble et al., 1997), Attar et al. examined the entire enzyme cascade of estradiol synthesis from cholesterol in ovarian endometrioma (Attar et al., 2009). When compared to normal endometrial stromal tissue, the endometriotic cells expressed significantly higher mRNA levels of StAR, CYP11A1, HSD3B2, CYP17A1 and CYP19A1 once stimulated by PGE2. Furthermore, PGE2 significantly increased the StAR promoter activity in both the endometriotic and the endometrial stromal cells; in another series of overexpression studies, SF-1 induced, whereas COUP-TFII or WT1

suppressed StAR promoter activity. Consistent with such positive and negative roles were in situ assessments of the transcription factors binding by ChIP; these studies showed that COUP-TFII or WT1 binding to both promoters was significantly higher in endometrial compared with endometriotic cells; in the latter, PGE2 induced coordinate binding of SF1 to StAR and aromatase promoters but decreased COUP-TFII binding. Collectively, this study strongly supported the possibility that endometriotic cells contain a full complement of the gene products required for prostaglandin regulated estrogen synthesis from cholesterol. 4.4. Ovarian carcinoma The search for StAR and de novo steroidogenesis in the transformed ovarian epithelium may sound counterintuitive since the normal epithelium is never steroidogenic. Ovarian cancer is the leading cause of death from all gynecologic malignancies. Germ line mutations in BRCA1/2 have been implicated in a small fraction of the cases, while four risk-filled patterns are considered in the etiology of the bulk ovarian neoplasia, namely, the ‘‘incessant ovulation’’ (‘tear-and-repair’ hypothesis) (Fathalla, 1971), the on-going debate on the role of repeated exposure to gonadotropins (Gadducci et al., 2012), the inflammation hypothesis (Maccio and Madeddu, 2012) and the intriguing possibility that ovarian carcinoma may evolve from fallopian tube epithelial cells (King et al., 2011). Similar to the observed response of endometrial cancer to the sex hormones mentioned above, intra-tumor synthesis and/or metabolism of steroid hormones have been considered to play important roles in the etiology of ovarian cancer, where estrogen is considered mitogenic (Sasano and Harada, 1998) and progesterone inhibits growth of the tumor cells (Risch, 1998). Assuming that sex steroids can nevertheless play a role in ovarian tumor development, Abd-Elaziz et al. (2005) examined, for unclear reasons, whether the genes responsible for progesterone synthesis are locally expressed during different stages of tumor development. Using immunohistochemistry and qRT-PCR measurements of StAR, CYP11A1 and HSD3B, these investigators determined the gene product profiles in frozen tissues of 20 cases of epithelial ovarian carcinoma and histological samples of 100 such patients. The results were subjected to detailed statistical analyses and suggested an inverse correlation between the expression of steroidogenic genes and the tumor size, proliferative activity and tumor grade. In light of their findings, the presence of StAR in epithelial ovarian carcinoma cells was found to be ‘‘a marker for a good prognosis, possibly due to the local production of progesterone and subsequent hormone induced inhibition of cell proliferation’’. 4.5. Intestine/colon Consistent with a chronological perspective, StAR expression was never explored in the thymus where local glucocorticoid synthesis was one of the earlier models for extra-adrenal steroidogenesis (Ashwell et al., 2000; Vacchio et al., 1994). Inspired by the thymus studies, Brunner and co-workers unraveled the bi-directional cross-talk between steroidogenic cells of the intestine and the immune cells passing the gut, when activation of the latter induced intestine cortisol synthesis, which in turn performed immunomodulatory functions (Cima et al., 2004). In a murine model, these investigators showed that corticosterone synthesis was dramatically induced in organ culture of intestinal sections if the sections were taken from animals injected 4 h earlier with anti-CD3 to activate passing intestinal T-cells. These studies showed that the transcript and protein levels of CYP11A1 were considerably lower when compared to adrenal cell positive controls. However, such differences were explained by in situ hybridizations showing that the transcripts of the steroidogenic genes CYP11A1, CYP11B1 and

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HSD11B1 (corticosterone synthesis) were specifically confined to the relatively minor population of the intestinal crypt cells (Cima et al., 2004). Further studies with cell lines showed that transcription of the steroidogenic genes in the intestinal epithelial cells is controlled by phorbol ester activated liver receptor homolog-1 (NR5A2), whereas ACTH/cAMP and SF-1 (NR5A1) controlled the steroidogenic genes in the Y1 adrenal cell line that were used by the investigators for comparison (Mueller et al., 2007). Recently, this group described cortisol synthesis, readily produced de novo by StAR and the relevant enzyme cascade, in colorectal cancer cell lines and primary tumors, (Sidler et al., 2011). Furthermore, the primary colon carcinoma samples expressed more StAR, and steroidogenic mRNAs than normal colonic mucosa. Consistent with their earlier notions, the findings of Sidler et al. suggested that the tumor-derived cortisol is immunomodulatory and suppresses activation of T-lymphocytes, which may provide a mechanism of tumor immune escape. 4.6. Adrenal tumors Unlike the ovary, tumors of non-functional cell origin (carcinoma) in the adrenal cortex indeed met the expectations and lacked significant expression of StAR and CYP11A1 as shown by Zenkert et al. (2000). These investigators also concluded that StAR alone cannot be a sole marker for steroidogenesis in adrenal adenomas (benign tumors); for example, their studies showed that non-functional adenomas express unexplained high StAR mRNA levels without a matching expression of the CYP11A1 transcript (Liu et al., 1996; Zenkert et al., 2000). Hence, no special role could be assigned to StAR expression in the adrenal tumors. 5. Conclusions The literature survey presented herein suggests that significant revision for the present definition of the role of StAR may be in order considering the multiple tasks that StAR may be able to perform within many physiological contexts. In the vast majority of the searches for StAR expression in extra-adrenal or extra-gonadal tissues, StAR was considered as a marker of choice for local de novo steroidogenesis. In other instances, StAR was studied as a result of it being the most logical provider of the substrate, cholesterol, for an enzyme system similar to CYP11A1, namely, CYP27A1; the oxysterol product of the CYP27A1 activity was then expected to lead to either bile acid synthesis in the liver, or regulate lipid homeostasis by modulating gene transcription. Therefore, in potential future studies, this niche of StAR function is expected to cast an impact on cholesterol synthesis, mobilization, deposition, obesity and atherosclerosis (monocytes/foam cells). Not less intriguing is the expression of StAR in malignancies, claimed now to endow chemotherapy resistance on tumor cells (hepatoma cells), to help bypass body surveillance (prostate cancer), to allow escape from tumor immunity and tumor progression (colorectal cancer), or to play protective roles under a cytotoxic environment (brain, kidney). Yet, it is worth noting that some of the conclusions mentioned above are not necessarily based on indisputable evidence, hence, they invite further studies utilizing attractive working hypotheses. For example, in more than a few cases, the physiological relevance of StAR expression remained unclear, due to an inability to detect protein levels in extracts containing 102–105 of the transcript levels found in the adrenal. Yet, one cannot exclude the possibility that in such cases StAR-rich cells account for 1% or less of the tissue mass. Therefore, it is likely that more studies focused on single cell information will provide a better understanding of novel StAR activities. Then, once the exact cell types expressing StAR are elucidated, design of cell-specific knockouts of StAR should be able to

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provide clearer information on the role of this protein under specific locational and physiological circumstances. Collectively, the information gathered to date suggests that local production of classical steroid hormones, sterol derivatives, or other outcomes of mitochondrial cholesterol mobilization by StAR, probably play significant physiological roles more prevalent than presently known. 6. Support This research was supported by the Israel Science Foundation Grants 1558/07 and 677/12 (to J.O.) and by funding from NIH Grant HD-17481, and grant B1-0028 from the Robert A. Welch Foundation (to D.M.S.). Acknowledgments The authors would like to dedicate this review to the memory of Dr. Keith Parker who was a dear friend and valued collaborator to us. Keith was a true scholar whose monumental contributions to this field were of the highest order and whose input into the understanding of steroidogenic function in the adrenal gland and other steroidogenic tissues remain sorely missed to this day. References Abd-Elaziz, M., Moriya, T., Akahira, J., Suzuki, T., Sasano, H., 2005. StAR and progesterone producing enzymes (3beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochromes P450) in human epithelial ovarian carcinoma: immunohistochemical and real-time PCR studies. Cancer science 96, 232–239. Adcock, I.M., Gilbey, T., Gelder, C.M., Chung, K.F., Barnes, P.J., 1996. Glucocorticoid receptor localization in normal and asthmatic lung. American Journal of Respiratory and Critical Care Medicine 154, 771–782. Alberta, J.A., Epstein, L.F., Pon, L.A., Orme-Johnson, N.R., 1989. Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP. The Journal of Biological Chemistry 264, 2368–2372. Andersson, S., Davis, D.L., Dahlback, H., Jornvall, H., Russell, D.W., 1989. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26hydroxylase, a bile acid biosynthetic enzyme. The Journal of Biological Chemistry 264, 8222–8229. Arakane, F., Sugawara, T., Nishino, H., Liu, Z., Holt, J.A., Pain, D., Stocco, D.M., Miller, W.L., Strauss III, J.F., 1996. Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence. implications for the mechanism of StAR action. Proceedings of the National Academy of Sciences of the United States of America 93, 13731–13736. Arakane, F., Kallen, C.B., Watari, H., Foster, J.A., Sepuri, N.B., Pain, D., Stayrook, S.E., Lewis, M., Gerton, G.L., Strauss III, J.F., 1998. The mechanism of action of steroidogenic acute regulatory protein (StAR). StAR acts on the outside of mitochondria to stimulate steroidogenesis. The Journal of Biological Chemistry 273, 16339–16345. Arensburg, J., Payne, A.H., Orly, J., 1999. Expression of steroidogenic genes in maternal and extraembryonic cells during early pregnancy in mice. Endocrinology 140, 5220–5232. Ashwell, J.D., Lu, F.W., Vacchio, M.S., 2000. Glucocorticoids in T cell development and function⁄. Annual Review of Immunology 18, 309–345. Atsuta, Y., Okuda, K., 1981. On the stereospecificity of cholestanetriol 26monooxygenase. The Journal of Biological Chemistry 256, 9144–9146. Attar, E., Tokunaga, H., Imir, G., Yilmaz, M.B., Redwine, D., Putman, M., Gurates, B., Attar, R., Yaegashi, N., Hales, D.B., Bulun, S.E., 2009. Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis. The Journal of Clinical Endocrinology and Metabolism 94, 623–631. Azhar, S., Reaven, E., 2002. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis. Molecular and Cellular Endocrinology 195, 1–26. Bai, Q., Li, X., Ning, Y., Zhao, F., Yin, L., 2010. Mitochondrial cholesterol transporter, StAR, inhibits human THP-1 monocyte-derived macrophage apoptosis. Lipids 45, 29–36. Ballard, P.L., Ning, Y., Polk, D., Ikegami, M., Jobe, A.H., 1997. Glucocorticoid regulation of surfactant components in immature lambs. The American Journal of Physiology 273, L1048–L1057. Barnett, C.A., Pritchett, E.L., 1988. Detection of corticosteroid type I binding sites in heart. Molecular and Cellular Endocrinology 56, 191–198. Baulieu, E.E., 1998. Neurosteroids: a novel function of the brain. Psychoneuroendocrinology 23, 963–987.

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