Role of the steroidogenic acute regulatory protein in health and disease.

The deposition of excess lipids/CEs in the arterial walls is a hallmark of atherosclerosis in which macrophages play a vital role to form the foam cells. Atherosclerosis is the most prevalent event within cardiovascular diseases and is the leading cause of morbidity and mortality worldwide. Accumulating evidence indicates that stimulation of CE hydrolysis following overexpression of HSL correlates with ABCA1 expression in macrophages, a crucial event in cellular lipid transport and atherosclerosis [ 141 – 145 ]. ABCA1, a key protein in cholesterol efflux, transports cellular cholesterol from macrophages to apolipoprotein A1 (Apo-A1). The importance of ABCA1 has been documented by its absence in patients afflicted with Tangier disease, which is linked to HDL deficiency and premature atherosclerosis [ 146 ]. Removal of excess CEs from macrophage-derived foam cells is critical for the progression of atherosclerotic lesions [ 145 , 147 – 149 ]. Studies have shown that overexpression of STAR increases macrophage cholesterol efflux and decreases intracellular lipids and the secretion of inflammatory factors [ 148 , 149 ]. LXR-RXR/RAR and their heterodimerization partners, especially lipid homeostasis-related transcription factors, SREBPs (particularly SREBP-1c) and peroxisome proliferator-activated receptors (PPARs), are targets for intervention in atherosclerosis [ 144 , 150 – 152 ]. Our recent findings demonstrate that retinoids strikingly increase cAMP/PKA responsive STAR and steroid levels in a variety of steroidogenic cell models [ 9 , 34 , 35 ]. These results suggest that retinoid signaling is capable of enhancing cholesterol clearance from cells, an approach that may be effective in limiting plaque stability and progression of atherosclerotic cardiovascular disease. Consistent with this, in a recent study [ 153 ] we have reported that retinoids enhance cholesterol efflux to Apo-A1 in mouse RAW 264.7 macrophages, and this effect was further augmented in the presence of cAMP/PKA signaling ( Figure 3 ). Besides, macrophages overexpressing HSL increased the hydrolysis of CEs resulting in a depletion of CE content and elevated StAR mRNA expression. Concurrently, HSL overexpression was capable of enhancing the efficacy of RAR and LXR ligands on StAR and ABCA1 protein levels [ 153 ]. These findings imply that an increase in HSL levels promotes oxysterol production, which, in turn, activates LXR and results in up-regulation of retinoid mediated macrophage cholesterol efflux. In support of this, previous studies have demonstrated the role of LXRs and PPARs in the control of cholesterol trafficking in macrophages [ 142 , 154 ]. Taken together, it is plausible that LXR activation enhances plasma membrane cholesterol trafficking and efflux, and modulates cholesterol esterification, thus contributing to the effects of retinoids in controlling cholesterol balance for limiting/stabilizing atherosclerotic cardiovascular disease. An understanding of the process of macrophage foam cell formation and its connection to HSL-LXR regulated events will help develop novel therapeutic interventions for atherosclerosis.

Complex endocrine changes, affecting the morphology and function of a multitude of organs, occur as life progresses from adulthood into senescence. This aging process results in a decline of various hormones and, as a consequence, affects a number of physiological functions [ 155 – 160 ]. The occurrence of hormone deficiencies (or endocrinosenescence) is constituted to be the major cause of human senescence and it is associated with numerous complications and disabilities [ 156 , 160 – 162 ]. Endocrinosenescence includes growth hormone/insulin-like growth factor-1 axis (somatopause), hypothalamic-pituitary gonadal axis (hypogonadism), testosterone (andropause), estradiol (menopause), and dehydroepiandrosterone (adrenopause) [ 159 , 160 , 163 – 166 ]. The manifestations of these deficiencies include, but are not limited to, inefficient hypothalamic-pituitary-thyroidal-adrenal-gonadal (HPTAG) axes, neurodegenerative disorders, diminished eyesight, impaired memory and cognitive function, decreased muscle mass and bone density, decreased steroid biosynthesis, sexual dysfunction and depression, increased risk in cardiovascular disease, and skin disorders [ 65 , 139 , 155 , 159 , 162 , 167 , 168 ]. These conditions profoundly affect geriatric populations worldwide within the context of a substantial rise in life expectancy. Preservation of hormonal balance is the key to proper functioning of various biological activities during aging. We have demonstrated that retinoids, especially RAs, up-regulate STAR expression and steroid biosynthesis in adrenal, gonadal, glial, and epidermal cells [ 34 , 35 ], indicating that retinoid signaling is capable of influencing a number of cholesterol/steroid coupled physiological activities that are frequently impaired in geriatric populations. Aging is an inevitable heterogeneous phenomenon involving the whole organism and results in a decline of the central nervous system and the endocrine system [ 156 , 169 – 172 ]. The maintenance of a well-balanced endocrine circadian rhythmicity is critical to health. Age-associated hormonal imbalance, involving a reduction in the steroidogenic output, is linked to numerous health complications along with a host of pathologies [ 138 , 173 – 177 ]. There is increasing evidence that aging is connected to the progressive accumulation of dysfunctional mitochondria and oxidative damage, which modulate the immune system and contribute to increased morbidity and mortality [ 177 – 181 ]. With aging, excessive production of free radicals and reactive oxygen species (ROS) occurs in the mitochondria, which affects the function of the HTPAG axis auto-regulating system. The increase in free radicals is inversely correlated with antioxidant capacity in the central nervous system and its associated glands [ 179 , 182 ]. It is conceivable that an imbalance between production of free radicals/ROS and protective antioxidant systems, affecting cellular oxidative damage, might induce age-related complications and diseases [ 179 , 180 , 182 ]. Previous studies have reported that ROS disrupts mitochondria and decreases in STAR expression and steroidogenesis in a variety of steroid producing cells, and is tightly connected with age-related decline in steroid biosynthesis [ 177 , 178 , 183 , 184 ]. As such, oxidative damage induced by ROS is deleterious to the functional efficiency of various cellular processes and is implicated in the pathogenesis of numerous conditions including age-related complications and disabilities. Physiological aging results in most of the phenotypic changes observed in skin. The latter forms an essential barrier between the external environment and the biological milieu, and it is tightly networked to central regulatory systems [ 34 , 139 , 176 , 185 – 187 ]. Several lines of evidence demonstrate that regulation of glucocorticosteroidogenesis in the skin is similar to those operating in classical steroidogenic tissues [ 188 – 191 ]. Human skin cells express STAR, synthesize cholesterol, and possess the functional biochemical apparatus for the synthesis of glucocorticoids, androgens, and estrogens, which play vital roles in epidermal homeostasis [ 139 , 188 , 190 , 192 – 194 ]. It should be noted that in human skin CYP11A1 can also use 7-dehydrocholesterol (precursor to cholesterol and vitamin D) as an alternative substrate leading to production of 7Δ-steroids [ 176 , 195 ]. Expression levels of STAR and aromatase have been correlated with androgen and estrogens in male and female skin tissues, respectively, demonstrating the relevance of STAR and sex steroids in homeostasis of the human skin [ 194 ]. In contrast, malfunction in skin cholesterol synthesis, involving a global reduction in steroids, is associated with down-regulation of epidermal differentiation, leading to many skin complications/disorders [ 34 , 139 , 187 , 193 , 196 ]. We and others have demonstrated that expression of STAR mRNA is decreased or aberrant in several inflammatory skin diseases, including eczema, intertrigo, atopic dermatitis, signifying that steroid biosynthesis is disrupted in these diseased conditions [ 34 , 139 , 197 – 199 ]. As mentioned above, retinoids influence an array of functions, ranging from vision to reproduction to homeostasis [ 35 , 139 , 199 – 202 ]. The therapeutic and preventive effects of retinoids in numerous skin conditions and diseases, including premature skin aging, skin cancer prevention, squamous cell carcinoma, and skin rejuvenation and hyperpigmentation have long been established [ 137 , 191 , 203 – 205 ]. Retinoid metabolism and signaling also decreases in a variety of complications and diseases [ 138 , 140 , 168 , 206 ]. The systemic administration of RAs has been shown to reverse most reproductive and developmental blocks in vitamin A deficient (VAD) rats and mice, demonstrating that retinoid signaling rescues reproductive defects as well as steroidogenesis in VAD animals [ 201 , 207 , 208 ]. An unanswered question is if retinoids are able to reverse the decline in steroid biosynthesis in target tissues and thereby restore steroid coupled impaired biological activities that particularly evolve during aging. It has been shown that retinoids, especially RAs, elevate expression of steroidogenic enzymes, STAR, and steroid biosynthesis in a variety of target tissues [ 35 , 209 – 213 ]. Recently, we observed that retinoids were capable of enhancing and/or restoring STAR expression and pregnenolone synthesis in isolated epidermal keratinocytes of elderly (64–83 years) individuals (Manna PR et al., unpublished observations). These de-identified human skin tissues were obtained upon various surgeries from the Department of Dermatology clinic, Texas Tech University Health Sciences Center (IRB# L14-085). This implies that retinoid signaling is capable of reversing the decline in steroid biosynthesis and relevant skin complications and disorders in aging populations. Hence, it is conceivable that therapeutic strategies involving the use of retinoids will have benefits in the restoration of many impaired physiological activities that are important for healthy aging. Future studies on tissue-specific regulation of retinoid mediated restoration of cholesterol/steroid dependent events provide better understanding on many important physiological and/or patho-physiological processes.

The maintenance of normal reproductive development and function, and bodily homeostasis, is dependent on steroid hormones. Although the steroid hormones are diverse, they are synthesized from a common precursor substrate, cholesterol, which can be derived from a number of sources, i.e., de novo synthesis of cellular cholesterol, lipoprotein-derived cholesteryl esters (CEs), and hydrolysis of CEs stored in lipid droplets [ 1 – 3 ]. Even so, the conversion of CEs into free cholesterol serves as a crucial step in controlling cholesterol availability for steroidogenesis. Regulation of steroid biosynthesis is primarily mediated by trophic hormones, although multiple intracellular events and signaling pathways have been demonstrated to play permissive roles [ 3 – 10 ]. Hormonal control of steroid biosynthesis occurs within minutes (acute) to hours (chronic) and is mediated by cAMP signaling. The biosynthesis of steroid hormones is initiated upon mobilization of cholesterol from cellular stores to the mitochondrial inner membrane and to the site of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc or CYP11A1) (reviewed in Refs. [ 6 , 10 – 12 ]). The precise mechanism by which cholesterol is transported to the mitochondria for steroidogenesis remains unknown; however, considerable evidence suggests the involvement of a dynamic mitochondrial protein complex in this process. These proteins include acyl-CoA synthetase 4, steroidogenic acute regulatory protein (STAR), peripheral benzodiazepine receptor/translocator protein, and AAA domain containing protein 3 [ 12 – 20 ]. Among them, the STAR protein has essentially all of the characteristics to become an acute regulator of steroid biosynthesis in steroidogenic tissues [ 6 , 8 , 10 , 11 , 13 , 21 – 24 ]. STAR consists of several forms of a newly synthesized 30 kDa protein which has a 37 kDa precursor form containing an N-terminal mitochondrial targeting sequence. In describing STAR’s role in cholesterol transport, the preponderance of evidence indicates that the 37 kDa STAR acts on the outer mitochondrial membrane [ 13 , 25 ]. Alternatively, it has been demonstrated that the 30 kDa phosphorylated form acting on the inner mitochondrial membrane allowing for the transfer of the majority of cholesterol [ 14 ]. The compelling evidence for the critical role of STAR in the regulation of steroidogenesis has been demonstrated in patients suffering from lipoid congenital adrenal hyperplasia (lipoid CAH), an autosomal recessive disorder in which both adrenal and gonadal steroid biosyntheses are severely impaired due to mutations in the STAR gene [ 23 , 26 – 29 ]. Targeted disruption of the STAR gene in mouse results in an essentially identical phenotype to that found in lipoid CAH in humans [ 30 – 32 ]. In fact, the STAR protein plays an important role in the regulation of steroid hormones required for life itself, in the case of adrenal steroids, and for maintaining reproductive capacity, in the case of gonadal steroids [ 3 , 4 , 6 , 8 , 21 , 25 ]. Nonetheless, in virtually every system studied, agents that influence STAR expression also influence steroid biosynthesis through endocrine, autocrine and paracrine regulation. Regardless of the regulatory events, studies have demonstrated a tight correlation between the synthesis of STAR protein and the synthesis of steroids in a variety of classical (e.g. adrenal and gonadal) and non-classical (e.g. glial and skin) steroidogenic tissues [ 3 , 6 , 33 , 34 ]. Whereas STAR plays an indispensable role in controlling steroid biosynthesis, a complete understanding of the regulation of its expression and function in steroidogenesis is not available. Recent findings have demonstrated that hormone-sensitive lipase (HSL), a neutral cholesteryl ester hydrolase (NCEH), plays a vital role in regulating STAR expression in adrenal and gonadal cells [ 3 , 9 , 35 ]. Of note, HSL catalyzes the hydrolysis of CEs in steroidogenic tissues and macrophages. In addition, the hydrolysis of CEs has been shown to be influenced by several enzymes, including acyl coenzyme A:cholesterol acyltransferase-1, neutral CE hydrolase 1 (also known as KIAA1363 or arylacetamide deacetylase-like 1), carboxylesterase 3, and CE hydrolase (identical to either human liver carboxylesterase 1 or macrophage serine esterase 1) [ 36 – 41 ]. Studies have demonstrated that regulation of HSL mediated STAR expression and steroid biosynthesis involves the liver X receptor (LXR) pathway [ 3 , 9 , 34 ]. Oxysterols act as ligands for LXRs (LXRα and LXRβ; also known as NR1H3 and NR1H2, respectively), which are members of the nuclear receptor superfamily of ligand activated transcription factors [ 42 , 43 ]. LXRs form obligate heterodimers with retinoid X receptors (RXRs), which also dimerize with retinoid acid receptors (RARs), and regulate the transcription of a number of genes involved in cholesterol utilization, metabolism, and balance, including sterol regulatory element-binding proteins (SREBPs), ATP-binding cassette transporter 1 (ABCA1), and STAR [ 9 , 42 – 44 ]. In this review, we will summarize the significant findings that have been made with regards to expression of the STAR protein and, thus, steroid biosynthesis, and their relevance to a number of endocrinological health issues and/or relevant abnormalities.

Mutations in the STAR gene results in a protein that is non-functional and inactive, resulting in lipoid CAH, the rarest and most severe form of CAH [ 8 , 26 – 28 ]. This potentially life-threatening disorder is characterized by an inborn error of steroid hormone biosynthesis resulting in near complete inability of the newborn to synthesize steroids. Clinical manifestations of lipoid CAH include a marked adrenocortical insufficiency, hypergonadotropic hypogonadism, and severe salt wasting, and affected newborns die shortly after birth as a result of glucocorticoid and mineralocorticoid deficiencies. Individuals afflicted with lipoid CAH are phenotypically female irrespective of chromosomal sex, have large adrenals containing high CE and cholesterol levels, and also have cholesterol deposition in steroidogenic cells [ 8 , 26 , 27 , 73 – 77 ]. Affected individuals (who fail to metabolize cholesterol in mitochondria of the adrenal glands and gonads) die shortly after birth due to glucocorticoid and mineralocorticoid insufficiencies; however, appropriate hormone replacement therapy results in the survival of lipoid CAH patients to adulthood. Analysis of the STAR gene isolated from testicular tissue of lipoid CAH patients demonstrated the presence of nonsense and deletion mutations, which demonstrated initial proof of this disease and the critical role of STAR in steroid biosynthesis [ 23 ]. Whereas overexpression of wild type STAR in monkey kidney COS-1 cells (rendered steroidogenic by transfection with the P450scc system) increased steroid production, cells expressing mutant STAR were completely inactive in promoting steroidogenesis [ 23 , 26 , 78 ]. Therefore, expression of the STAR protein was an obligatory requirement for intracellular trafficking of cholesterol and that lipoid CAH represents a natural knockout of STAR with consequences consistent with the role of STAR on steroid biosynthesis. Molecular genetic analyses have identified approximately three-dozen mutations in the STAR gene producing lipoid CAH and include nonsense and missense mutations, splicing errors, and frameshifts causing deletions/insertions [ 27 , 28 , 32 , 76 , 77 , 79 ]. Notably, two patients diagnosed with lipoid CAH lacked mutations in the STAR gene. However, these patients have heterozygous mutations in the CYP11A1 gene, allowing afflicted individuals to survive longer periods without hormone replacement therapy. Thus, haploinsufficiency of CYP11A1 can lead to a late onset form of lipoid CAH or these patients may harbor mutations in other gene(s) whose function is dependent on STAR action. Noteworthy, however, CAH can be induced by the deficiency of one of four steroidogenic enzymes involved in cortisol biosynthesis, i.e., 21-hydroxylase, 11-hydroxylase, 3β-hydroxysteroid dehydrogenase, and 17α hydroxylase/17,20-lyase [ 29 , 80 – 82 ]. All of the STAR mutations identified in lipoid CAH are found in the C-terminus of the STAR protein, alter its structure/function, and result in a biologically inactive non-functional STAR that lacks the ability to deliver cholesterol to CYP11A1 in supporting steroidogenesis. While a correlation between STAR mutations and lipoid CAH is documented, molecular analyses of female subjects led to the formulation of the two hit model [ 8 , 26 , 28 , 76 ]. The first hit is caused by the inability of STAR to transfer cholesterol to the inner mitochondrial membrane for acute steroid synthesis due to mutations in the STAR gene. However, cells can continue to make small amounts of steroid by a STAR independent mechanism, which allows for the survival of some lipoid CAH patients for 1 to 2 months without treatment [ 26 , 83 ]. The second hit results in the prolonged and massive accumulation of lipids in affected cells that eventually interferes with normal cellular processes and results in death of cells. Thus, the feminization occurring at puberty with lipoid CAH is due to the small amount of STAR independent steroids synthesized by ovarian follicles recruited during each cycle. Since STAR is not present in the granulosa cells of preovulatory follicles, the first hit cannot occur. In the second hit, which utilizes a STAR-independent process, recruited follicles synthesize small amounts of estrogen but ultimately accumulate excessive amounts of lipids resulting in destruction of the follicle prior to luteinization and progesterone production [ 26 , 76 , 84 – 86 ]. Factors responsible for the production of small amount of steroids include members of the START (STAR-related lipid-transfer) domain family (that have been reported to be involved in steroidogenesis) and oxysterols [ 43 , 87 – 92 ]. Further insights into these mechanisms have been documented with targeted disruption of the STAR gene in a mouse model [ 30 , 31 , 93 ]. Similar to lipoid CAH in humans, STAR null mice have female external genitalia, fail to grow normally and die shortly after birth as a result of adrenocortical insufficiency. The adrenal glands were smaller in STAR knockout mice than those of wild type littermates and demonstrated profound morphological anomalies in the cortex [ 8 , 30 , 31 ]. Moreover, mice lacking the STAR gene had multiple abnormalities in adrenal and gonadal functions caused by the massive lipid accumulation in these tissues. Serum corticosterone and aldosterone levels were low with elevated ACTH and corticotropin-releasing hormone levels, representing impaired adrenal steroid production and loss of feedback regulation at the hypothalamic-pituitary level. Despite the dramatic effects of the absence of STAR on adrenal and gonadal steroid formation, prepubertal serum testosterone levels in STAR null mice did not differ from wild type littermates [ 30 ]. The physio-pathological characteristics of STAR knockout mice were similar to those seen in lipoid CAH in humans, supporting the two hit model, demonstrating the consequences of a non-functional STAR, and reinforcing the crucial role of this protein in the regulation of steroid biosynthesis (reviewed in Refs. [ 6 , 8 , 26 – 28 ]).