Epigenetic Dysregulation and Its Correlation with the Steroidogenic Machinery Impacting Breast Pathogenesis: Data Mining and Molecular Insights into Therapeutics.

Breast cancer (BC) is a heterogeneous condition and comprises molecularly distinct subtypes. An imbalance in the levels of epigenetic histone deacetylases (HDACs), modulating estrogen accumulation, especially 17ß-estradiol (E2), promotes breast tumorigenesis. In the present study, analyses of The Cancer Genome Atlas (TCGA) pan-cancer normalized RNA-Seq datasets revealed the dysregulation of 16 epigenetic enzymes (among a total of 18 members) in luminal BC subtypes, in comparison to their non-cancerous counterparts. Explicitly, genomic profiling of these epigenetic enzymes displayed increases in HDAC1, 2, 8, 10, 11, and Sirtuins (SIRTs) 6 and 7, and decreases in HDAC4-7, -9, and SIRT1-4 levels, respectively, in TCGA breast tumors. Kaplan-Meier plot analyses showed that these HDACs, with the exception of HDAC2 and SIRT2, were not correlated with the overall survival of BC patients. Additionally, disruption of the epigenetic signaling in TCGA BC subtypes, as assessed using both heatmaps and boxplots, was associated with the genomic expression of factors that are instrumental for cholesterol trafficking/utilization for accelerating estrogen/E2 levels, in which steroidogenic acute regulatory protein (STAR) mediates the rate-limiting step in steroid biosynthesis. TCGA breast samples showed diverse expression patterns of a variety of key steroidogenic markers and hormone receptors, including LIPE, CYP27A1, STAR, STARD3, CYP11A1, CYP19A1, ER, PGR, and ERBB2. Moreover, regulation of STAR-governed steroidogenic machinery was found to be influenced by various transcription factors, i.e., CREB1, CREM, SF1, NR4A1, CEBPB, SREBF1, SREBF2, SP1, FOS, JUN, NR0B1, and YY1. Along these lines, ingenuity pathway analysis (IPA) recognized a number of new targets and downstream effectors influencing BCs. Of note, genomic, epigenomic, transcriptional, and hormonal anomalies observed in human primary breast tumors were qualitatively similar in pertinent BC cell lines. These findings identify the functional correlation between dysregulated epigenetic enzymes and estrogen/E2 accumulation in human breast tumors, providing the molecular insights into more targeted therapeutic approaches involving the inhibition of HDACs for combating this life-threatening disease.

Expression and Function of StAR in Cancerous and Non-Cancerous Human and Mouse Breast Tissues: New Insights into Diagnosis and Treatment of Hormone-Sensitive Breast Cancer.

Breast cancer (BC) is primarily triggered by estrogens, especially 17ß-estradiol (E2), which are synthesized by the aromatase enzyme. While all steroid hormones are derived from cholesterol, the rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. Herein, we demonstrate that StAR mRNA expression was aberrantly high in human hormone-dependent BC (MCF7, MDA-MB-361, and T-47D), modest in hormone-independent triple negative BC (TNBC; MDA-MB-468, BT-549, and MDA-MB-231), and had little to none in non-cancerous mammary epithelial (HMEC, MCF10A, and MCF12F) cells. In contrast, these cell lines showed abundant expression of aromatase (CYP19A1) mRNA. Immunofluorescence displayed qualitatively similar patterns of both StAR and aromatase expression in various breast cells. Additionally, three different transgenic (Tg) mouse models of spontaneous breast tumors, i.e., MMTV-Neu, MMTV-HRAS, and MMTV-PyMT, demonstrated markedly higher expression of StAR mRNA/protein in breast tumors than in normal mammary tissue. While breast tumors in these mouse models exhibited higher expression of ERα, ERß, and PR mRNAs, their levels were undetected in TNBC tumors. Accumulation of E2 in plasma and breast tissues, from MMTV-PyMT and non-cancerous Tg mice, correlated with StAR, but not with aromatase, signifying the importance of StAR in governing E2 biosynthesis in mammary tissue. Treatment with a variety of histone deacetylase inhibitors (HDACIs) in primary cultures of enriched breast tumor epithelial cells, from MMTV-PyMT mice, resulted in suppression of StAR and E2 levels. Importantly, inhibition of StAR, concomitant with E2 synthesis, by various HDACIs, at clinical and preclinical doses, in MCF7 cells, indicated therapeutic relevance of StAR in hormone-dependent BCs. These findings provide insights into the molecular events underlying the differential expression of StAR in human and mouse cancerous and non-cancerous breast cells/tissues, highlighting StAR could serve not only as a novel diagnostic maker but also as a therapeutic target for the most prevalent hormone-sensitive BCs.

Extra-adrenal glucocorticoid biosynthesis: implications for autoimmune and inflammatory disorders.

Glucocorticoid synthesis is a complex, multistep process that starts with cholesterol being delivered to the inner membrane of mitochondria by StAR and StAR-related proteins. Here its side chain is cleaved by CYP11A1 producing pregnenolone. Pregnenolone is converted to cortisol by the enzymes 3-ßHSD, CYP17A1, CYP21A2, and CYP11B1. Glucocorticoids play a critical role in the regulation of the immune system and exert their action through the glucocorticoid receptor (GR). Although corticosteroids are primarily produced in the adrenal gland, they can also be produced in a number of extra-adrenal tissue including the immune system, skin, brain, and intestine. Glucocorticoid production is regulated by ACTH, CRH, and cytokines such as IL-1, IL-6, and TNFα. The bioavailability of cortisol is also dependent on its interconversion to cortisone, which is inactive, by 11ßHSD1/2. Local and systemic glucocorticoid biosynthesis can be stimulated by ultraviolet B, explaining its immunosuppressive activity. In this review, we want to emphasize that dysregulation of extra-adrenal glucocorticoid production can play a key role in a variety of autoimmune diseases including multiple sclerosis (MS), lupus erythematosus (LE), rheumatoid arthritis (RA), and skin inflammatory disorders such as psoriasis and atopic dermatitis (AD). Further research on local glucocorticoid production and its bioavailability may open doors into new therapies for autoimmune diseases.

Overexpression of the steroidogenic acute regulatory protein in breast cancer: Regulation by histone deacetylase inhibition.

Dysregulation of steroid biosynthesis has been implicated in the pathophysiology of a variety of cancers. One such common malignancy in women is breast cancer that is frequently promoted by estrogen overproduction. All steroid hormones are made from cholesterol, and the rate-limiting step in steroid biosynthesis is primarily mediated by the steroidogenic acute regulatory (StAR) protein. Whereas the involvement of StAR in the regulation steroid hormone biosynthesis is well established, its association to breast cancer remains obscure. Herein, we report that estrogen receptor positive breast cancer cell lines (MCF7, MDA-MB-361, and T-47D) displayed aberrant high expression of the StAR protein, concomitant with 17ß-estradiol (E2) synthesis, when compared their levels with normal mammary epithelial (MCF10A and MCF12F) and triple negative breast cancer (MDA-MB-468, MDA-MB-231, and BT-549) cells. StAR was identified as a novel acetylated protein in MCF7 cells, in which liquid chromatography-tandem mass spectrometry analysis identified seven StAR acetyl lysine residues under basal and in response to histone deacetylase (HDAC) inhibition. A number of HDAC inhibitors were capable of diminishing StAR expression and E2 synthesis in MCF7 cells. The validity of StAR protein acetylation and its correlation to HDAC inhibition mediated steroid synthesis was demonstrated in adrenocortical tumor H295R cells. These findings provide novel insights that StAR protein is abundantly expressed in the most prevalent hormone sensitive breast cancer subtype, wherein inhibition of HDACs altered StAR acetylation patterns and decreased E2 levels, which may have important therapeutic implications in the prevention and treatment of this devastating disease.

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy.

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

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

Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease.

Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

Cutaneous glucocorticosteroidogenesis: securing local homeostasis and the skin integrity.

Human skin has the ability to synthesize glucocorticoids de novo from cholesterol or from steroid intermediates of systemic origin. By interacting with glucocorticoid receptors, they regulate skin immune functions as well as functions and phenotype of the epidermal, dermal and adnexal compartments. Most of the biochemical (enzyme and transporter activities) and regulatory (neuropeptides mediated activation of cAMP and protein kinase A dependent pathways) principles of steroidogenesis in the skin are similar to those operating in classical steroidogenic organs. However, there are also significant differences determined by the close proximity of synthesis and action (even within the same cells) allowing para-, auto- or intracrine modes of regulation. We also propose that ultraviolet light B (UVB) can regulate the availability of 7-dehydrocholesterol for transformation to cholesterol with its further metabolism to steroids, oxysterols or ∆7 steroids, because of its transformation to vitamin D3. In addition, UVB can rearrange locally produced ∆7 steroids to the corresponding secosteroids with a short- or no-side chain. Thus, different mechanisms of regulation occur in the skin that can be either stochastic or structuralized. We propose that local glucocorticosteroidogenic systems and their regulators, in concert with cognate receptors operate to stabilize skin homeostasis and prevent or attenuate skin pathology.

Cell-Free DNA As Peripheral Biomarker of Alzheimer's Disease.

Alzheimer's disease (AD) and Alzheimer's disease-related disorders (ADRD) are progressive neurodegenerative diseases without cure. Alzheimer's disease occurs in 2 forms, early-onset familial AD and late-onset sporadic AD. Early-onset AD is a rare (~1%), autosomal dominant, caused by mutations in presenilin-1, presenilin-2, and amyloid precursor protein genes and the other is a late-onset, prevalent and is evolved due to age-associated complex interactions between environmental and genetic factors, in addition to apolipoprotein E4 polymorphism. Cellular senescence, promoting the impairment of physical and mental functions is constituted to be the main cause of aging, the primary risk factor for AD, which results in progressive loss of cognitive function, memory, and visual-spatial skills for an individual to live or act independently. Despite significant progress in the understanding of the biology and pathophysiology of AD, we continue to lack definitive early detectable biomarkers and/or drug targets that can be used to delay the development of AD and ADRD in elderly populations. However, recent developments in the studies of DNA double-strand breaks result in the release of fragmented DNA into the bloodstream and contribute to higher levels of cell-free DNA (cf-DNA). This fragmented cf-DNA can be released into the bloodstream from various cell types, including normal cells and cells undergoing apoptosis or necrosis and elevated levels of cf-DNA in the blood have the potential to serve as blood blood-based biomarker for early detection of AD and ADRD. The overall goal of our study is to discuss the latest developments in circulating cell-free DNA into the blood in the progression of AD and ADRD. Our article summarized the status of research on double-strand breaks and circulating cell-free DNA in both healthy and disease states and how these recent developments can be used to develop early detectable biomarkers for AD and ADRD. Our article also discussed the impact of lifestyle and epigenetic factors that are involved in DNA double-strand breaks and circulating cell-free DNA in AD and ADRD.

Regulation of retinoid mediated StAR transcription and steroidogenesis in hippocampal neuronal cells: Implications for StAR in protecting Alzheimer's disease.

Retinoids (vitamin A and its derivatives) play pivotal roles in diverse processes, ranging from homeostasis to neurodegeneration, which are also influenced by steroid hormones. The rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. In the present study, we demonstrate that retinoids enhanced StAR expression and pregnenolone biosynthesis, and these parameters were markedly augmented by activation of the PKA pathway in mouse hippocampal neuronal HT22 cells. Deletion and mutational analyses of the 5'-flanking regions of the StAR gene revealed the importance of a retinoic acid receptor (RAR)/retinoid X receptor (RXR)-liver X receptor (LXR) heterodimeric motif at -200/-185 bp region in retinoid responsiveness. The RAR/RXR-LXR sequence motif can bind RARα and RXRα, and retinoid regulated transcription of the StAR gene was found to be influenced by the LXR pathway, representing signaling cross-talk in hippocampal neurosteroid biosynthesis. Steroidogenesis decreases during senescence due to declines in the central nervous system and the endocrine system, and results in hormone deficiencies, inferring the need for hormonal balance for healthy aging. Loss of neuronal cells, involving accumulation of amyloid beta (Aß) and/or phosphorylated Tau within the brain, is the pathological hallmark of Alzheimer's disease (AD). HT22 cells overexpressing either mutant APP (mAPP) or mutant Tau (mTau), conditions mimetic to AD, enhanced toxicities, and resulted in attenuation of both basal and retinoid-responsive StAR and pregnenolone levels. Co-expression of StAR with either mAPP or mTau diminished cytotoxicity, and concomitantly elevated neurosteroid biosynthesis, pointing to a protective role of StAR in AD. These findings provide insights into the molecular events by which retinoid signaling upregulates StAR and steroid levels in hippocampal neuronal cells, and StAR, by rescuing mAPP and/or mTau-induced toxicities, modulates neurosteroidogenesis and restores hormonal balance, which may have important implications in protecting AD and age-related complications and diseases.

Protective function of StAR in amyloid-ß accumulated hippocampal neurotoxicity and neurosteroidogenesis: Mechanistic insights into Alzheimer's disease.

The steroidogenic acute regulatory (StAR) protein principally mediates steroid hormone biosynthesis by governing the transport of intramitochondrial cholesterol. Neurosteroids progressively decrease during aging, the key risk factor for Alzheimer's disease (AD), which is triggered by brain-region specific accumulation of amyloid beta (Aß) precursor protein (APP), a key pathological factor. We demonstrate that hippocampal neuronal cells overexpressing wild-type (WtAPP) and mutant APP (mAPP) plasmids, conditions mimetic to AD, resulted in decreases in StAR mRNA, free cholesterol, and pregnenolone levels. The magnitude of suppression of the steroidogenic response was more pronounced with mAPP than that of WtAPP. While mAPP-waned assorted anomalies correlate to AD pathology, deterioration of APP/Aß laden StAR expression and neurosteroid biosynthesis was enhanced by retinoid signaling. An abundance of mitochondrially targeted StAR expression partially restored APP/Aß accumulated diverse neurodegenerative vulnerabilities. Immunofluorescence analyses revealed that overexpression of StAR diminishes mAPP provoked Aß aggregation. Co-expression of StAR and mAPP in hippocampal neurons substantially reversed the declines in mAPP mediated cell survival, mitochondrial oxygen consumption rate, and ATP production. Concurrently, induction of mAPP induced Aß loading showed an increase in cholesterol esters, but decrease in free cholesterol, concomitant with pregnenolone biosynthesis, events that were inversely regulated by StAR. Moreover, retinoid signaling was found to augment cholesterol content for facilitating neurosteroid biosynthesis in an AD mimetic condition. These findings provide novel insights into the molecular events by which StAR acts to protect mAPP-induced hippocampal neurotoxicity, mitochondrial dysfunction, and neurosteroidogenesis, and these measures are fundamental for ameliorating and/or delaying dementia in individuals with AD.

Healthy Immunity on Preventive Medicine for Combating COVID-19.

Immunomodulation is influenced by the consumption of nutrients, and healthy immunity is pivotal to defending an individual from a variety of pathogens. The immune system is a network of intricately regulated biological processes that is comprised of many organs, cellular structures, and signaling molecules. A balanced diet, rich in vitamins, minerals, and antioxidants, is key to a strengthened immune system and, thus, crucial to proper functioning of various physiological activities. Conversely, deficiencies of these micronutrients, involving impaired immunity, are linked to numerous health complications, along with a host of pathologies. Coronavirus disease 2019 (COVID-19) is a dangerous infectious disease caused by a ß-form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its genomic variants, which enter host cells upon binding to the angiotensin converting enzyme 2 receptors, and is associated with substantial morbidities and mortalities globally. Patients afflicted with COVID-19 display asymptomatic to severe symptoms, occurrences of which are multifactorial and include diverse immune responses, sex and gender differences, aging, and underlying medical conditions. Geriatric populations, especially men in comparison to women, regardless of their states, are most vulnerable to severe COVID-19-associated infections and complications, with fatal outcomes. Advances in genomic and proteomic technologies help one understand molecular events, including host-pathogen interactions and pathogenesis of COVID-19 and, subsequently, have developed a variety of preventive measures urgently, ranging from mask wearing to vaccination to medication. Despite these approaches, no unique strategy is available today that can effectively prevent and/or treat this hostile disease. As a consequence, the maintenance of a boosted immune system could be considered a high priority of preventive medicine for combating COVID-19. Herein, we discuss the current level of understanding underlining the contribution of healthy immunity and its relevance to COVID-19 molecular pathogenesis, and potential therapeutic strategies, in the management of this devastating disease.

COVID-19 and its genomic variants: Molecular pathogenesis and therapeutic interventions.

Coronavirus disease-19 (COVID-19), caused by a ß-coronavirus and its genomic variants, is associated with substantial morbidities and mortalities globally. The COVID-19 virus and its genomic variants enter host cells upon binding to the angiotensin converting enzyme 2 receptors that are expressed in a variety of tissues, but predominantly in the lungs, heart, and blood vessels. Patients afflicted with COVID-19 may be asymptomatic or present with critical symptoms possibly due to diverse lifestyles, immune responses, aging, and underlying medical conditions. Geriatric populations, especially men in comparison to women, with immunocompromised conditions, are most vulnerable to severe COVID-19 associated infections, complications, and mortalities. Notably, whereas immunomodulation, involving nutritional consumption, is essential to protecting an individual from COVID-19, immunosuppression is detrimental to a person with this aggressive disease. As such, immune health is inversely correlated to COVID-19 severity and resulting consequences. Advances in genomic and proteomic technologies have helped us to understand the molecular events underlying symptomatology, transmission and, pathogenesis of COVID-19 and its genomic variants. Accordingly, there has been development of a variety of therapeutic interventions, ranging from mask wearing to vaccination to medication. This review summarizes the current understanding of molecular pathogenesis of COVID-19, effects of comorbidities on COVID-19, and prospective therapeutic strategies for the prevention and treatment of this contagious disease.

Hormonal and Genetic Regulatory Events in Breast Cancer and Its Therapeutics: Importance of the Steroidogenic Acute Regulatory Protein.

Estrogen promotes the development and survival of the majority of breast cancers (BCs). Aromatase is the rate-limiting enzyme in estrogen biosynthesis, and it is immensely expressed in both cancerous and non-cancerous breast tissues. Endocrine therapy based on estrogen blockade, by aromatase inhibitors, has been the mainstay of BC treatment in post-menopausal women; however, resistance to hormone therapy is the leading cause of cancer death. An improved understanding of the molecular underpinnings is the key to develop therapeutic strategies for countering the most prevalent hormone receptor positive BCs. Of note, cholesterol is the precursor of all steroid hormones that are synthesized in a variety of tissues and play crucial roles in diverse processes, ranging from organogenesis to homeostasis to carcinogenesis. The rate-limiting step in steroid biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process that is primarily mediated by the steroidogenic acute regulatory (StAR) protein. Advances in genomic and proteomic technologies have revealed a dynamic link between histone deacetylases (HDACs) and StAR, aromatase, and estrogen regulation. We were the first to report that StAR is abundantly expressed, along with large amounts of 17ß-estradiol (E2), in hormone-dependent, but not hormone-independent, BCs, in which StAR was also identified as a novel acetylated protein. Our in-silico analyses of The Cancer Genome Atlas (TCGA) datasets, for StAR and steroidogenic enzyme genes, revealed an inverse correlation between the amplification of the StAR gene and the poor survival of BC patients. Additionally, we reported that a number of HDAC inhibitors, by altering StAR acetylation patterns, repress E2 synthesis in hormone-sensitive BC cells. This review highlights the current understanding of molecular pathogenesis of BCs, especially for luminal subtypes, and their therapeutics, underlining that StAR could serve not only as a prognostic marker, but also as a therapeutic candidate, in the prevention and treatment of this life-threatening disease.

Role of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 in protein kinase A- and protein kinase C-mediated regulation of the steroidogenic acute regulatory protein expression in mouse Leydig tumor cells: mechanism of action.

Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position -44/-20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor cells.

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

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

Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene.

The regulation of steroidogenic acute regulatory protein (StAR) gene transcription by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP response element (CRE, TGACGTGA). This regulation is coordinated by multiple transcription factors that bind to sequence-specific elements located approximately 150 bp upstream of the transcription start site. Among the proteins that bind within this region, the basic leucine zipper (bZIP) family of transcription factors, i.e. CRE binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF), activator protein 1 (AP-1; Fos/Jun), and CCAAT enhancer binding protein beta (C/EBPbeta), interact with an overlapping region (-81/-72 bp) in the StAR promoter, mediate stimulus-transcription coupling of cAMP signaling and play integral roles in regulating StAR gene expression. These bZIP proteins are structurally similar and bind to DNA sequences as dimers; however, they exhibit discrete transcriptional activities, interact with several transcription factors and other properties that contribute in their regulatory functions. The 5'-flanking -81/-72 bp region of the StAR gene appears to function as a key element within a complex cAMP response unit by binding to different bZIP members, and the StAR promoter displays variable states of cAMP responsivity contingent upon the occupancy of these cis-elements with these transcription factors. The expression and activities of CREB/CREM/ATF, Fos/Jun and C/EBPbeta have been demonstrated to be mediated by a plethora of extracellular signals, and the phosphorylation of these proteins at several Ser and Thr residues allows recruitment of the transcriptional coactivator CREB binding protein (CBP) or its functional homolog p300 to the StAR promoter. This review will focus on the current level of understanding of the roles of selective bZIP family proteins within the complex series of processes involved in regulating StAR gene transcription.

The differential regulation of steroidogenic acute regulatory protein-mediated steroidogenesis by type I and type II PKA in MA-10 cells.

Following tropic hormone challenge, steroidogenic tissues utilize PKA to phosphorylate unique subsets of proteins necessary to facilitate steroidogenesis. This includes the PKA-dependent expression and activation of the steroidogenic acute regulatory protein (STAR), which mediates the rate-limiting step of steroidogenesis by inducing the transfer of cholesterol from the outer to the inner mitochondrial membrane. Since both type I and type II PKA are present in steroidogenic tissues, we have utilized cAMP analog pairs that preferentially activate each PKA subtype in order to examine their impact on STAR synthesis and activity. In MA-10 mouse Leydig tumor cells Star gene expression is more dependent upon type I PKA, while the post-transcriptional regulation of STAR appears subject to type II PKA. These experiments delineate the discrete effects that type I and type II PKA exert on STAR-mediated steroidogenesis, and suggest complimentary roles for each subtype in coordinating steroidogenesis.

Involvement of peroxisome proliferator-activated receptor gamma in gonadal steroidogenesis and steroidogenic acute regulatory protein expression.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to the PPAR family of nuclear transcription factors whose ligands, such as eicosanoids, fatty acids and prostaglandins, are known to affect gonadal function. Although several of these enhance the expression of the steroidogenic acute regulatory protein (STAR) and steroid production, the role of PPARgamma in regulating STAR-mediated steroidogenesis remains unclear. In the present study, we used ciglitazone to selectively activate PPARgamma and examine its role in STAR-mediated steroidogenesis in immortalised KK1 mouse granulosa cells and MA-10 mouse Leydig tumour cells. Cotreatment with both dibutyryl-cAMP and ciglitazone revealed a dose-dependent, significant increase in progesterone synthesis, Star promoter activity, Star mRNA and STAR protein relative to either compound alone. The overexpression of PPARgamma further increased Star-promoter activity. The ciglitazone-induced activity of the Star-promoter appears to be mediated through the cAMP-response element half-sites located within its proximal 151 bp. Combined treatment with ciglitazone and dibutyryl-cAMP significantly increased the expression and activity of transcriptional pathways impacted by the activator protein-1 family member c-JUN. The present study demonstrates that ciglitazone and dibutyryl-cAMP synergistically enhance STAR expression in MA-10 and KK1 cells. Ciglitazone-activated PPARgamma appears to increase the sensitivity of Leydig and granulosa cells to cAMP stimulation, possibly via upregulation of c-JUN expression.