Acid Receptor-related Orphan Receptor Alpha Research Articles

An RORα agonist, ODH-08, inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3

AimsHepatic fibrosis is a dynamic process characterized by the net accumulation of an extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. Activation of hepatic stellate cells (HSCs) plays a role in transdifferentiation of quiescent cells into fibrogenic myofibroblasts. We aimed to examine the function of retinoic acid receptor-related orphan receptor alpha (RORα) and its novel agonistic ligand, 1-(4-benzyloxybenzyl)-3-(2-dimethylaminoethyl)-thiourea (ODH-08) against activation of HSCs using hepatic fibrosis mouse models. Main methodsChemical synthesis, a reporter gene assay, surface plasmon resonance analysis, and a docking study were performed to evaluate ODH-08 as a ligand of RORα. In vivo experiments with mice fed a Western diet were performed to evaluate the effect of ODH-08. The human HSC line, Lx-2, and primary mouse HSCs were employed to identify the molecular mechanisms underlying the antifibrogenic effect of ODH-08. Key findingsA novel RORα-selective ligand, ODH-08, was developed based on modification of JC1-40, an analog of N-methylthiourea. Administration of ODH-08 to the Western diet-fed mice reduced hepatic collagen deposition and expression levels of fibrogenic markers such as α-smooth muscle actin and collagen type I alpha 1 chain. Activation of RORα—either by transient overexpression of RORα or treatment with ODH-08—suppressed the expression of fibrogenic proteins in HSCs. The activation of RORα suppressed the activity of SMAD2 and 3, which are the primary downstream proteins of transforming growth factor β. SignificanceRORα and its agonist ODH-08 have a potent antifibrotic effect, which could provide a novel antifibrotic strategy against hepatic fibrosis.

Neuroprotective role for RORA in Parkinson’s disease revealed by analysis of post-mortem brain and a dopaminergic cell line

Parkinson’s disease (PD) is almost twice as prevalent in men, which has largely been attributed to neuroprotective effect of oestradiol in women. RORA (retinoic acid receptor-related orphan receptor alpha) regulates the transcription of central aromatase, the enzyme responsible for local oestradiol synthesis, simultaneously, RORA expression is regulated by sex hormones. Moreover, RORA protects neurones against oxidative stress, a key mechanism contributing to the loss of dopaminergic neurones in PD. Therefore, we hypothesized that there would be sex differences in RORA expression in the substantia nigra pars compacta (SNpc), which could contribute to sex differences observed in PD prevalence and pathogenesis. In a case control study, qPCR and western blot analyses were used to quantify gene and protein expression in the SNpc of post-mortem brains (n = 14 late-stage PD and 11 age and sex matched controls). The neuroprotective properties of a RORA agonist were then investigated directly using a cell culture toxin-based model of PD coupled with measures of viability, mitochondrial function and apoptosis. RORA was expressed at significantly higher levels in the SNpc from control females’ brains compared to males. In PD, we found a significant increase in SNpc RORA expression in male PD compared to female PD. Treatment with a RORA agonist showed a significant neuroprotection in our cell culture model of PD and revealed significant effects on intracellular factors involved in neuronal survival and demise. This study is the first to demonstrate a sex specific pattern of RORA protein and gene expression in the SNpc of controls post-mortem human brains, and to show that this is differentially altered in male and female PD subjects, thus supporting a role for RORA in sex-specific aspects of PD. Furthermore, our in vitro PD model indicates mechanisms whereby a RORA agonist exerts its neuroprotective effect, thereby highlighting the translational potential for RORA ligands in PD.

Is obstructive sleep apnea a circadian rhythm disorder?

Obstructive sleep apnea is the most common sleep-related breathing disorder worldwide and remains underdiagnosed. Its multiple associated comorbidities contribute to a decreased quality of life and work performance as well as an increased risk of death. Standard treatment seems to have limited effects on cardiovascular and metabolic aspects of the disease, emphasising the need for early diagnosis and additional therapeutic approaches. Recent evidence suggests that the dysregulation of circadian rhythms, processes with endogenous rhythmicity that are adjusted to the environment through various cues, is involved in the pathogenesis of comorbidities. In patients with obstructive sleep apnea, altered circadian gene expression patterns have been demonstrated. Obstructive respiratory events may promote circadian dysregulation through the effects of sleep disturbance and intermittent hypoxia, with subsequent inflammation and disruption of neural and hormonal homeostasis. In this review, current knowledge on obstructive sleep apnea, circadian rhythm regulation, and circadian rhythm sleep disorders is summarised. Studies that connect obstructive sleep apnea to circadian rhythm abnormalities are critically evaluated. Furthermore, pathogenetic mechanisms that may underlie this association, most notably hypoxia signalling, are presented. A bidirectional relationship between obstructive sleep apnea and circadian rhythm dysregulation is proposed. Approaching obstructive sleep apnea as a circadian rhythm disorder may prove beneficial for the development of new, personalised diagnostic, therapeutic and prognostic tools. However, further studies are needed before the clinical approach to obstructive sleep apnea includes targeting the circadian system.

Intestinal Microflora Altered by Vancomycin Exposure in Early Life Up-regulates Type 2 Innate Lymphocyte and Aggravates Airway Inflammation in Asthmatic Mice.

Allergic asthma is a chronic inflammatory disease primarily mediated by Th2 immune mechanisms. Exposure to antibiotics during early life is associated with an increased risk of allergic asthma, although the exact mechanism is not fully understood. In this study, mice were randomly divided into a normal saline control group (NS group), an OVA-induced asthma group (OVA group), a vancomycin treatment control group (VAN.NS group), and a vancomycin treatment the OVA-induced asthma group (VAN.OVA group). The results showed that vancomycin altered dominant species in experimental mice. The phylum level histogram showed that Bacteroides abundance was increased, and Firmicutes abundance was decreased in the OVA group. Airway inflammation and airway hyperresponsiveness (AHR) were aggravated in the vancomycin-exposed group. Enzyme-linked immunosorbent assay (ELISA) showed that the serum levels of IL-5, IL-13, and IL-33 in the OVA group were higher than those in the NS group, especially in the VAN.OVA group. The expression of GATA binding protein-3(GATA3) and retinoid acid receptor-related orphan receptor alpha (RORa) increased in the OVA group, even more so in the VAN.OVA group. Group 2 innate lymphoid cells (ILC2s) in the lung detected by flow cytometry was increased in OVA mice more than those in control mice, with a more remarkable increase in the VAN.OVA. Our results demonstrated that vancomycin used in early life could alter the intestinal microecology of mice, which, in turn, aggravates airway inflammation and upregulate type 2 innate lymphocytes.

The nuclear retinoid-related orphan receptor RORα controls adipose tissue inflammation in patients with morbid obesity and diabetes.

Inflammation governs adipose tissue (AT) dysfunction in obesity. Retinoic acid receptor-related orphan receptor alpha (RORα) is associated with inflammation and insulin resistance in animal studies, but its role in human obesity remains elusive. We investigated the expression and function of RORα on AT inflammation in patients with morbid obesity with/without diabetes. We assessed RORα expression in paired biopsies of subcutaneous and omental AT from 41 patients (body mass index (BMI) 43.3 ± 0.8 kg/m2) during Roux-en-Y-gastric surgery and explored the functional consequences of pharmacological RORα blockade in AT ex vivo. RORα expression was significantly higher in omental AT than in subcutaneous AT (p = 0.03) and was positively associated with BMI (r = 0.344, p = 0.027) and homeostasis model assessment of insulin resistance (r = 0.319, p = 0.041). In ex vivo assays, IL-8/CXCL8 and MCP-1/CCL2 chemokine release was significantly higher in omental fat explants from diabetic patients than from non-diabetics and was significantly diminished by RORα blockade (p < 0.05). Inhibition of RORα improved protein kinase B signaling and decreased NF-κB activity in omental AT from patients with diabetes (p < 0.05). Under dynamic flow conditions, RORα blockade prevented mononuclear cell attachment to human dysfunctional endothelial cells. RORα blockade represents a potential therapy to prevent AT dysfunction and inflammation associated with insulin resistance in human obesity.

Deletion of the nuclear receptor ROR\u03b1 in macrophages does not modify the development of obesity, insulin resistance and NASH

Retinoic acid receptor-related orphan receptor-alpha (RORα) is a transcription factor from the nuclear receptor family expressed by immune cells and involved in the development of obesity, insulin resistance (IR) and non-alcoholic steatohepatitis (NASH). It was recently reported that mice deficient for RORα in macrophages develop more severe NASH upon high fat diet (HFD) feeding due to altered Kupffer cell function. To better understand the role of RORα in obesity and IR, we independently generated a macrophage RORα-deficient mouse line. We report that RORα deletion in macrophages does not impact on HFD-induced obesity and IR. Surprisingly, we did not confirm an effect on NASH development upon HFD feeding nor in the more severe and obesity-independent choline-deficient, L-amino acid-defined diet model. Our results therefore show that RORα deletion in macrophages does not alter the development of obesity and IR and question its role in NASH.

Lack of retinoid acid receptor-related orphan receptor alpha accelerates and melatonin supplementation prevents testicular aging.

The role of retinoid acid receptor-related orphan receptor alpha (RORα) on male reproductive functions during aging is unclear. Here, we analyze the morphological changes in the testis of both young and aged RORα-deficient mice, with and without melatonin supplementation. Young mutants showed vacuolation, degeneration and pyknosis of spermatogenic epithelium and Sertoli cells. Aged mutants showed atrophy of the seminiferous tubules and absence of mitotic spermatogenic cells. Absence of sperms in many tubules, loss of acrosomal cap, vacuolation and hypertrophy of Sertoli cells were detected in aged mice, with a significant reduction in the number of seminiferous tubules and a significant increase in the number of Leydig cells and telocytes. Repair in seminiferous tubules and interstitial tissues with enhancement of spermatogenesis was observed in melatonin-treated aged mice. Young mutants overexpressed VEGF that was weaker in aged animals and observed only in the spermatocytes, while melatonin increased VEGF expression in spermatocytes and spermatids. Caspase 3 increased in both young and aged mutant mice in all seminiferous tubules and interstitium; caspase 3 immunostaining in seminiferous tubules, however, showed a normal pattern of apoptosis with melatonin supplementation. The present study reports that age-dependent testicular changes in RORα mutant mice were recovered by melatonin treatment.

Loss of RAR-related orphan receptor alpha (RORα) selectively lowers docosahexaenoic acid in developing cerebellum

Deficiency in retinoid acid receptor-related orphan receptor alpha (RORα) of staggerer mice results in extensive granule and Purkinje cell loss in the cerebellum as well as in learned motor deficits, cognition impairments and perseverative tendencies that are commonly observed in autistic spectrum disorder (ASD). The effects of RORα on brain lipid metabolism associated with cerebellar atrophy remain unexplored. The aim of this study is to examine the effects of RORα deficiency on brain phospholipid fatty acid concentrations and compositions. Staggerer mice (Rorasg/sg) and wildtype littermates (Rora+/+) were fed n-3 polyunsaturated fatty acids (PUFA) containing diets ad libitum. At 2 months and 7 or more months old, brain total phospholipid fatty acids were quantified by gas chromatography-flame ionization detection. In the cerebellum, all fatty acid concentrations were reduced in 2 months old mice. Since total fatty acid concentrations were significantly different at 2-month-old, we examined changes in fatty acid composition. The composition of ARA was not significantly different between genotypes; though DHA composition remained significantly lowered. Despite cerebellar atrophy at >7-months-old, cerebellar fatty acid concentrations had recovered comparably to wildtype control. Therefore, RORα may be necessary for fatty acid accretions during neurodevelopment. Specifically, the effects of RORα on PUFA metabolisms are region-specific and age-dependent.

Retinoic Acid Receptor-Related Receptor Alpha Ameliorates Autoimmune Arthritis via Inhibiting of Th17 Cells and Osteoclastogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis characterized by progressive joint destruction. IL-17-producing CD4+ T (Th17) cells play pivotal roles in RA development and progression. Retinoic acid receptor-related orphan receptor alpha (RORα) is a negative regulator of inflammatory responses, whereas RORγt, another member of the ROR family, is a Th17 lineage-specific transcription factor. Here, we investigated the immunoregulatory potential of RORα in collagen-induced arthritis (CIA) mice, an experimental model of RA. Cholesterol sulfate (CS) or SR1078, a ligand of RORα, inhibited RORγt expression and Th17 differentiation in vitro. In addition, fortification of RORα in T cells inhibited the expression levels of glycolysis-associated genes. We found that RORα overexpression in CIA mice attenuated the clinical and histological severities of inflammatory arthritis. The anti-arthritic effect of RORα was associated with suppressed Th17 differentiation and attenuated mTOR-STAT3 signaling in T cells. Furthermore, altered RORα activity could directly affect osteoclastogenesis implicated in progressive bone destruction in human RA. Our findings defined a critical role of RORα in the pathogenesis of RA. These data suggest that RORα may have novel therapeutic uses in the treatment of RA.

RORA Overexpression Alleviates Nasal Mucosal Injury and Enhances Red Blood Cell Immune Adhesion Function in a Mouse Model of Allergic Rhinitis via Inactivation of the Wnt/β-Catenin Signaling Pathway

Background: In this study, we examined whether RORA (retinoic acid receptor-related orphan receptor alpha) was capable of alleviating the progression of allergic rhinitis (AR). Methods: In order to elucidate the possible effects of RORA and the regulatory mechanism between RORA and the Wnt/β-catenin signaling pathway, mouse AR models were established and treated with RORA vector, siRNA against RORA, or the Wnt/β-catenin pathway inhibitor WIF-1. Subsequently, the serum levels of inflammatory cytokines (IgE, INF-γ, IL-1β, IL-4, and IL-17), red blood cell (RBC) immune adhesion function, the levels of RORA, β-catenin, and GSK3β, as well as the extent of β-catenin and GSK-3β phosphorylation were evaluated and measured. Results: The OVA-induced AR mouse model exhibited obvious nasal mucosal injury and inflammatory cell infiltration. RORA overexpression or the inactivation of the Wnt/β-catenin signaling pathway was uncovered as a way to ameliorate nasal mucosal injury and eosinophil infiltration of the OVA-induced AR mouse model. On the other hand, it reduced the number of eosinophils and mast cells, which also resulted in downregulated expression of IgE, INF-γ, IL-1β, IL-4, IL-17, β-catenin, and GSK-3β. Moreover, this led to a decreased extent of β-catenin and GSK-3β phosphorylation, while the rates of C₃b receptor rosette and Ic rosette were elevated. Conclusion: Taken together, the key findings provided evidence suggesting that the elevated RORA could potentially alleviate nasal mucosal injury and simultaneously enhance RBC immune adhesion function through the inhibition of the Wnt/β-catenin signaling pathway activation in an OVA-induced AR mouse model. This emphasizes a novel therapeutic target for the treatment of AR.

Estradiol-induced RORα expression positively regulates osteoblast differentiation

The retinoic acid receptor-related orphan receptor alpha (RORα) is a member of the nuclear hormone receptor superfamily. Several studies show that estradiol is related to RORα expression. However, the link between estradiol and RORα in osteoblast differentiation remains unknown. Here, we showed that estradiol induces RORα expression in C3H10T1/2 and MC3T3-E1 cells. RORα overexpression increased the expression of osteogenic genes including bone morphogenetic protein 2 (BMP2), distal-less homeobox 5, inhibitor of DNA binding, runt-related transcription factor 2 (Runx2), and osteocalcin. In addition, RORα increased phosphorylation of smad1/5/9. Furthermore, RORα knockdown suppressed estradiol-induced BMP2 and Runx2 protein level. Also, we confirmed that estradiol-induced ALP staining and matrix mineralization was attenuated in RORα knockdown. Summarily, these results suggest that estradiol-induced RORα promotes osteoblast differentiation.

BMAL1 Links Bevacizumab Resistance in Colorectal Cancer to Circadian Rhythm and Heme Receptor REVERBA

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

Nocturnal asthma is affected by genetic interactions between RORA and NPSR1.

Neuropeptide S Receptor 1 ( NPSR1) and Retinoid Acid Receptor-Related Orphan Receptor Alpha (RORA ) interact biologically, are both known candidate genes for asthma, and are involved in controlling circadian rhythm. Thus, we assessed (1) whether interactions between RORA and NPSR1 specifically affect the nocturnal asthma phenotype and (2) how this may differ from other asthma phenotypes. Interaction effects between 24 single-nucleotide polymorphisms (SNPs) in RORA and 35 SNPs in NPSR1 on asthma and nocturnal asthma symptoms were determined in 1432 subjects (763 asthmatics [192 with nocturnal asthma symptoms]; 669 controls) from the Multicenter Asthma Genetic in Childhood/International Study of Asthma and Allergies in Childhood studies. The results were validated and extended in children from the Manchester Asthma and Allergy Study (N = 723) and the Children Allergy Milieu Stockholm and Epidemiological cohort (N = 1646). RORA* NPSR1 interactions seemed to affect both asthma and nocturnal asthma. In stratified analyses, however, interactions mainly affected nocturnal asthma and less so asthma without nocturnal symptoms or asthma severity. Results were replicated in two independent cohorts and seemed to remain constant over time throughout youth. RORA* NPSR1 interactions appear to be involved in mechanisms specific for nocturnal asthma. In contrast to previous studies focusing on the role of beta 2 receptor polymorphisms in nocturnal asthma as a feature of asthma control or severity in general, our data suggest that changes in circadian rhythm control are associated with nighttime asthma symptoms.

Comparative analysis of RORA expression in brain tissue from multiple sclerosis and normal individuals:a pilot study

The key to understanding the autoimmune disease multiple sclerosis (MS) lies in a deeper analysis of the genes that play a role in the pathology of the disease. A new gene of interest encodes the retinoic acid receptor‐related orphan receptor alpha (RORA). This receptor is expressed in several tissues and plays a critical role in regulating inflammatory response, neuronal cell development, bone metabolism, and has been proposed to promote the differentiation of Th17 cells. Th17 cells play a role in initiating autoimmune central nervous system inflammation, which is the cause of this debilitating disease. To better understand the significance of RORA in MS, the focus of this study was to observe the expression of this receptor and determine its role in susceptibility of multiple sclerosis. We did so by isolating RNA from 10 brain samples, 5 from multiple sclerosis patients and 5 from healthy controls. We then subjected the RNA to quantitative RT‐PCR and normalized the samples to control RNAs TBP and GAPDH. We found that there was a significant increase in expression of RORA (P&lt;0.05) in brain samples from MS patients compared with healthy controls. This novel finding could be used as diagnostic criteria for susceptibility of multiple sclerosis and could potentially be a target for new treatments.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A role for the clock period circadian regulator 2 gene in regulating the clock gene network in human oral squamous cell carcinoma cells.

Clock genes are the core of the circadian rhythms in the human body and are important in regulating normal physiological functions. To date, research has indicated that the clock gene, period circadian clock 2 (PER2), is downregulated in numerous types of cancer, and that it is associated with cancer occurrence and progression via the regulation of various downstream cell cycle genes. However, it remains unclear whether the decreased expression of PER2 influences the expression of other clock genes in cancer cells. In the present study, short hairpin RNA interference was used to knockdown PER2 effectively in human oral squamous cell carcinoma SCC15 cells. Quantitative polymerase chain reaction was used to assess the mRNA expression levels of various clock genes and revealed that, following the knockdown of PER2 in SCC15 cells, the mRNA expression levels of PER3, brain and muscle ARNT-like 1, deleted in esophageal cancer (DEC)1, DEC2, cryptochrome circadian clock (CRY)2, timeless circadian clock, retinoic acid receptor-related orphan receptor-alpha and neuronal PAS domain protein 2 were significantly downregulated, while the mRNA expression levels of PER1 and nuclear receptor subfamily 1 group D member 1 were significantly upregulated. In addition, flow cytometric analysis demonstrated that proliferation was enhanced and apoptosis was reduced following PER2 knockdown in SCC15 cells (P<0.05). To the best of our knowledge, the present study is the first to report that PER2 is important for the regulation of other clock genes of the clock gene network in cancer cells. This is of great significance in elucidating the molecular function and tumor suppression mechanism of PER2.

Melatonin up-regulates the expression of the GATA-4 transcription factor and increases testosterone secretion from Leydig cells through RORα signaling in an in vitro goat spermatogonial stem cell differentiation culture system.

Because androgen function is regulated by its receptors, androgen-androgen receptor signaling is crucial for regulating spermatogenesis. Androgen is mainly testosterone secreted by testis. Based on the results of early studies in goats, the administration of melatonin over an extended period of time increases steroid production, but the underlying mechanism remains unclear. Here, we report the expression of the melatonin membrane receptors MT1 and MT2 and the retinoic acid receptor-related orphan receptor-alpha (RORα) in the goat testis. An in vitro differentiation system using spermatogonial stem cells (SSCs) cultured in the presence of testicular somatic cells was able to support the formation of sperm-like cells with a single flagellum. The addition of 10-7 M melatonin to the in vitro culture system increased RORα expression and considerably improved the efficiency of haploid cell differentiation, and the addition of the RORα agonist CGP52608 significantly increased the testosterone concentration and expression of GATA binding factor 4 (GATA-4). Furthermore, inhibitors of melatonin membrane receptors and a RORα antagonist (T0901317) also led to a considerable reduction in the efficiency of haploid spermatid formation, which was coupled with the suppression of GATA-4 expression. Based on these results, RORα may play a crucial role in enhancing melatonin-regulated GATA-4 transcription and steroid hormone synthesis in the goat spermatogonial stem cell differentiation culture system.

Melatonin promotes sheep Leydig cell testosterone secretion in a co-culture with Sertoli cells

Leydig cells synthesize and secrete testosterone, and are regulated by Sertoli cells. These two cell types may work together to regulate testicular androgen production. Studies have shown that Leydig cell androgen synthesis can be dramatically enhanced by Sertoli cells in the presence of melatonin, which can regulate the secretory function of Leydig and Sertoli cells. However, the molecular mechanism of melatonin-regulated Leydig cell androgen production via Sertoli cells remains unclear. Here, we found that 10−7 M melatonin increased testosterone production in co-cultured Leydig and Sertoli cells isolated from sheep. Melatonin increased the expression of stem cell factor and insulin-like growth factor-1 and decreased estrogen synthesis in Sertoli cells. Melatonin promoted insulin-like growth factor-1 and decreased estrogen content via the membrane melatonin receptor 1. It also enhanced stem cell factor expression via the retinoic acid receptor-related orphan receptor alpha. Addition of PD98059, a MEK inhibitor, to Sertoli cell culture demonstrated that the melatonin upregulation of insulin-like growth factor-1 and downregulation of estrogen may be through the MEK/extracellular signal-regulated kinase pathway. Together, these results suggest that melatonin may function through modulating melatonin receptor 1-regulated insulin-like growth factor-1 expression, as well as melatonin receptor 1-induced suppression of estrogen synthesis to increase androgen production in co-cultured Leydig and Sertoli cells.

Retinoic acid receptor-related orphan receptor RORα regulates differentiation and survival of keratinocytes during hypoxia.

Low O2 pressures present in the microenvironment of epidermis control keratinocyte differentiation and epidermal barrier function through hypoxia inducible factors (HIFs) dependent gene expression. This study focuses on investigating relations of the retinoic acid receptor-related orphan receptor alpha (RORα) to HIF-1α in keratinocytes under hypoxic conditions. The expression level of RORα is significantly elevated under hypoxia in both human and murine keratinocytes. Gene silencing of RORA attenuates hypoxia-stimulated expression of genes related to late differentiation and epidermal barrier function, and leads to an enhanced apoptotic response. While the hypoxic induction of RORα is dependent on HIF-1α, RORα is in turn critical for nuclear accumulation of HIF-1α and activation of HIF transcriptional activity. These results collectively suggest that RORα functions as an important mediator of HIF-1α activities in regulating keratinocyte differentiation/survival and epidermal barrier function during the oxygen sensing stage.

Role of simvastatin and RORα activity in the macrophage apoptotic pathway.

Objective:Atherosclerosis is a chronic inflammatory condition and is one of the main causes of death worldwide. Macrophages play important roles in the formation of atherosclerotic plaques. Apoptosis is progressively observed while plaques develop, although the precise mechanisms and outcomes of apoptosis in atherosclerosis development and progression are still contradictory. This study was conducted to explore the effects of simvastatin and retinoic acid receptor-related orphan receptor alpha (RORa) ligands on apoptosis in human acute monocytic leukemia (THP-1) macrophage cells.Methods:Briefly, the occupancy of RORa in the promoter regions of apoptotic pathway genes was demonstrated in THP-1 cell lines using chromatin immunoprecipitation (ChIP) analysis. In order to modulate RORa activity, THP-1 macrophage cells were treated with specific ligands (CPG52608 and SR1001) and then viability as well as count of THP-1 macrophage cells were analyzed.Results:We observed that simvastatin and both RORa ligands had a tendency to decrease THP-1 macrophage cell viability in culture. When compared with non-treated controls, simvastatin significantly decreased cell viability (p=0.04) and cell count (p=0.03). However, this negative effect of simvastatin seemed to be partly prevented by RORa ligands. In addition, bioinformatics analysis of ChIP-on-chip data demonstrated that several genes that are involved in the apoptotic pathway were likely RORa target genes. These genes were involved in the regulation of apoptosis through various pathways.Conclusion:In summary, our study suggest that simvastatin-mediated macrophage apoptosis might be modulated by SR1001 administration. However, involvement of RORa in this modulation through potential apoptotic target genes remains elusive.

Retinoid acid receptor-related orphan receptor alpha (RORα) regulates macrophage M2 polarization via activation of AMPKα.

Macrophages are able to polarize to pro-inflammatory M1 or anti-inflammatory M2 states with distinct phenotypes and physiological functions. RORα is a member of the nuclear receptor super family and plays important roles in lipid, glucose metabolism, as well as the inflammatory response. In this study, we examined the potential function of RORα in the regulation of macrophage polarization. Treatment of RAW264.7 macrophages with RORα agonist cholesterol sulfate (CH-S) and overexpression of RORα increased M2 macrophage markers expressions (Arg1, Ym1 and Fizz1) both on mRNA and protein levels. Conversely, selective antagonism (SR1001) abrogated the induction of M2 macrophage markers which induced by CH-S. In addition, CH-S induced phosphorylation of Adenosine monophosphate (AMP)-activated protein kinase α (AMPKα), which was accompanied by the activation of acetyl-CoA carboxylase 1 (ACC). However, SR1001 abolished the activation of AMPKα and ACC induced by CH-S. Meanwhile, inactivation of AMPKα by its inhibitor Compound C (CompC) abrogated the mRNA and protein levels of CH-S-induced M2 macrophage markers expressions. Together these findings reveal that RORα regulates macrophage M2 polarization via activation of AMPKα, which may provide a novel beneficial effect of RORα against inflammation.