Inhibition of nuclear receptor RORα attenuates cartilage damage in osteoarthritis by modulating IL-6/STAT3 pathway.

Osteoarthritis (OA) is characterized by cartilage destruction, chronic inflammation, and local pain. Evidence showed that retinoic acid receptor-related orphan receptor-α (RORα) is crucial in cartilage development and OA pathogenesis. Here, we investigated the role and molecular mechanism of RORα, an important member of the nuclear receptor family, in regulating the development of OA pathologic features. Investigation into clinical cartilage specimens showed that RORα expression level is positively correlated with the severity of OA and cartilage damage. In an in vivo OA model induced by anterior crucial ligament transaction, intra-articular injection of si-Rora adenovirus reversed the cartilage damage. The expression of cartilage matrix components type II collagen and aggrecan were elevated upon RORα blockade. RNA-seq data suggested that the IL-6/STAT3 pathway is significantly downregulated, manifesting the reduced expression level of both IL-6 and phosphorylated STAT3. RORα exerted its effect on IL-6/STAT3 signaling in two different ways, including interaction with STAT3 and IL-6 promoter. Taken together, our findings indicated the pivotal role of the RORα/IL-6/STAT3 axis in OA progression and confirmed that RORα blockade improved the matrix catabolism in OA chondrocytes. These results may provide a potential treatment target in OA therapy.

Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.

Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that RORα, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of RORα is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of RORα, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD in vitro, tumor necrosis factor alpha (TNF-α) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF-α treatment. Next, TNF-α treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.

Pharmacological modulation of RORα controls fat browning, adaptive thermogenesis, and body weight in mice.

Beiging is an attractive therapeutic strategy to fight against obesity and its side metabolic complications. The loss of function of the nuclear transcription factor RORα has been related to a lean phenotype with higher thermogenesis in sg/sg mice lacking this protein. Here we show that pharmacological modulation of RORα activity exerts reciprocal and cell-autonomous effect on UCP1 expression ex vivo, in cellulo, and in vivo. The RORα inverse-agonist SR3335 upregulated UCP1 expression in brown and subcutaneous white adipose tissue (scWAT) explants of wild-type (WT) mice, whereas the RORα agonist SR1078 had the opposite effect. We confirmed the reciprocal action of these synthetic RORα ligands on gene expression, mitochondrial mass, and uncoupled oxygen consumption rate in cultured murine and human adipocytes. Time course analysis revealed stepwise variation in gene expression, first of TLE3, an inhibitor of the thermogenic program, followed by a reciprocal effect on PRDM16 and UCP1. Finally, RORα ligands were shown to be useful tools to modulate in vivo UCP1 expression in scWAT with associated changes in this fat depot mass. SR3335 and SR1078 provoked the opposite effects on the WT mice body weight, but without any effect on sg/sg mice. This slimming effect of SR3335 was related to an increased adaptive thermogenesis of the mice, as assessed by the rectal temperature of cold-stressed mice and induction of UCP1 in scWAT, as well as by indirect calorimetry in presence or not of a ß3-adrenoceptor agonist. These data confirmed that RORα ligands could be useful tools to modulate thermogenesis and energy homeostasis.NEW & NOTEWORTHY The regulation of adipose tissue browning was not fully deciphered and required further studies explaining how the regulation of this process may be of interest for tackling obesity and related metabolic disorders. Our data confirmed the involvement of the transcription factor RORα in the regulation of nonshivering thermogenesis, and importantly, revealed the possibility to in vivo modulate its activity by synthetic ligands with beneficial consequences on fat mass and body weight of the mice.

RORα autoregulates its transcription via MLL4-associated enhancer remodeling in the liver.

AIMS: In hepatocytes, the retinoic acid receptor-related orphan receptor α (RORα) regulates the transcription of diverse genes encoding lipogenic enzymes, antioxidant enzymes, and mitochondrial factors via the regulation of the transcriptional activity of their promoters. The coordination of the expression of RORα by driving its transcription would provide better aspects for managing liver homeostasis. MAIN METHODS: The transcriptional expression of RORα was measured after treatment of RORα agonists on primary hepatocytes and liver. The histone status of Rora gene bodies was examined by analyzing ChIP-seq database. To elucidate molecular mechanism for RORα autoregulation, broad ChIP assays for promoters and enhancers with histone and RORα antibodies were performed. KEY FINDINGS: We report that natural and synthetic RORα agonists, cholesterol sulfate and JC1-40, respectively, increased the transcriptional expression of RORα in primary hepatocytes. An analysis of histone status around the Rora gene body identified promoter and enhancer regions of RORα. We found that RORα indirectly increased histone acetylation of H3K9 at the promoter region and directly enhanced histone monomethylation of H3K4 by binding to enhancer regions. Interestingly, disturbance of mixed-lineage leukemia 4 (MLL4), a histone methyltransferase for enhancers, abolished the JC1-40-induced activation of RORα via a decrease in H3K4me1. Finally, we observed that the MLL4-mediated autoregulation of RORα also occurred in human liver cancer cell lines. SIGNIFICANCE: The ability of RORα to modulate its own transcription is crucial for liver homeostasis, and ligand-dependent autoregulation could amplify the therapeutic effects of RORα in fatty liver diseases.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced ß-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

Discovery and Optimization of a Series of Sulfonamide Inverse Agonists for the Retinoic Acid Receptor-Related Orphan Receptor-α.

BACKGROUND: Despite a massive industry endeavor to develop RORγ-modulators for autoimmune disorders, there has been no indication of efforts to target the close family member RORα for similar indications. This may be due to the misconception that RORα is redundant to RORγ, or the inherent difficulty in cultivating tractable starting points for RORα. RORα-selective modulators would be useful tools to interrogate the biology of this understudied orphan nuclear receptor. OBJECTIVE: The goal of this research effort was to identify and optimize synthetic ligands for RORα starting from the known LXR agonist T0901317. METHODS: Fourty-five analogs of the sulfonamide lead (1) were synthesized and evaluated for their ability to suppress the transcriptional activity of RORα, RORγ, and LXRα in cell-based assays. Analogs were characterized by 1H-NMR, 13C-NMR, and LC-MS analysis. The pharmacokinetic profile of the most selective RORα inverse agonist was evaluated in rats with intraperitoneal (i.p.) and per oral (p.o.)dosing. RESULTS: Structure-activity relationship studies led to potent dual RORα/RORγ inverse agonists as well as RORα-selective inverse agonists (20, 28). LXR activity could be reduced by removing the sulfonamide nitrogen substituent. Attempts to improve the potency of these selective leads by varying substitution patterns throughout the molecule proved challenging. CONCLUSION: The synthetic RORα-selective inverse agonists identified (20, 28) can be utilized as chemical tools to probe the function of RORα in vitro and in vivo.

Topical ROR Inverse Agonists Suppress Inflammation in Mouse Models of Atopic Dermatitis and Acute Irritant Dermatitis.

The retinoic acid receptor-related orphan receptors RORα and RORγ are critical for the functions of specific subsets of T cells and innate lymphoid cells, which are key drivers of inflammatory disease in barrier tissues. Here, we investigate the anti-inflammatory potential of SR1001, a synthetic RORα/γ inverse agonist, in mouse models of atopic dermatitis and acute irritant dermatitis. Topical treatment with SR1001 reduces epidermal and dermal features of MC903-induced atopic dermatitis-like disease and suppresses the production of type 2 cytokines and other inflammatory mediators in lesional skin. In the epidermis, SR1001 treatment blocks MC903-induced expression of TSLP and reverses impaired keratinocyte differentiation. SR1001 is also effective in alleviating acute dermatitis triggered by 12-O-tetradecanoylphorbol-13-acetate. Overall, our results suggest that RORα/γ are important therapeutic targets for cutaneous inflammation and suggest topical usage of inhibitory ligands as an approach to treating skin diseases of inflammatory etiology.

Retinoic acid receptor-related orphan receptor α stimulates adipose tissue inflammation by modulating endoplasmic reticulum stress.

Adipose tissue inflammation has been linked to metabolic diseases such as obesity and type 2 diabetes. However, the molecules that mediate inflammation in adipose tissue have not been addressed. Although retinoic acid receptor-related orphan receptor α (RORα) is known to be involved in the regulation of inflammatory response in some tissues, its role is largely unknown in adipose tissue. Conversely, it is known that endoplasmic reticulum (ER) stress and unfolding protein response (UPR) signaling affect the inflammatory response in obese adipose tissue, but whether RORα regulates these processes remains unknown. In this study, we investigate the link between RORα and adipose tissue inflammation. We showed that the inflammatory response in macrophages or 3T3-L1 adipocytes stimulated by lipopolysaccharide, as well as adipose tissue in obese mice, markedly increased the expression of RORα. Adenovirus-mediated overexpression of RORα or treatment with the RORα-specific agonist SR1078 enhanced the expression of inflammatory cytokines and increased the number of infiltrated macrophages into adipose tissue. Furthermore, SR1078 up-regulated the mRNA expression of ER stress response genes and enhanced phosphorylations of two of the three mediators of major UPR signaling pathways, PERK and IRE1α. Finally, we found that alleviation of ER stress using a chemical chaperone followed by the suppression of RORα induced inflammation in adipose tissue. Our data suggest that RORα-induced ER stress response potentially contributes to the adipose tissue inflammation that can be mitigated by treatment with chemical chaperones. The relationships established here between RORα expression, inflammation, and UPR signaling may have implications for therapeutic targeting of obesity-related metabolic diseases.

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as "biased" agonists on VDR and inverse agonists on RORα and RORγ.

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)2D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)2D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)2D3 have been tested in a cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)2D3 and VDR.

Novel protective role of the circadian nuclear receptor retinoic acid-related orphan receptor-α in diabetic cardiomyopathy.

Diabetic cardiomyopathy is a major complication that significantly contributes to morbidity and mortality in diabetics with few therapies. Moreover, antidiabetic drugs reported inconsistent or even adverse cardiovascular effects, suggesting that it is important to exploit novel therapeutic targets against diabetic cardiomyopathy. Here, we observed that the nuclear melatonin receptor, the retinoic acid-related orphan receptor-α (RORα), was downregulated in diabetic hearts. By utilizing a mouse line with RORα disruption, we demonstrated that RORα deficiency led to significantly augmented diastolic dysfunction and cardiac remodeling induced by diabetes. Microscopic and molecular analyses further indicated that the detrimental effects of RORα deficiency were associated with aggravated myocardial apoptosis, autophagy dysfunction, and oxidative stress by disrupting antioxidant gene expression. By contrast, restoration of cardiac RORα levels in transgenic mice significantly improved cardiac functional and structural parameters at 8 weeks after diabetes induction. Consistent with genetic manipulation, pharmacological activation of RORα by melatonin and SR1078 (a synthetic agonist) showed beneficial effects against diabetic cardiomyopathy, while the RORα inhibitor SR3335 significantly exacerbated cardiac impairments in diabetic mice. Collectively, these findings suggest that cardiac-targeted manipulation of nuclear melatonin receptor RORα may hold promise for delaying diabetic cardiomyopathy development.

Discovery of oxa-sultams as RORc inverse agonists showing reduced lipophilicity, improved selectivity and favorable ADME properties.

Modification of the δ-sultam ring of RORc inverse agonist 2 led to the discovery of more polar oxa-sultam 65. The less lipophilic inverse agonist (65) displayed high potency in a biochemical assay, which translated into inhibition of IL-17 production in human peripheral blood mononuclear cells. The successful reduction of lipophilicity of this new analog gave rise to additional improvements in ROR selectivity and aqueous kinetic solubility, as well as reduction in plasma protein binding, while maintaining high cellular permeability.

Therapeutic Effect of a Synthetic RORα/γ Agonist in an Animal Model of Autism.

Autism is a developmental disorder of the nervous system associated with impaired social communication and interactions as well excessive repetitive behaviors. There are no drug therapies that directly target the pathology of this disease. The retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor that has been demonstrated to have reduced expression in many individuals with autism spectrum disorder (ASD). Several genes that have been shown to be downregulated in individuals with ASD have also been identified as putative RORα target genes. Utilizing a synthetic RORα/γ agonist, SR1078, that we identified previously, we demonstrate that treatment of BTBR mice (a model of autism) with SR1078 results in reduced repetitive behavior. Furthermore, these mice display increased expression of ASD-associated RORα target genes in both the brains of the BTBR mice and in a human neuroblastoma cell line treated with SR1078. These data suggest that pharmacological activation of RORα may be a method for treatment of autism.

ROR inverse agonist suppresses insulitis and prevents hyperglycemia in a mouse model of type 1 diabetes.

Hyperglycemia associated with type 1 diabetes is a consequence of immune-mediated destruction of insulin producing pancreatic ß-cells. Although it is apparent that both CD8(+) T cells and TH1 cells are key contributors to type 1 diabetes, the function of TH17 cells in disease onset and progression remains unclear. The nuclear receptors retinoic acid receptor-related orphan receptors-α and γt (RORα and RORγt) play critical roles in the development of TH17 cells and ROR-specific synthetic ligands have proven efficacy in several mouse models of autoimmunity. To investigate the roles and therapeutic potential for targeting the RORs in type 1 diabetes, we administered SR1001, a selective RORα/γ inverse agonist, to nonobese diabetic mice. SR1001 significantly reduced diabetes incidence and insulitis in the treated mice. Furthermore, SR1001 reduced proinflammatory cytokine expression, particularly TH17-mediated cytokines, reduced autoantibody production, and increased the frequency of CD4(+)Foxp3(+) T regulatory cells. These data suggest that TH17 cells may have a pathological role in the development of type 1 diabetes, and use of ROR-specific synthetic ligands targeting this cell type may prove utility as a novel treatment for type 1 diabetes.

Development of novel silicon-containing inverse agonists of retinoic acid receptor-related orphan receptors.

Retinoic acid receptor (RAR)-related orphan receptors (RORs) regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm and immune function. The RAR agonist: all-trans retinoic acid was reported to be an RORß inverse agonist, but no information is available regarding ROR activity of its synthetic analogue Am580. Therefore, we screened Am580 and some related tetramethyltetrahydronaphthalene derivatives and carried out structural development studies, including substitution of carbon atoms with silicon, with the aim of creating a potent ROR transcriptional inhibitor. The phenyl amide disila compound 22 showed the most potent ROR-inhibitory activity among the compounds examined. Its activity towards RORα, RORß and RORγ was increased compared to that of Am580. The IC50 values for RORα, RORß and RORγ are 1.3, >10 and 4.5 µM, respectively.

The identification of naturally occurring neoruscogenin as a bioavailable, potent, and high-affinity agonist of the nuclear receptor RORα (NR1F1).

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.

N-methylthioureas as new agonists of retinoic acid receptor-related orphan receptor.

Thirty two thiourea derivatives were prepared and their agonistic activities on the retinoic acid receptor-related orphan receptor α (RORα) were evaluated. The replacement of the 3-allyl-2-imino-thiazolidin-4-one moiety of the lead compound CGP52608 (1) with various functional group substituted aromatic rings, improved the agonistic activity of RORα. Among the prepared derivatives, 1-methyl-3-(4-phenoxy-benzyl)-thiourea (32) showed 2.6-fold higher agonistic activity than CGP52608 in the RORα-activation assay.

Regulation of p53 stability and apoptosis by a ROR agonist.

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anti-cancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORα. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer.

Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis.

Many nuclear hormone receptors (NRs) control lipid, glucose and energy homeostasis in an organ specific manner. Concordantly, dysfunctional NR signalling results in metabolic disease. The Retinoic acid receptor-related orphan receptor alpha (RORα), a member of the NR1F subgroup, is expressed in metabolic tissues. Previous studies identified the role of this NR in dyslipidemia, apo-lipoprotein metabolism and atherosclerosis. Recent data is underscoring the significant role of this orphan NR in the regulation of phase I/II metabolism (bile acids, xenobiotics, steroids etc.), adiposity, insulin signalling, and glucose tolerance. Moreover, oxygenated sterols, have been demonstrated to function as native ligands and inverse agonists. This review focuses on the rapidly emerging and evolving role of RORα in the control of lipid and glucose homeostasis in major mass metabolic tissues. Article from the special issue orphan receptors.

Protein kinase A activates and phosphorylates RORα4 in vitro and takes part in RORα activation by CaMK-IV.

The retinoic acid related orphan receptor RORα positively regulates the transcription of genes important for cerebellar development, immune function, lipid metabolism, and circadian rhythm. In the present study, we identified protein kinase A (PKA) as RORα4 phosphorylating kinase in vitro. The primary sequence of RORα4 contains a PKA recognition motif (R-D-S99) within the c-terminal extension of the DNA-binding domain, and mutation of Ser-99 to Ala prevents RORα4 phosphorylation by PKA. Activation of PKA by dBcAMP results in a marked induction of RORα4 activity. Inhibition of PKA with the selective kinase inhibitor H89 inhibits dBcAMP mediated as well as CaMK-IV triggered increase in RORα4 transcriptional activity. The regulation of RORα activity by PKA as well as CaMK-IV provides a new link in the signalling network that regulates metabolic processes such as glycogen and lipid metabolism.