Retinoic Acid Receptor-related Receptor Research Articles

050 Loss of retinoic acid receptor-related receptor alpha (Rorα) promotes the progression of UV-induced cSCC

Cutaneous squamous cell carcinoma (cSCC) is prevalent in the world, accounting a huge part in non-melanoma skin cancer. Most cSCCs are associated with a distinct pre-cancerous lesion, the actinic keratosis (AK). However, the progression trajectory from normal skinto AK and cSCC has not fully demonstrated yet. To identify genes involved in this progression trajectory and possible threptic targets for cSCC, here we constructed a UV-induced cSCC mouse model covering the progression from normal skinto AK to cSCC, which mimicked the solar UV radiation perfectly using the solar-like ratio of UVA and UVB, firstly. Then, transcriptome analysis and a series of bioinformatics analysesand cell experiments proved that Rorα is a key transcript factor during cSCC progression.Rorα could down-regulate the expressions of S100a9 and Sprr2f in cSCC cells, which can inhibit theproliferation and migration in cSCC cells, but not the normal keratinocyte. Finally, further animal experiment confirmed the inhibitory effect of cSCC growth by Rorα in vivo.Our findings showed that Rorα would serve as a potential novel target for cSCC, which will facilitate the treatment of cSCC in the future.

Loss of retinoic acid receptor-related receptor alpha (Ror\u03b1) promotes the progression of UV-induced cSCC

Cutaneous squamous cell carcinoma (cSCC) is prevalent in the world, accounting for a huge part of non-melanoma skin cancer. Most cSCCs are associated with a distinct pre-cancerous lesion, the actinic keratosis (AK). However, the progression trajectory from normal skin to AK and cSCC has not been fully demonstrated yet. To identify genes involved in this progression trajectory and possible therapeutic targets for cSCC, here we constructed a UV-induced cSCC mouse model covering the progression from normal skin to AK to cSCC, which mimicked the solar UV radiation perfectly using the solar-like ratio of UVA and UVB, firstly. Then, transcriptome analysis and a series of bioinformatics analyses and cell experiments proved that Rorα is a key transcript factor during cSCC progression. Rorα could downregulate the expressions of S100a9 and Sprr2f in cSCC cells, which can inhibit the proliferation and migration in cSCC cells, but not the normal keratinocyte. Finally, further animal experiments confirmed the inhibitory effect of cSCC growth by Rorα in vivo. Our findings showed that Rorα would serve as a potential novel target for cSCC, which will facilitate the treatment of cSCC in the future.

Metabolic Characterization of a Novel RORα Knockout Mouse Model without Ataxia.

The retinoic acid receptor-related receptor α (RORα) is a nuclear receptor that plays an important role in regulation of metabolism and the immune system. Genetic deletion of the receptor yields mice with significant cerebellar developmental issues associated with severe ataxia. Although many metabolic studies have been performed in these models, the impaired locomotor activity of these mice is known to affect their normal mobility and feeding behaviors. This creates some difficulty in interpretation of the role of RORα in models of metabolic disease where feeding and muscle function is a critical component of the pathophysiology. We generated a mouse with a floxed Rora allele that we crossed with a mouse line expressing Cre recombinase under the control of the EIIa promoter to obtain a full body deletion of Rora. This cross led to a partial deletion of the Rora locus likely due to mosaic expression of the EIIa-Cre transgene. These mice lack any signs of ataxia but display an improved metabolic profile relative to normal WT mice. The mice were resistant to diet- and age-induced metabolic syndrome and exhibited improved glucose tolerance and increased insulin sensitivity. Decreased RORα expression in the mice was also associated with reduced inflammation in models of metabolic syndrome. These data indicate that suppression of RORα activity improves metabolic function and reduces inflammation.

Retinoic acid receptor-related receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma.

Retinoic acid receptor-related receptor alpha (RORalpha) has been proven to play a tumor suppressive role in certain types of solid tumors. However, the clinical characteristic of RORalpha has not been reported by far. This study investigated the expression of RORalpha in hepatocellular carcinoma (HCC) and evaluated its relationship with clinical parameters and prognosis in HCC patients. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to detect RORalpha expression levels in 20 paired HCC and corresponding adjacent non-cancerous tissues. Immunohistochemistry was performed on 100 archived paraffin-embedded HCC samples. Statistical analyses evaluated the correlations between RORalpha expression and clinicopathological features. qRT-PCR showed that RORalpha mRNA expression was significantly down-regulated in tumors compared to the adjacent non-cancerous tissues, and Western blots found that RORalpha protein expression was also reduced in tumor tissues. Immunohistochemical assays revealed that decreased RORalpha expression was present in 65 % of HCC patients. Correlation analyses showed that RORalpha expression was significantly correlated with serum alpha fetoprotein (AFP, p = 0.005), pathology grade (p < 0.001), tumor recurrence (p = 0.008), and vascular invasion (p < 0.001). Kaplan-Meier analysis revealed that patients with low RORalpha expression levels had a shorter overall and disease-free survival than patients with high expression (p < 0.001 and p = 0.002, respectively). Multivariate regression analysis indicated that RORalpha was an independent predictor for overall survival and disease-free survival. In conclusion, the results of our study showed that down-regulated RORalpha expression was associated with poorer prognosis in HCC patients. RORalpha may be a new potential prognostic marker for HCC patients.

Retinoic Acid Receptor β Stimulates Hepatic Induction of Fibroblast Growth Factor 21 to Promote Fatty Acid Oxidation and Control Whole-body Energy Homeostasis in Mice

Activation of retinoic acid receptor (RAR) with all-trans-retinoic acid (RA) ameliorates glucose intolerance and insulin resistance in obese mice. The recently discovered fibroblast growth factor 21 (FGF21) is a hepatocyte-derived hormone that restores glucose and lipid homeostasis in obesity-induced diabetes. However, whether hepatic RAR is linked to FGF21 in the control of lipid metabolism and energy homeostasis remains elusive. Here we identify FGF21 as a direct target gene of RARβ. The gene transcription of Fgf21 is increased by the RAR agonist RA and by overexpression of RARα and RARβ, but it is unaffected by RARγ in HepG2 cells. Promoter deletion analysis characterizes a putative RA-responsive element (RARE) primarily located in the 5'-flanking region of the Fgf21 gene. Disruption of the RARE sequence abolishes RA responsiveness. In vivo adenoviral overexpression of RARβ in the liver enhances production and secretion of FGF21, which in turn promotes hepatic fatty acid oxidation and ketogenesis and ultimately leads to increased energy expenditure in mice. The metabolic effects of RA or RARβ are mimicked by FGF21 overexpression and largely abolished by FGF21 knockdown. Moreover, hepatic RARβ is bound to the putative RAREs of the Fgf21 promoter in a fasting-inducible manner in vivo, which contributes to FGF21 induction and the metabolic adaptation to prolonged fasting. In addition to other nuclear receptors, such as peroxisome proliferator-activated receptor α and retinoic acid receptor-related receptor α, RAR may act as a novel component to induce hepatic FGF21 in the regulation of lipid metabolism. The hepatic RAR-FGF21 pathway may represent a potential drug target for treating metabolic disorders.

Identification of SR3335 (ML-176): A Synthetic RORα Selective Inverse Agonist

Several nuclear receptors (NRs) are still character-ized as orphan receptors because ligands have not yet been identified for these proteins. The retinoic acid receptor-related receptors (RORs) have no well-defined physiological ligands. Here, we describe the identification of a selective RORα synthetic ligand, SR3335 (ML-176). SR3335 directly binds to RORα, but not other RORs, and functions as a selective partial inverse agonist of RORα in cell-based assays. Furthermore, SR3335 suppresses the expression of endogenous RORα target genes in HepG2 involved in hepatic gluconeogenesis including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Pharmacokinetic studies indicate that SR3335 displays reasonable exposure following an ip injection into mice. We assess the ability of SR3335 to suppress gluconeogenesis in vivo using a diet-induced obesity (DIO) mouse model where the mice where treated with 15 mg/kg b.i.d., ip for 6 days followed by a pyruvate tolerance test. SR3335-treated mice displayed lower plasma glucose levels following the pyruvate challenge consistent with suppression of gluconeogenesis. Thus, we have identified the first selective synthetic RORα inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both in vitro and in vivo. Additionally, our data suggests that RORα inverse agonists may hold utility for suppression of elevated hepatic glucose production in type 2 diabetics.

Identification of SR1078, a Synthetic Agonist for the Orphan Nuclear Receptors RORα and RORγ

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors because ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice, and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist, and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo.

Regulation of FGF21 Expression and Secretion by Retinoic Acid Receptor-related Orphan Receptor α

Fibroblast growth factor 21 (FGF21) is a hormone produced by fat and the liver that plays an important role in lipid metabolism. FGF21 expression is induced by peroxisome proliferator-actived receptor alpha in response to physiological conditions requiring increased fatty acid oxidation. Retinoic acid receptor-related receptor alpha (RORalpha) is another nuclear receptor that plays a critical role in lipid metabolism as well as in regulation of the circadian rhythm. In this study we demonstrate that RORalpha directly regulates the expression and secretion of FGF21. A canonical ROR response element was identified in the proximal promoter of the FGF21 gene and shown to exhibit functional activity. Overexpression of RORalpha in HepG2 cells resulted in increased expression and secretion of FGF21. Suppression of RORalpha expression caused a decrease in FGF21 expression and secretion, suggesting that RORalpha contributes to the basal expression of FGF21. These data suggest that one mechanism by which RORalpha regulates lipid metabolism may be by modulation of FGF21 secretion. Furthermore, this study identifies a clear link between RORalpha, a key regulator of the mammalian clock, and FGF21, an important hormone regulating glucose and lipid homeostasis.

The BmE75 Nuclear Receptors Function as Dominant Repressors of the Nuclear Receptor BmHR3A

The orphan nuclear receptors BmE75 and BmHR3 are induced by 20-hydroxyecdysone in the ovary of the silk moth Bombyx mori at the beginning of pupation and show stage-specific expression in ovarian follicles during pharate adult development. To analyze the function of these receptors, we have developed a transactivation assay based on the transcriptional stimulation of a retinoic acid receptor-related receptor response element (RORE)-linked promoter-reporter construct. Co-transfection of a Bombyx cell line with a BmHR3A expression construct results in constitutive activation of the reporter, whereas expression of BmE75 has no measurable effects on reporter expression. However, when the BmE75 receptors are co-introduced with BmHR3A into the cells, the BmHR3A-mediated transactivation is repressed. Repression of BmHR3A by BmE75 occurs by two distinct mechanisms. Increasing doses of BmE75 efficiently displace BmHR3A bound to the RORE target site in gel retardation assays, indicating that both receptors compete for common DNA target sites. However, analysis of the function of deletion mutants of BmE75 in the transactivation assay indicates that repression can also occur in the absence of the DNA-binding domain and that the C-terminal F domain is sufficient for repression. In gel retardation assays, the two receptor types form a ternary complex on a single RORE, suggesting that repression is also mediated by protein interactions on the DNA target site. Yeast two-hybrid assays show that BmHR3A interacts with BmE75 and that this interaction is dependent on the C terminus of BmHR3A and the F domain of BmE75. Because the C terminus of BmHR3A contains a strong activation domain, we predict that BmE75 blocks activation by BmHR3A through competition for co-activator binding sites located at the C terminus of BmHR3A. Our data also indicate that the transcriptional activities of BmHR3A and BmE75 are integrated in such a way that activation of RORE-linked target genes depends on the relative expression levels of the two receptor types.