Maternal diabetes-mediated RORA suppression in mice contributes to autism-like offspring through inhibition of aromatase.

Retinoic acid-related orphan receptor alpha (RORA) suppression is associated with autism spectrum disorder (ASD) development, although the mechanism remains unclear. In this study, we aim to investigate the potential effect and mechanisms of RORA suppression on autism-like behavior (ALB) through maternal diabetes-mediated mouse model. Our in vitro study in human neural progenitor cells shows that transient hyperglycemia induces persistent RORA suppression through oxidative stress-mediated epigenetic modifications and subsequent dissociation of octamer-binding transcription factor 3/4 from the RORA promoter, subsequently suppressing the expression of aromatase and superoxide dismutase 2. The in vivo mouse study shows that prenatal RORA deficiency in neuron-specific RORA null mice mimics maternal diabetes-mediated ALB; postnatal RORA expression in the amygdala ameliorates, while postnatal RORA knockdown mimics, maternal diabetes-mediated ALB in offspring. In addition, RORA mRNA levels in peripheral blood mononuclear cells decrease to 34.2% in ASD patients (n = 121) compared to the typically developing group (n = 118), and the related Receiver Operating Characteristic curve shows good sensitivity and specificity with a calculated 84.1% of Area Under the Curve for ASD diagnosis. We conclude that maternal diabetes contributes to ALB in offspring through suppression of RORA and aromatase, RORA expression in PBMC could be a potential marker for ASD screening.

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.

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.

Maresin-1 induces cardiomyocyte hypertrophy through IGF-1 paracrine pathway.

The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.

RORα suppresses interleukin-6-mediated hepatic acute phase response.

Acute liver failure (ALF) is characterized by loss of liver function in response to sustained augmentation of the acute-phase response (APR) in the liver, which can progress even to death. Although the inflammatory interleukin-6 (IL-6)-axis is a crucial factor that drives the hepatic APR by releasing diverse acute-phase proteins (APPs), therapeutic strategies to block the IL-6-STAT3-mediated APR are not well developed. Here, we show that the nuclear receptor retinoic acid-related orphan receptor α (RORα) limits APR-mediated liver injury by inhibiting the hepatic IL-6-STAT3 signaling pathway. Administration of JC1-40, an RORα activator, diminished diethylnitrosamine-induced acute liver injury and repressed transcriptional expression of APPs such as CXCL1 and LCN2 in mice. IL-6-mediated activation of STAT3 was repressed after RORα activation by either adenoviral infusion of RORα or JC1-40 treatment in primary hepatocytes. Activation of RORα decreased transcriptional expression of IL-6 receptor α, an upstream activator of STAT3, both in vitro and in vivo. This may be one mechanism underlying the RORα-mediated inhibition of STAT3. Taken together, our results suggest that RORα is a regulator of the hepatic IL-6-STAT3 signaling pathway and may be a new therapeutic target for treating APR-associated inflammatory ALF.

Synthesis of dihydroimidazole tethered imidazolinethiones and their activity as novel antagonists of the nuclear retinoic acid receptor-related orphan receptors (RORs).

Targeting the transcriptional activity of nuclear hormone receptors has proven an effective strategy to treat certain human diseases, and they have become a major focus point to develop novel therapies for the treatment of cancer, inflammation, autoimmune diseases, metabolic disorders, and others. One family of nuclear receptors that has attracted most interest in recent years is the retinoic acid receptor-related orphan receptors (RORs), in particular RORγ. RORγ is a critical regulator of the immune system and RORγ antagonists have shown activity in animal models of inflammatory autoimmune diseases. Here we present the synthesis and biological evaluation of dihydroimidazole tethered imidazolinethiones. We have identified several dual RORγ/α and pan-ROR antagonists with significant activity in cellular assays that could serve as starting points for future optimization efforts to generate potent and selective RORγ modulators.

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells.

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

RORα inhibits adipocyte-conditioned medium-induced colorectal cancer cell proliferation and migration and chick embryo chorioallantoic membrane angiopoiesis.

Lipid metabolic disturbances are related to many diseases, such as obesity, diabetes, and certain cancers. Notably, lipid metabolic disturbances have been reported to be a risk factor for colorectal cancer. Nuclear receptors act as ligand-dependent transcription regulators and play key roles in the regulation of body lipid metabolism and the development of many cancers. Retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor and can regulate several lipid metabolism genes in certain cancers. Herein, we demonstrate that the conditioned medium from adipocytes has a proproliferative and promigratory effect on colorectal cancer cells and enhances angiogenesis in chicken embryonic chorioallantoic membranes. In addition, the conditioned medium leads to a decrease in the expression of RORα and its target genes. Meanwhile, RORα and its target gene expressions are lower in human colorectal tumor tissue compared with control colorectal tissue. Activation of RORα inhibits the effect of conditioned medium on the proliferation and migration of colorectal cancer cells as well as the angiogenesis in chicken embryonic allantoic membranes. In colorectal cancer cells, the putative ligand of RORα, cholesterol sulfate (CS), prevents cell cycle progression at the G1/S boundary and concurrently modulates the expression of cell cycle-regulatory genes in colorectal cancer cell. CS inhibits angiogenesis in chicken embryonic chorioallantoic membranes and concurrently decreases the mRNA expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α as well as the secretion of VEGF. In addition, lipogenic gene expression is higher in human colorectal tumor tissue compared with control colorectal tissue. CS inhibits the expression of lipogenic genes in colorectal cancer cells. These results suggest that RORα could represent a direct link between local lipid metabolism of colorectal tissue and colorectal cancer. Therefore, the reduction of the expression of RORα could represent a potential warning sign of colorectal cancer.

The hidden switches underlying RORα-mediated circuits that critically regulate uncontrolled cell proliferation.

Prostaglandin E2 (PGE2) is known to have a key role in the development of colorectal cancer, but previous experiments showed its contrasting (i.e. tumor-promoting or tumor-suppressive) roles depending on experimental conditions. To elucidate the mechanisms underlying such contrasting roles of PGE2 in tumorigenesis, we investigated all the previous experiments and found a new signal transduction pathway mediated by retinoic acid receptor-related orphan receptor (ROR)α, in which PGE2/PKCα-dependent phosphorylation of RORα attenuates Wnt target gene expression in colon cancer cells. From mathematical simulations combined with biochemical experimentation, we revealed that RORα induces a biphasic response of Wnt target genes to PGE2 stimulation through a regulatory switch formed by an incoherent feedforward loop, which provides a mechanistic explanation on the contrasting roles of PGE2 observed in previous experiments. More interestingly, we found that RORα constitutes another regulatory switch formed by coupled positive and negative feedback loops, which regulates the hysteretic response of Wnt signaling and eventually converts a proliferative cellular state into an anti-proliferative state in a very delicate way. Our results indicate that RORα is the key regulator at the center of these hidden switches that critically regulate cancer cell proliferation and thereby being a promising anti-cancer therapeutic target.

REV-ERB and ROR nuclear receptors as drug targets.

The nuclear receptors REV-ERB (consisting of REV-ERBα and REV-ERBß) and retinoic acid receptor-related orphan receptors (RORs; consisting of RORα, RORß and RORγ) are involved in many physiological processes, including regulation of metabolism, development and immunity as well as the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and opened up the possibility of targeting these receptors to treat several diseases, including diabetes, atherosclerosis, autoimmunity and cancer. This Review focuses on the latest developments in ROR and REV-ERB pharmacology indicating that these nuclear receptors are druggable targets and that ligands targeting these receptors may be useful in the treatment of several disorders.

Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics.

PURPOSE OF REVIEW: In the late 1980s, the cloning of several nuclear receptors led to the intense search and isolation of new members of this superfamily. Despite their identification, many of these receptors were dubbed 'orphan' receptors, as their physiological ligands remained unknown. Recent reports have presented evidence for one family of orphan receptors, the retinoic acid receptor-related orphan receptors (RORs), in several pathologies, including osteoporosis, several autoimmune diseases, asthma, cancer, diabetes and obesity. The present review summarizes the studies identifying ligands for the RORs and evaluates their role as targets for potential therapeutics. RECENT FINDINGS: Significant progress was made in the initial identification of ligands for the RORs when X-ray crystallographic studies identified several molecules within the ligand-binding pockets of RORalpha and RORbeta. Recently, we identified endogenous and synthetic ligands for RORalpha and RORgamma, thereby solidifying their function as ligand-dependent transcription factors. SUMMARY: Recent studies have established roles for the RORs in physiological development and the advent of disease. Identification of ligands for the RORs, both endogenous and synthetic, has established these receptors as attractive new therapeutic targets for the treatment of ROR-related diseases.

A second class of nuclear receptors for oxysterols: Regulation of RORalpha and RORgamma activity by 24S-hydroxycholesterol (cerebrosterol).

The retinoic acid receptor-related orphan receptors alpha and gamma (RORalpha [NR1F1] and RORgamma [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORalpha and RORgamma with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORalpha and RORgamma (K(i) approximately 25 nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORalpha/gamma inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORalpha target genes including BMAL1 and REV-ERBalpha in a ROR-dependent manner. We also demonstrate that 24S-OHC decreases the ability of RORalpha to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORgamma. These data indicate that RORalpha and RORgamma may serve as sensors of oxsterols. Thus, RORalpha and RORgamma display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRalpha [NR1H3] and LXRbeta [NR1H2]).