RORα is critical for mTORC1 activity in T cell-mediated colitis.

Inflammatory bowel disease (IBD) is multi-factorial chronic intestinal inflammation driven by pathogenic T cells, among which a large portion of patients are resistant to current anti-inflammatory regimes. The mechanisms underlying colitis pathogenicity and drug resistance are not fully understood. Here, we demonstrate that RORα is highly expressed in active UC patients, particularly in those non-responsive to anti-TNF treatment. Rorα deficiency in CD4+ T cells greatly reduced colitis development. Mechanistically, RORα regulated T cell infiltration in colon and inhibited T cell apoptosis. Meanwhile, genome-wide occupancy and transcriptome analysis revealed that RORα promoted mTORC1 activation. mTORC1 signaling, also hyperactivated in active UC patients, is necessary for T cell-mediated colitis. Our results thus demonstrate a crucial role of the RORα-mTORC1 axis in CD4+ T cells in promoting IBD, which may be targeted in human patients.

Functions for Retinoic Acid-Related Orphan Receptor Alpha (RORα) in the Activation of Macrophages During Lipopolysaccharide-Induced Septic Shock.

The transcription factor Related Orphan Receptor Alpha (RORα) plays an important role in regulating circadian rhythm, inflammation, metabolism and cellular development. Herein we show that in the absence of functional RORα in mice there is reduced susceptibility to LPS-induced endotoxic shock, with selective decreases in release of pro-inflammatory cytokines. Treatment of mice with a RORα selective synthetic inhibitor also reduced the severity of LPS-induced endotoxemia. The reduction in responses in Rora deficient mice was associated with an alterations in metabolic and pro-inflammatory functions of macrophages, both in vivo peritoneal macrophages and in vitro generated bone marrow derived macrophages. Using LysMCreRorafl/sg mice the reduced susceptibility to LPS was shown to be specific to Rora expression in the macrophages. This study identifies that Rora-mediated regulation of macrophages impacts on the pro-inflammatory responses elicited by LPS.

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 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.

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.

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.

Nuclear receptor retinoid-related orphan receptor α deficiency exacerbates high-fat diet-induced cardiac dysfunction despite improving metabolic abnormality.

Retinoid-related orphan receptor α (RORα), a member of the metabolic nuclear receptor superfamily, plays a vital regulatory role in circadian rhythm and metabolism. Here, we investigated the role of RORα in high-fat diet (HFD)-induced cardiac impairments and the underlying mechanisms involved. RORα-deficient stagger mice (sg/sg) and wild type (WT) littermates were fed with either standard diet or HFD. At 20weeks after HFD treatment, RORα deficiency resulted in significantly decreased body weight gain, improved dyslipidemia and ameliorated insulin resistance (evaluated by blood biochemical and glucose/insulin tolerance tests) compared with WT control. However, compared with HFD-treated WT mice, HFD-treated sg/sg mice exhibited significantly augmented myocardial hypertrophy, cardiac fibrosis (wheat germ agglutinin, masson trichrome and sirius red staining) and cardiac dysfunction (echocardiography and hemodynamics). Mechanistically, RORα deficiency impaired mitochondrial biogenesis and function. Additionally, RORα deficiency resulted in inhibition of the AMPK-PGC1α signaling pathway. In contrast, cardiomyocyte-specific RORα overexpression ameliorated myocardial hypertrophy, fibrosis and dysfunction by restoring AMPK-PGC1α signaling, and subsequently normalizing mitochondrial biogenesis. These findings demonstrated for the first time that nuclear receptor RORα deficiency aggravated HFD-induced myocardial dysfunction at least in part by impairing mitochondrial biogenesis in association with disrupting AMPK-PGC1α signaling. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren and Megan Yingmei Zhang.

Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.

By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

The retinoid-related orphan receptor alpha is essential for the end-stage effector phase of experimental epidermolysis bullosa acquisita.

Genetic studies have added to the understanding of complex diseases. Here, we used a combined genetic approach for risk-loci identification in a prototypic, organ-specific, autoimmune disease, namely experimental epidermolysis bullosa acquisita (EBA), in which autoantibodies to type VII collagen (COL7) and neutrophil activation cause mucocutaneous blisters. Anti-COL7 IgG induced moderate blistering in most inbred mouse strains, while some showed severe disease or were completely protected. Using publicly available genotyping data, we identified haplotype blocks that control blistering and confirmed two haplotype blocks in outbred mice. To identify the blistering-associated genes, haplotype blocks encoding genes that are differentially expressed in EBA-affected skin were considered. This procedure identified nine genes, including retinoid-related orphan receptor alpha (RORα), known to be involved in neurological development and function. After anti-COL7 IgG injection, RORα+/- mice showed reduced blistering and homozygous mice were completely resistant to EBA induction. Furthermore, pharmacological RORα inhibition dose-dependently blocked reactive oxygen species (ROS) release from activated neutrophils but did not affect migration or phagocytosis. Thus, forward genomics combined with multiple validation steps identifies RORα to be essential to drive inflammation in experimental EBA.

Critical role of p38 and GATA3 in natural helper cell function.

Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.

Is retinoic acid-related orphan receptor-alpha (RORA) a target for gene-environment interactions contributing to autism?

It is becoming increasingly clear that gene-environment interactions are risk factors for autism. However, there is limited information regarding the susceptibility of specific autism candidate genes to dysregulation by environmental factors, and even less information on the types of environmental agents that may lead to increased risk for autism. Based on our published studies, I propose that the demonstrated responsiveness of RORA to sex hormones makes it a prime target for disruption by endocrine disrupting compounds.

Transcriptional profiling reveals a role for RORalpha in regulating gene expression in obesity-associated inflammation and hepatic steatosis.

Retinoid-related orphan receptor (ROR)α4 is the major RORα isoform expressed in adipose tissues and liver. In this study we demonstrate that RORα-deficient staggerer mice (RORα(sg/sg)) fed with a high-fat diet (HFD) exhibited reduced adiposity and hepatic triglyceride levels compared with wild-type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORα(sg/sg) mice. In contrast, overexpression of RORα in mouse hepatoma Hepa1-6 cells significantly increased the expression of genes that were repressed in RORα(sg/sg) liver, including Sult1b1, Adfp, Cidea, and ApoA4. ChIP and promoter analysis suggested that several of these genes were regulated directly by RORα. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORα(sg/sg) mice fed with an HFD. The infiltration of macrophages and the expression of many immune response and proinflammatory genes, including those encoding various chemo/cytokines, Toll-like receptors, and TNF signaling proteins, were significantly reduced in RORα(sg/sg) WAT. Moreover, RORα(sg/sg) mice fed with an HFD were protected from the development of insulin resistance. RORα(sg/sg) mice consumed more oxygen and produced more carbon dioxide, suggesting increased energy expenditure in this genotype. Our study indicates that RORα plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORα may provide a novel therapeutic target in the management of obesity and associated metabolic diseases.

Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain.

Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.