Retinoic Acid Receptor-related Orphan Research Articles

Melatonin-Mediated Circadian Rhythm Signaling Exhibits Bidirectional Regulatory Effects on the State of Hair Follicle Stem Cells

The development and regulation of hair are widely influenced by biological rhythm signals. Melatonin plays a crucial role as a messenger in transmitting biological rhythm signals, and its impact on hair development has been well documented. During the process of hair follicle reconstruction, hair follicle stem cells (HFSCs) are the most important cell type, but the regulatory effect of melatonin on the state of HFSCs is still not fully understood. Therefore, it is necessary to conduct a more comprehensive characterization of the effects of melatonin on the state of hair follicle stem cells. The research results indicate that HFSCs express retinoic acid receptor-related orphan receptor alpha (Rorα), and melatonin inhibits the expression level of RORA. Experimental results from CUT&Tag, CUT&RUN, and dual luciferase reporter assays demonstrate that Foxc1 is a downstream target gene of RORA, with RORA regulating Foxc1 expression by binding to the promoter region of Foxc1. The CCK-8 assay results show that low doses of melatonin upregulate the survival rate of hair follicle stem cells, while high doses have the opposite effect. The knockdown of Foxc1 reverses the inhibitory effect of high-dose melatonin on the survival rate of hair follicle stem cells. Based on these findings, we believe that melatonin-mediated circadian signals exert a bidirectional regulatory effect on the state of HFSCs.

Roles of retinoic acid-related orphan receptor α in high glucose-induced cardiac fibroblasts proliferation

Roles of retinoic acid-related orphan receptor α in high glucose-induced cardiac fibroblasts proliferation

Abstract B006: Targeting REV-ERB/RORα/IRF3 axis to enhance radiation-induced immune response in cancer radiotherapy

Abstract Radiotherapy is an important component of the therapeutics for cancer. Although the primary objective of RT is the local control of tumors, RT-mediated antitumor adaptive immunity is emerging as a promising tool in cancer treatment. Increasing evidence indicates that RT enhances adaptive anti-tumor immunity by augmenting the innate immune sensing of tumors in a type I IFN-dependent matter. Both retinoic acid-related orphan receptor α (RORα) and nuclear heme receptor Rev-erbα, are core components of the circadian rhythm, all have critical roles in inflammation and immune process. Our preliminary data showed that both RORα inverse agonist (antagonist) SR1001 and Rev-erbα agonist GSK4112 significantly enhanced radiation-induced type I IFN activation. We also found that both RORα and Rev-erbα could associate with IRF3 and regulate IRF3 phosphorylation, which enhance radiation-induced type I IFN activation. Furthermore, our data indicated that radiation induced Type I IFN production may depend on circadian cycle, and SR1001 treatment contribute to antitumor immunity of radiation. These results usher the hypothesis that RORα/Rev-erbα work together to regulate radiation-induced type I IFN response, and contribute to antitumor immunity of RT. Specifically, our data shown the critical role of RORα/Rev-erbα/IRF3 axis in regulating radiation-induced type I IFN activation and targeting RORα/Rev-erbα will synergize with radiotherapy. Altogether, the preliminary data generated from this proposal opens additional avenues of research, both preclinically and clinically, to help better understand the relationship of the circadian rhythm proteins on the RT-immune response. Citation Format: Nan LI, Chen Bruan, Steven Hsesheng Lin. Targeting REV-ERB/RORα/IRF3 axis to enhance radiation-induced immune response in cancer radiotherapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B006.

The cyclin-dependent kinase inhibitor AT7519 is a human RORγt agonist.

AT7519, which inhibits multiple cyclin-dependent kinases, has been extensively investigated in various types of cancer cells. Previous studies have demonstrated the ability of this molecule to suppress the expression of the nuclear receptor retinoic acid-related orphan receptor gamma (RORγ) and several genes involved in hepatocellular carcinoma progression. In this study, we identified a distinct agonistic effect of AT7519 on RORγt, an isoform expressed by various immune cells, including T helper 17 lymphocytes. These immune cells play pivotal roles in shaping the tumor microenvironment and promoting the anticancer response of the immune system. After exposure to AT7519 during differentiation, primary human CD4+ Tcells presented increased expression of IL17A/F, IFNG and GZMB and decreased expression of PDCD1 and CTLA4. These findings elucidate a previously unrecognized facet of AT7519 activity and suggest the potential incorporation of this molecule into immune therapies to augment the effectiveness of diverse anticancer strategies involving anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen 4 (anti-CTLA4) regimens.

Phase II Randomized Trial of BI 730357, a Novel Oral RORγt Inhibitor, for Moderate-to-Severe Plaque Psoriasis.

This two-part, double-blinded trial assessed the truncated retinoic acid-related orphan receptor γ (RORγt) inhibitor BI 730357 in plaque psoriasis. Part 1: patients were randomized 2:2:2:2:1 to BI 730357 25, 50, 100, 200 mg, or placebo once daily (qd; fasting conditions); non-responders switched to higher doses. Part 2: a separate patient set was randomized 4:4:1 to BI 730357 400 mg qd, 200 mg twice daily, or placebo (fed conditions). Patients from Parts 1 and 2 could enter a long-term extension (LTE) trial. Co-primary endpoints: ≥75% reduction from baseline in Psoriasis Area Severity Index (PASI 75) and static physician's global assessment (sPGA) score 0/1 (clear/almost clear) at week 12. 274 patients were treated (178 [Part 1]; 96 [Part 2]). Part 1: 12 (30.0%) patients achieved PASI 75 (P=0.0062) and 11 (27.5%) achieved sPGA 0/1 (P=0.0095) with BI 730357 200 mg versus none receiving placebo. Exposure-response plateaued at BI 730357 ≥200 mg qd. Drug-related adverse events occurred in ≤15.8% of patients. Of 165 patients who entered the LTE, 93 (56.4%) achieved PASI 75 during treatment and ≤18.5% experienced a drug-related adverse event. BI 730357 was well-tolerated with moderate efficacy versus placebo in plaque reduction.

Neuro-Behçet's disease: an update of clinical diagnosis, biomarkers, and immunopathogenesis.

Neuro-Behçet's disease (NBD) is a more severe but rare symptom of Behçet's disease, which is mainly divided into parenchymal NBD (p-NBD) involving brain stem, spinal cord, and cerebral cortex. Non-p-NBD manifests as intracranial aneurysm, cerebral venous thrombosis, peripheral nervous system injuries, and mixed parenchymal and non-parenchymal disease. p-NBD is pathologically characterized by perivasculitis presenting with cerebrospinal fluid (CSF) pleocytosis, elevated total protein, and central nervous system (CNS) infiltration of macrophages and neutrophils, which are subdivided into acute and chronic progressive stages according to relapsing-remitting courses and responses to steroids. The diagnosis of NBD depends heavily on clinical features and magnetic resonance imaging (MRI) findings. The lack of laboratory biomarkers has hindered standard diagnostics. CSF interleukin (IL)-6 is the most investigated dimension of NBD and correlates with NBD activity, therapeutic responses, and prognosis. Further investigations have focused on inflammatory biomarkers that reflect the activation of innate and adaptive immune responses. Higher levels of CSF migration inhibitory factor and immunosuppressive acidic protein indicated the activation of macrophages in the CNS; increased IL-17, IL-10, T-bet/GATA-3, and retinoic acid related orphan receptor (ROR)-γt/Foxp3 ratios, marking the disrupted scale of the Th1/Th2 and Th17/Treg axis; and elevated B-cell activating factor of the TNF family (BAFF) and IgA/IgM intrathecal synthesis, suggesting that B cells play a dominant role in NBD. CNS destruction and degeneration as a consequence of neuroinflammatory cascades were confirmed by elevated CSF levels of NFL, β2MG, and MBP. Autoantibodies, including anti-STIP-1, anti-Mtch1, anti-B-Crystallin, and anti-m-Hsp65, provide substantial evidence for autoimmune essence and underlying microbiological infections in NBD immunopathogenesis. We summarized opinions on the clinical diagnosis, biomarkers, and pathological findings of NBD.

JAK/STAT3 signaling promotes pain and depression-like behaviors in rats with bone cancer pain by regulating Th17 cell differentiation.

Pain and depression are common complications in patients with advanced cancer, which significantly affects their quality of life and survival. Dysregulation of the JAK/STAT3 pathway in the central nervous system is associated with pain and brain inflammatory disorders, but its role in bone cancer pain (BCP) remains unclear. This study aimed to investigate the specific role of the JAK/STAT3 pathway in the amygdala in BCP. A BCP rat model was established by intratibial injection of MRMT-1 carcinoma cells. Pain behavior was assessed using the mechanical withdrawal threshold, while depression-like behavior was assessed using the sucrose preference and forced swim test. Changes in inflammatory factors and related protein expression levels in the amygdala were detected using western blotting, immunofluorescence, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of intra-amygdala injections of a lentivirus targeting retinoic acid-related orphan receptor γt (RORγt) (LV-shRORγt), nifuroxazide (a STAT3 antagonist), and colivelin (a STAT3 agonist) were evaluated. Rats with BCP demonstrated increased microglial activation in the amygdala. Rats experiencing RORγt knockout in the amygdala showed reduced microglial activation levels. Nifuroxazide reduced Th17 cell differentiation, potentially alleviating pain and depression-like behaviors. To further explore the underlying relationship between the JAK/STAT3 pathway and Th17 cells, LV-shRORγt and a STAT3 agonist were co-administered. The inhibitory effect of LV-shRORγt counteracted the STAT3 agonist's active effects. Our study showed that targeting JAK/STAT3 signaling alleviated pain- and depression-like behaviors in rats with BCP by inhibiting Th17 cell differentiation.

The Pro-inflammatory Functions of Type 1 CD 8+ T Cells and Interleukin-17-producing Cluster of Differentiation 8+ T Cells are Exhausted by Cholesterol in Atherosclerosis.

CD8+ T cells have been found to accumulate in atherosclerotic plaques. However, the specific role of CD8+ T cell subsets in the development of atherosclerosis is still not fully understood. To investigate the presence and functions of type 1 CD8+ T (Tc1) cells and interleukin-17 (IL-17)-producing CD8+ T (Tc17) cells. Apolipoprotein E-deficient mice were fed a high-fat diet to induce atherosclerosis. Flow cytometry was used to identify and isolate aortic CD8+ T cell subsets, which were then cultured in vitro to assess their pro-inflammatory activities. The cholesterol content of the CD8+ T cell subsets was quantified. T-box expressed in T cells (T-bet)+ Tc1 cells and retinoic acid-related orphan receptor gamma t (RORγt)+ Tc17 cells were found in the atherosclerotic aorta. Aortic CD8+ T cells showed lower pro-inflammatory activity compared to splenic counterparts, with less interferon-gamma (IFN-γ) (P <0.01) and tumor necrosis factor-alpha (TNF-α) production (P <0.01). Surprisingly, aortic CD8+ T cells expressed little IL-17 and interleukin-21 (IL-21) despite the presence of Tc17 cells. Aortic Tc1 and Tc17 cells expressed high levels of 2B4 and programmed cell death protein 1 (PD-1). Furthermore, aortic Tc1 and Tc17 cells had higher cholesterol contents than splenic CD8+ T cells (P <0.05, respectively). Cholesterol treatment decreased IFN-γ expression in Tc1 cells (P <0.001) and reduced IL-17 expression in Tc17 cells (P <0.001). Additionally, cholesterol up-regulated 2B4 and PD-1 on Tc1 (P <0.001) and Tc17 cells (P <0.001). Aortic CD8+ T cells, particularly aortic Tc17 cells, are functionally exhausted in atherosclerosis, possibly due to the influence of cholesterol.

Retinoic Acid-Related Orphan Receptor Alpha May Regulate the State of Hair Follicle Stem Cells by Upregulating the Expression of BNIP3.

The hair, an exclusive keratinized dermal appendage in mammals, stands as a quintessential outcome of adaptive evolution, conferring resilience against adverse environmental conditions. The ontogenesis of the coat displays a pronounced rhythmic pattern, with hair follicle stem cells (HFSCs) emerging as pivotal facilitators of hair follicle reconstitution. The retinoic acid-related orphan receptor alpha, a nuclear receptor with extensive involvement in the regulation of cellular physiological states, exerts its functions predominantly through the modulation of downstream target gene transcription. The Bnip3 gene exhibits a robust correlation with cellular apoptosis and autophagy, which are indispensable physiological mechanisms underlying the maintenance of HFSC homeostasis. Consequently, the expression level of Bnip3 may be intimately linked to the status of HFSCs. In this investigative endeavor, we employed rat HFSCs as a model system to validate the regulatory impact of RORA on Bnip3 gene expression. Our findings unequivocally demonstrate that Bnip3 serves as a direct downstream target of RORA. Specifically, RORA binds to the motif within the Bnip3 promoter region, thereby upregulating Bnip3 expression levels. In light of our research findings, we propose that RORA holds potential as a target for modulating the status of HFSCs.

Discovery of 1-(Phenylsulfonyl)-1,2,3,4-tetrahydroquinoline Derivative as Orally Bioavailable and Safe RORγt Inverse Agonists for Potential Treatment of Rheumatoid Arthritis.

The retinoic acid receptor-related orphan receptor γt (RORγt) is a key regulator of Th17 cells, associated with autoimmune diseases, making it a significant drug target. Herein, we designed and synthesized 1-(phenylsulfonyl)-1,2,3,4-tetrahydroquinoline derivatives, improving upon GSK2981278 to enhance bioavailability. Of which, D4 exhibited superior bioavailability (F = 48.1 and 32.9% in mice and rats, respectively) compared to GSK2981278 (F = 6.2 and 4.1%, respectively), and effectively treated psoriasis in mice at lower doses. Moreover, for the first time, we report the efficacies of a RORγt inverse agonist in mouse models of rheumatoid arthritis. (R)-D4, the eutomer of D4, matched or exceeded GSK2981278's therapeutic effects at lower doses, with no adverse effects observed after 2 weeks of administration. These results position (R)-D4 as a promising and orally bioavailable candidate for Th17-mediated autoimmune disease treatment.

NR1D1 activation alleviates inflammatory response through inhibition of IL-6 expression in bovine endometrial epithelial cells

Endometritis, an inflammatory disease affecting dairy cattle, causes substantial economic losses in the dairy industry. Conventional treatment using uterine infusion of antibiotics often results in bacterial resistance and antibiotic residues in milk. Thus, identifying novel, effective therapeutic targets for endometritis in dairy cows is necessary. Nuclear receptor subfamily 1 group D member 1 (NR1D1) activation attenuates inflammatory responses in various diseases through transcriptional repression; however, its role in treating bovine endometritis remains unclear. This study investigated the role and underlying mechanisms of NR1D1 in endometritis using a bovine endometrial epithelial cell line (BENDs) and primary bovine endometrial epithelial cells, both induced with Escherichia coli lipopolysaccharide (LPS). Immunofluorescence staining revealed the predominant nuclear localization of NR1D1 in endometrial epithelial cells. LPS treatment (1 μg/mL for 12 h) significantly increased the expression levels of NR1D1 and proinflammatory cytokines (IL-6, IL-1β, IL-8, and CCL5) in BENDs. Immunohistochemical staining showed elevated NR1D1 expression in uterine tissues of cows with endometritis. Deletion of NR1D1 significantly increased IL-6 mRNA expression; NR1D1 overexpression substantially repressed IL-6 expression in BENDs. NR1D1 agonist SR9009 attenuated LPS-induced mRNA expression of proinflammatory cytokines (IL-6, IL-1β, CCL5) in both BENDs and primary endometrial epithelial cells. Additionally, SR9009 treatment attenuated LPS-induced inflammatory responses in the endometrium of mice. Dual-luciferase reporter assays and real-time monitoring via luminescence assays showed that NR1D1 overexpression significantly repressed luciferase activity driven by the IL-6 promoter region, which was abolished by deletion of the retinoic acid receptor-related orphan receptor-responsive element (−473 to −479) within the IL-6 promoter fragment. In summary, NR1D1 activation alleviates the inflammatory response of BENDs by repressing the expression of proinflammatory cytokines, at least partly via IL-6, suggesting NR1D1 is a promising therapeutic target for endometritis prevention and treatment.

Effects of different monochromatic light on growth performance and liver circadian rhythm of Yangzhou geese

The objective of this study is to examine the impact of monochromatic light on the circadian rhythms of blood melatonin and insulin-like growth factor 1 (IGF-1) levels, liver clock genes, and melatonin receptors. A total of 144 male Yangzhou geese were randomly assigned to four groups based on light color, with each group consisting of 36 geese. The geese were provided with ad libitum access to food and water, and were raised for 70 days under a photoperiod of 16 hours of light and 8 hours of darkness. They were weighed every 10 days, and blood, liver, and pituitary gland samples were collected at six time points every four hours when the geese reached 70 days of age. The findings indicated that exposure to green light (GL) had a stimulating impact on weight gain in Yangzhou geese, while not significantly affecting the feed-to-weight ratio. After undergoing the four photochromic treatments, both plasma melatonin levels and liver positive feedback clock gene expression displayed a diurnal low-night high pattern, reaching their peak in the evening. Conversely, plasma IGF-1 and negative feedback clock genes exhibited an opposite trend. However, monochromatic light significantly down-regulated the gene expression, peak and amplitude of retinoic acid receptor-related orphan receptor α (RORα), as well as advancing or delaying the phase of the circadian rhythm. Among them, GL significantly up-regulated the gene expression of the melatonin receptors 1C (MEL1C); blue light (BL) significantly increased plasma melatonin concentration and IGF-1 concentration and significantly decreased the peak and amplitude of period 3 gene (PER3), resulting in almost no difference in the expression of PER3 at the six times; and red light (RL) significantly down-regulated the expression and the peak of MEL1C as well as the peaks and amplitudes of the seven liver clock genes, especially circadian locomotor output cycles kaput factor (CLOCK). Moreover, the peaks and amplitudes of the clock genes for BL and GL are almost identical, except for PER3. The findings of this study offer a theoretical framework for facilitating the development of meat geese and implementing a logical approach to managing lighting conditions.

A High-Quality Photoswitchable Probe that Selectively and Potently Regulates the Transcription Factor RORγ.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a nuclear hormone receptor with multiple biological functions in circadian clock regulation, inflammation, and immunity. Its cyclic temporal role in circadian rhythms, and cell-specific activity in the immune system, make it an intriguing target for spatially and temporally localised pharmacology. To create tools that can study RORγ biology with appropriate spatiotemporal resolution, we designed light-dependent inverse RORγ agonists by building azobenzene photoswitches into ligand consensus structures. Optimizations gave photoswitchable RORγ inhibitors combining a large degree of potency photocontrol, with remarkable on-target potency, and excellent selectivity over related off-target receptors. This still rare combination of performance features distinguishes them as high quality photopharmaceutical probes, which can now serve as high precision tools to study the spatial and dynamic intricacies of RORγ action in signaling and in inflammatory disorders.

The Role of Retinoic-Acid-Related Orphan Receptor (RORs) in Cellular Homeostasis.

Retinoic-acid-related orphan receptors (RORs) are transcription factors belonging to the nuclear receptor subfamily consisting of RORα, RORβ, and RORγ. By binding to the ROR response elements (ROREs) on target gene promoters, RORs regulate a wide variety of cellular processes, including autophagy, mitophagy, oxidative stress, and inflammation. The regulatory roles of RORs are observed in cardiac cells, hepatocytes, pulmonary epithelial cells, renal cells, immune cells, and cancer cells. A growing body of clinical and experimental evidence suggests that ROR expression levels are markedly reduced under different pathological and stress conditions, suggesting that RORs may play a critical role in the pathogenesis of a variety of disease states, including myocardial infarction, immune disorders, cancer, and metabolic syndrome. Reductions in RORs are also associated with inhibition of autophagy, increased reactive oxygen species (ROS), and increased cell death, underscoring the importance of RORs in the regulation of these processes. Herein, we highlight the relationship between RORs and homeostatic processes that influence cell viability. Understanding how these intricate processes are governed at the cellular level is of high scientific and clinical importance to develop new therapeutic strategies that modulate ROR expression and disease progression.

Unraveling the long-term gastrointestinal impact of perinatal perfluorobutane sulfonate exposure on rat offspring: Intestinal barrier dysfunction and Th17/Treg imbalance

Per- and polyfluoroalkyl substances (PFAS), especially long-chain perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), are increasingly acknowledged as a potential inflammatory bowel diseases (IBD) risk factor. Perfluorobutane sulfonate (PFBS), one kind of shorter chain alternative, has been reported to exhibit similar health hazards to those long-chain PFAS. However, the underlying mechanism underpinning PFBS-induced colonic inflammation has not been sufficiently elucidated. The T-helper-17 (Th17)/regulatory T (Treg) imbalance is a crucial event for the pathogenesis of colonic inflammation. In this study, we aimed to reveal whether and how perinatal PFBS exposure leads to the Th17/Treg imbalance and colonic inflammation in offspring. We firstly demonstrated in vivo that early-life PFBS exposure (0.5 mg/kg, 5 mg/kg) led to increased intestinal permeability and colonic inflammation accompanied by decreased expressions of tight junction protein 1 (Tjp1) and claudin-4 (Cldn4) and increased expressions of interleukin 17A (IL-17A) in colon of rat offspring. Further results indicated that PFBS exposure induces the Th17/Treg imbalance through upregulating the expression of retinoic acid receptor-related orphan receptor gamma t (Ror-γt) and transforming growth factor beta (TGF-β) and downregulating of forkhead box protein 3 (Foxp3) and IL-10 in colon. Moreover, metabolomics analyses indicated that bile secretion metabolism was significantly altered under PFBS exposure. The reduction of lithocholic acid and deoxycholic acid was closely related to the changes of TGF-β and IL-10 in colon, and may contribute to the perturbation of Th17/Treg balance and colonic inflammation. These results provide evidences for the immunotoxicity of PFBS and reveal the potential contribution to colonic inflammation, which raises concern on the health effects and risk assessment of short-chain PFAS.

Orally bioavailable RORγ/DHODH dual host-targeting small molecules with broad-spectrum antiviral activity

Host-directed antivirals (HDAs) represent an attractive treatment option and a strategy for pandemic preparedness, especially due to their potential broad-spectrum antiviral activity and high barrier to resistance development. Particularly, dual-targeting HDAs offer a promising approach for antiviral therapy by simultaneously disrupting multiple pathways essential for viral replication.Izumerogant (IMU-935) targets two host proteins, (i) the retinoic acid receptor-related orphan receptor γ isoform 1 (RORγ1), which modulates cellular cholesterol metabolism, and (ii) the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in de novo pyrimidine synthesis. Here, we synthesized optimized derivatives of izumerogant and characterized their antiviral activity in comparison to a recently described structurally distinct RORγ/DHODH dual inhibitor. Cell culture-based infection models for enveloped and non-enveloped DNA and RNA viruses, as well as a retrovirus, demonstrated high potency and broad-spectrum activity against human viral pathogens for RORγ/DHODH dual inhibitors at nanomolar concentrations. Comparative analyses with equipotent single-target inhibitors in metabolite supplementation approaches revealed that the dual-targeting mode represents the mechanistic basis for the potent antiviral activity. For SARS-CoV-2, an optimized dual inhibitor completely blocked viral replication in human airway epithelial cells at 5 nM and displayed a synergistic drug interaction with the nucleoside analog molnupiravir. In a SARS-CoV-2 mouse model, treatment with a dual inhibitor alone, or in combination with molnupiravir, reduced the viral load by 7- and 58-fold, respectively.Considering the clinical safety, oral bioavailability, and tolerability of izumerogant in a recent Phase I study, izumerogant-like drugs represent potent dual-targeting antiviral HDAs with pronounced broad-spectrum activity for further clinical development.

UBE2T promotes stage I lung adenocarcinoma progression through PBX1 ubiquitination and PBX1/RORA regulation

BackgroundPost-translational modification pathway of protein ubiquitination is intricately associated with tumorigenesis. We previously reported elevated ubiquitin-conjugating enzyme 2T (UBE2T) as an independent risk factor in stage I lung adenocarcinoma and promoting cellular proliferation. However, its underlying mechanisms needed further investigation.MethodsImmunohistochemistry was used to assess the expression of UBE2T and retinoic acid receptor-related orphan receptor α (RORA) in stage I LUAD. Cell proliferation, migration, and invasion of LUAD cell lines were measured by Cell Counting Kit-8 assay (CCK-8), Colony-forming assay and Transwell assay, respectively. Western blot analysis was performed to determine the expression of epithelial-mesenchymal transition (EMT) markers. A xenograft model was established to evaluate the proliferative capacity of UBE2T and its interaction with RORA in promoting LUAD. Mechanistic insights into the promotion of early-stage LUAD by UBE2T were obtained through luciferase reporter assay, chromatin immunoprecipitation and co-immunoprecipitation.ResultsUBE2T and RORA expression was significantly up- and down-regulated in early-stage LUAD patients which’s proved to be associated with unfavorable outcomes, strengthened cell proliferation, migration, EMT and invasion through its interaction with RORA both in vivo and in vitro. The growth NSCLC xenografts was reduced by down-expression of UBE2T but was suppressed by RORA knockout. Mechanistically, UBE2T mediated the ubiquitination of the intermediate transcription factor PBX1, which played a transcriptional role in downstream regulation of RORA.ConclusionThe oncogenic role of UBE2T and the UBE2T-PBX1-RORA axis in driving malignant progression in Stage I LUAD had been established. UBE2T might be a novel and promising therapeutic target for LUAD treatment.

Exosomal miR-205–5p contributes to the immune liver injury induced by trichloroethylene: Pivotal role of RORα mediating M1 Kupffer cell polarization

Trichloroethylene (TCE) is a common environmental contaminant that can induce occupational dermatitis medicamentosa-like TCE (ODMLT), where the liver damage is the most common complication. The study aims to uncover the underlying mechanism of TCE-sensitization-induced liver damage by targeting specific exosomal microRNAs (miRNAs). Among the enriched serum exosomal miRNAs of ODMLT patients, miR-205–5p had a significant correlation coefficient with the liver function damage indicators. Moreover, retinoic acid receptor-related orphan receptor α (RORα) was identified as a direct target of miR-205–5p via specific binding. Further experiments showed that kupffer cells (KCs) underwent M1 phenotypic and functional changes in liver injury induced by TCE which were alleviated by reducing the expression of miR-205–5p. However, this alleviation was reversed by the RORα antagonist SR1001. In vitro experiments showed that miR-205–5p promoted M1 polarization of macrophages and enhanced the secretion of inflammatory factors by regulating RORα. An increase in RORα reversed the polarization direction of M1-type macrophages and reduced the secretion of proinflammatory factors. In addition, pretreatment of mice with SR1078, a specific RORα agonist, effectively blocked M1 polarization of KCs and reduced the severity of TCE-induced liver injury. Our study uncovers that miR-205–5p regulates KC M1 polarization by targeting RORα in immune liver injury induced by TCE sensitization, providing new insight into the molecular mechanisms and new therapeutic targets for ODMLT.

A SAR Study on a Class of 6-(Trifluoromethyl)-pyridine Derivatives as RORγt Inverse Agonists

Background: The nuclear retinoic acid-related orphan receptor γt (RORγt) is an important transcription factor in immune cells. Functionally, RORγt plays an important role in promoting the differentiation of T helper 17 cells and regulating the expression of proinflammatory factors, such as interleukin 17. Therefore, RORγt is considered a promising target for the treatment of the autoimmune disorder. Currently, 21 RORγt inverse agonists with various scaffolds have entered clinical trials. Objective: To discover novel and potent RORγt inverse agonists, a series of novel 6-(trifluoromethyl) pyridine derivatives were designed and synthesized. Methods: We designed and synthesized a series of potent RORγt inverse agonists W1~W16 based on VTP-43742. Molecular docking, molecular dynamics (MD) simulation, and MM/GBSA were used to study the structure-activity relationship (SAR) of the derivatives. Results: The biological activity evaluation indicated that the target compounds showed potent RORγt inhibitory activity. The most active compound, W14, exhibited low nanomolar inhibitory activity (IC50 = 7.5 nM) in the luciferase reporter assay, which was superior to the clinical compound VTP-43742. Analysis of the binding mode of W14 demonstrated that the interaction of -CF3 with Leu324, Leu396, and His479 has an important contribution to the binding. Furthermore, W14 broke the H-bond formed by His479 and Tyr502 via a “push-pull” mechanism. Conclusion: Compound W14 could be used as a potential RORγt inverse agonist for further modification.

MiR-143-5p regulates the proangiogenic potential of human dental pulp stem cells by targeting HIF-1α/RORA under hypoxia: A laboratory investigation in pulp regeneration.

Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process. Human dental pulp stem cells were isolated, cultured and characterized invitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured invitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration. Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The invivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis invitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration. CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA.