miR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

Mechanism of lncRNA HOTAIR in attenuating cardiomyocyte pyroptosis in mice with heart failure via the miR-17-5p/RORA axis.

Heart failure (HF) is a complex clinical syndrome associated with significant morbidity and mortality. Dysregulation of long non-coding RNA (lncRNA) has been implicated in the pathogenesis of HF. The present study aims to investigate the role of lncRNA HOX transcript antisense RNA (HOTAIR) in cardiomyocyte pyroptosis in a murine HF model. A murine HF model was established through transverse aortic contraction surgery, and an in vitro HF cell model was developed by treating HL-1 cells with H2O2. HOTAIR was overexpressed in TAC mice and HL-1 cells via pcDNA3.1-HOTAIR transfection. Cardiac function was assessed in TAC mice, and myocardial changes were evaluated using HE staining. The expression of NLRP3 was examined by immunohistochemistry. Myocardial injury markers and pyroptosis-related inflammatory cytokines were quantified using ELISA. Protein levels of NLRP3, cleaved-caspase-1, and GSDMD-N were analyzed by Western blot. Dual-luciferase assays and RNA immunoprecipitation were employed to confirm the binding interactions between HOTAIR and miR-17-5p, miR-17-5p and RORA. Functional rescue experiments were conducted by overexpressing miR-17-5p or silencing RORA in HL-1 cells. HOTAIR exhibited reduced expression in TAC mice and H2O2-induced cardiomyocytes. Overexpression of HOTAIR ameliorated cardiac dysfunction, reduced myocardial pathological injury, enhanced cardiomyocyte viability, and decreased myocardial injury and pyroptosis. HOTAIR interacted with miR-17-5p to repress RORA transcription. Overexpression of miR-17-5p or silencing of RORA abolished the inhibitory effect of HOTAIR overexpression on cardiomyocyte pyroptosis. In conclusion, HOTAIR competitively bound to miR-17-5p, relieving its inhibition of RORA transcription and leading to increased RORA expression and suppressed cardiomyocyte pyroptosis in HF models.

The role of retinoid-related orphan receptor-α in cigarette smoke-induced autophagic response.

BACKGROUND: Retinoid-related orphan receptor-α (RORα) and autophagy dysregulation are involved in the pathophysiology of chronic obstructive pulmonary disease (COPD), but little is known regarding their association. We investigated the role of RORα in COPD-related autophagy. METHODS: The lung tissues and cells from a mouse model were analyzed for autophagy markers by using western blot analysis and transmission electron microscopy. RESULTS: Cigarette smoke increased the LC3-II level and decreased the p62 level in whole lung homogenates of a chronic cigarette smoking mouse model. Although cigarette smoke did not affect the levels of p62 in Staggerer mutant mice (RORαsg/sg), the baseline expression levels of p62 were significantly higher than those in wild type (WT) mice. Autophagy was induced by cigarette smoke extract (CSE) in Beas-2B cells and in primary fibroblasts from WT mice. In contrast, fibroblasts from RORαsg/sg mice failed to show CSE-induced autophagy and exhibited fewer autophagosomes, lower LC3-II levels, and higher p62 levels than fibroblasts from WT mice. Damage-regulated autophagy modulator (DRAM), a p53-induced modulator of autophagy, was expressed at significantly lower levels in the fibroblasts from RORαsg/sg mice than in those from WT mice. DRAM knockdown using siRNA in Beas-2B cells inhibited CSE-induced autophagy and cell death. Furthermore, RORα co-immunoprecipitated with p53 and the interaction increased p53 reporter gene activity. CONCLUSIONS: Our findings suggest that RORα promotes autophagy and contributes to COPD pathogenesis via regulation of the RORα-p53-DRAM pathway.

Retinoid orphan nuclear receptor alpha (RORα) suppresses the epithelial-mesenchymal transition (EMT) by directly repressing Snail transcription.

Retinoid orphan nuclear receptor alpha (RORα) is a member of the orphan nuclear factor family and regulates gene expression by binding to ROR response elements (ROREs). RORα has been identified as a potential tumor suppressor; however, how downregulation of RORα promotes cancer progression is not fully understood. Here, we showed that protein levels of RORα were downregulated during the Snail-, Twist-, or transforming growth factor-ß-induced epithelial-mesenchymal transition (EMT). We found that silencing of RORα induced expression of mesenchymal markers in MCF10A cells, accompanied by enhanced cell invasion, migration, and mammosphere formation. Furthermore, ectopic expression of RORα suppressed transforming growth factor-ß-induced EMT processes in MCF10A and HMLE cells. These results indicate that downregulation of RORα is crucial for the induction of EMT in mammary epithelial cells. By analyzing gene expression profiles in control and RORα-expressing cells, we also identified Snail, a key regulator of EMT, as a potential target of RORα. We show that RORα expression significantly inhibits Snail transcription in breast cancer cells. Chromatin immunoprecipitation analysis demonstrated that RORα bound to the ROREs in promoter region of SNAI1 gene, and using the luciferase reporter assay, we showed that binding to the ROREs was critical for RORα to repress Snail transcription. Finally, rescue experiments substantiated that Snail mediates RORα function in suppressing EMT and mammosphere formation. These results reveal a novel function of RORα in suppressing EMT and identify Snail as a direct target of RORα in mammary epithelial cells.

Hsa-miR-1290 is associated with stemness and invasiveness in prostate cancer cell lines by targeting RORA.

This work examined microRNA-1290 (miR-1290)'s effect on regulating the malignant phenotype of prostate cancer (PC) cells. We detected miR-1290 expression within PC based on open-sourced datasets as well as in cancer cells and tissues. Loss-of-function experiments by miR-1290 knockdown in PC cell lines were performed. We performed CCK-8, clone forming, Transwell, and sphere formation assays for examining PC cells' malignant phenotypes following miR-1290 knockdown. We estimated miR-1290's target genes using online resources including miRDB, miRbase, miRTarBase and TargetScan. We also performed in vivo studies for validating how miR-1290 affected tumour formation within the mouse model. According to findings in this work, miR-1290 showed overexpression within PC cells and tissues. miR-1290 was indispensable for PC cell growth, stemness and invasion as well as mesenchymal status. Further, we identified RORA (retinoic acid receptor-related orphan receptor A) as miR-1290's target gene for mediating miR-1290 within PC cells. To sum up, this work suggests that miR-1290 up-regulation enhances PC cell growth and invasion by regulating RORA expression.

Maternal diabetes-mediated RORA suppression contributes to gastrointestinal symptoms in autism-like mouse offspring.

BACKGROUND: Retinoic acid-related orphan receptor alpha (RORA) has been reported to be suppressed in autistic patients and is associated with autism spectrum disorders (ASD), although the potential role and mechanism of RORA on gastrointestinal (GI) symptoms in ASD patients is still not reported. In this study, we aim to investigate the contribution of RORA to GI symptoms through a maternal diabetes-mediated autism-like mouse model. RESULTS: Male offspring of diabetic dams were treated with either superoxide dismutase (SOD) mimetic MnTBAP or RORA agonist SR1078, or were crossbred with intestine epithelial cells (IEC)-specific RORA knockout (RORA-/-) mouse. Gene expression, oxidative stress and inflammation were measured in brain tissues, peripheral blood mononuclear cells (PBMC) and IEC, and GI symptoms were evaluated. Our results showed that SOD mimetic MnTBAP completely, while RORA agonist SR1078 partly, reversed maternal diabetes-mediated oxidative stress and inflammation in the brain, PBMC and IEC, as well as GI symptoms, including intestine permeability and altered gut microbiota compositions. IEC-specific RORA deficiency either mimicked or worsened maternal diabetes-mediated GI symptoms as well as oxidative stress and inflammation in IEC, while there was little effect on maternal diabetes-mediated autism-like behaviors. CONCLUSIONS: We conclude that RORA suppression contributes to maternal diabetes-mediated GI symptoms in autism-like mouse offspring, this study provides a potential therapeutical target for maternal diabetes-mediated GI symptoms in offspring through RORA activation.

RORA alleviates LPS-induced apoptosis of renal epithelial cells by promoting PGC-1α transcription.

OBJECTIVE: To explore the effect of RORA on LPS-induced renal epithelial cell apoptosis and the underlying mechanism. METHODS: LPS-treated HK-2 cells were established as a cellular model of acute kidney injury. The expression of RORA or/and PGC-1α in LPS-induced HK-2 cells was altered by transfection. qRT-PCR and Western blotting were used to detect the expression changes of RORA and PGC-1α. ELISA was performed to detect the expression of IL-1ß and IL-6 and the activity of caspase-3. Western blotting was applied for visualization of cleaved caspase-3. CCK-8 and flow cytometry were used to assess cell proliferation and apoptosis. Dual-luciferase reporter and ChIP-qPCR were utilized to verify the binding of RORA to PGC-1α promoter. RESULTS: LPS treatment decreased the expression of RORA and PGC-1α and increased that of cleaved caspase-3 in HK-2 cells. Also, LPS treatment inhibited HK-2 cell proliferation and promoted HK-2 cell apoptosis and secretion of IL-1ß and IL-6. Overexpression of RORA or PGC-1α eliminated the adverse effects of LPS treatment in HK-2 cells. RORA drove the transcription of PGC-1α by binding PGC-1α promoter. Knockdown of PGC-1α offset the reduction in HK-2 cell injury caused by overexpression of RORA. CONCLUSION: RORA reduces LPS-induced apoptosis of renal epithelial cells by promoting PGC-1α transcription.

RORα contributes to the maintenance of genome ploidy in the liver of mice with diet-induced nonalcoholic steatohepatitis.

Hepatic polyploidization is closely linked to the progression of nonalcoholic fatty liver disease (NAFLD); however, the underlying molecular mechanism is not clearly understood. In this study, we demonstrated the role of retinoic acid-related orphan receptor α (RORα) in the maintenance of genomic integrity, particularly in the pathogenesis of NAFLD, using the high-fat diet (HFD)-fed liver-specific RORα knockout (RORα-LKO) mouse model. First, we observed that the loss of hepatic retinoic acid receptor-related orphan receptor α (RORα) accelerated hepatocyte nuclear polyploidization after HFD feeding. In 70% partial hepatectomy experiments, enrichment of hepatocyte polyploidy was more obvious in the RORα-LKO animals, which was accompanied by early progression to the S phase and blockade of the G2/M transition, suggesting a potential role of RORα in suppressing hepatocyte polyploidization in the regenerating liver. An analysis of a publicly available RNA sequencing (RNA-seq) and chromatin immunoprecipitation-seq dataset, together with the Search Tool of the Retrieval of Interacting Genes/Proteins database resource, revealed that DNA endoreplication was the top-enriched biological process Gene Ontology term. Furthermore, we found that E2f7 and E2f8, which encode key transcription factors for DNA endoreplication, were the downstream targets of RORα-induced transcriptional repression. Finally, we showed that the administration of JC1-40, an RORα activator (5 mg/kg body wt), significantly reduced hepatic nuclear polyploidization in the HFD-fed mice. Together, our observations suggest that the RORα-induced suppression of hepatic polyploidization may provide new insights into the pathological polyploidy of NAFLD and may contribute to the development of therapeutic strategies for the treatment of NAFLD.NEW & NOTEWORTHY It has been reported that hepatic polyploidization is closely linked to the progression of NAFLD. Here, we showed that the genetic depletion of hepatic RORα in mice accelerated hepatocyte polyploidization after high-fat diet feeding. The mechanism could be the RORα-mediated repression of E2f7 and E2f8, key transcription factors for DNA endoreplication. Thus, preservation of genome integrity by RORα could provide a new insight for developing therapeutics against the disease.

RORα and REV-ERBα are Associated With Clinicopathological Parameters and are Independent Biomarkers of Prognosis in Gastric Cancer.

Retinoid-related orphan receptor alpha (RORα) and nuclear receptor subfamily 1 group D member 1 (REV-ERBα) play critical roles in many human cancers. Whether RORα and REV-ERBα expression levels are associated with clinical characteristics are poorly understood, and they may be independent predictors of overall survival (OS) and progression-free survival (PFS) in gastric cancer (GC). This study aimed to investigate the correlation of RORα and REV-ERBα expression levels with clinicopathological parameters, OS, and PFS in GC. Immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were employed to assess the expression levels of RORα and REV-ERBα, which were downregulated in GC tissues compared with normal gastric tissues (P < .001; P < .001) and were associated with several clinicopathological parameters, including histological grade (P = .032; P < .001), preoperative carcinoembryonic antigen (CEA) levels (P = .004; P < .001), and tumor-node-metastasis (TNM) stage (P = .015; P < .001). Additionally, low RORα and REV-ERBα expression levels were associated with poor OS and PFS in GC patients, respectively (P < .001; P = .001). Furthermore, univariate Cox regression model analysis showed that histological grade (P < .001; P < .001), preoperative CEA levels (P < .001; P = .001), TNM stage (P < .001; P < .001), lymph node metastasis (P = .002; P = .002), RORα expression levels (P = .001; P < .001), and REV-ERBα expression levels (P < .001; P = .001) were associated with OS and PFS in GC. Multivariate Cox regression model analysis indicated that RORα expression levels and REV-ERBα expression levels are independent factors of OS and PFS in GC. Besides, RORα and REV-ERBα expression may be positively correlated (χ2 = 6.835; P = .009), and GC patients with both high RORα and REV-ERBα expression levels had the best prognosis. In conclusion, RORα and REV-ERBα may coparticipate in tumor activities and show potential to estimate the prognosis of GC.

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.

Identification of the RORα Transcriptional Network Contributes to the Search for Therapeutic Targets in Atherosclerosis.

The retinoic acid receptor-related orphan receptor α (RORα) is involved in the regulation of several physiological processes, including development, metabolism, and circadian rhythm. RORα-deficient mice display profound atherosclerosis, in which hypoalphalipoproteinemia is reportedly associated with decreased plasma levels of high-density lipoprotein, increased levels of inflammatory cytokines, and ischemia/reperfusion-induced damage. The recent characterization of endogenous ligands (including cholesterol, oxysterols, provitamin D3, and their derivatives), mediators, and initiation complexes associated with the transcriptional regulation of these orphan nuclear receptors has facilitated the development of synthetic ligands. These findings have also highlighted the potential of application of RORα as a therapeutic target for several diseases, including diabetes, dyslipidemia, and atherosclerosis. In this review, the current literature related to the structure and function of RORα, its genetic inter-individual differences, and its potential as a therapeutic target in atherosclerosis is discussed.

RORα Suppresses Cancer-Associated Inflammation by Repressing Respiratory Complex I-Dependent ROS Generation.

Breast cancer development is associated with macrophage infiltration and differentiation in the tumor microenvironment. Our previous study highlights the crucial function of reactive oxygen species (ROS) in enhancing macrophage infiltration during the disruption of mammary tissue polarity. However, the regulation of ROS and ROS-associated macrophage infiltration in breast cancer has not been fully determined. Previous studies identified retinoid orphan nuclear receptor alpha (RORα) as a potential tumor suppressor in human breast cancer. In the present study, we showed that retinoid orphan nuclear receptor alpha (RORα) significantly decreased ROS levels and inhibited ROS-mediated cytokine expression in breast cancer cells. RORα expression in mammary epithelial cells inhibited macrophage infiltration by repressing ROS generation in the co-culture assay. Using gene co-expression and chromatin immunoprecipitation (ChIP) analyses, we identified complex I subunits NDUFS6 and NDUFA11 as RORα targets that mediated its function in suppressing superoxide generation in mitochondria. Notably, the expression of RORα in 4T1 cells significantly inhibited cancer metastasis, reduced macrophage accumulation, and enhanced M1-like macrophage differentiation in tumor tissue. In addition, reduced RORα expression in breast cancer tissue was associated with an increased incidence of cancer metastasis. These results provide additional insights into cancer-associated inflammation, and identify RORα as a potential target to suppress ROS-induced mammary tumor progression.

MeCP2 duplication causes hyperandrogenism by upregulating LHCGR and downregulating RORα.

Duplication of MECP2 (methyl-CpG-binding protein 2) gene causes a serious neurological and developmental disorder called MECP2 duplication syndrome (MDS), which is usually found in males. A previous clinical study reported that MDS patient has precocious puberty with hyperandrogenism, suggesting increased MeCP2 may cause male hyperandrogenism. Here we use an MDS mouse model and confirm that MECP2 duplication significantly upregulates androgen levels. We show for the first time that MeCP2 is highly expressed in the Leydig cells of testis, where androgen is synthesized. Mechanistically, MECP2 duplication increases androgen synthesis and decreases androgen to estrogen conversion through either the upregulation of luteinizing hormone receptor (LHCGR) in testis, as a result of MeCP2 binds to G-quadruplex structure of Lhcgr promoter and recruits the transcription activator CREB1 or the downregulation of the expression of aromatase in testis by binding the CpG island of Rorα, an upstream regulator of aromatase. Taken together, we demonstrate that MeCP2 plays an important role in androgen synthesis, supporting a novel non-CNS function of MeCP2 in the process of sex hormone synthesis.

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.

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.

Sevoflurane blocks glioma malignant development by upregulating circRELN through circRELN-mediated miR-1290/RORA axis.

BACKGROUND: Sevoflurane (Sev) has been reported to inhibit cancer development, and sevoflurane treatment in cancers is implicated with the deregulation of specific non-coding RNAs (ncRNAs). This study aimed to investigate the relationship between sevoflurane and circular RNA reelin (circRELN) in glioma. METHODS: The expression of circRELN, microRNA-1290 (miR-1290) and RAR-related orphan receptor A (RORA) was measured by quantitative real-time PCR (qPCR). Cell proliferative capacity was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell apoptosis and cell cycle distribution were monitored by flow cytometry assay. Cell migration was assessed by wound healing assay and transwell assay, and cell invasion was assessed by transwell assay. The protein levels of matrix metalloproteinase-2 (MMP2), MMP9 and RORA were quantified by western blot. Tumor growth in vivo was assessed by Xenograft models. The binding relationship between miR-1290 and circRELN or RORA was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: We found that circRELN expression was declined in glioma tissues and cells, while Sev treatment enhanced circRELN expression. In function, Sev notably inhibited glioma cell proliferation, migration and invasion and promoted apoptosis and cell cycle arrest, while circRELN knockdown reversed these effects. MiR-1290 served as a target of circRELN, and glioma cell malignant phenotypes recovered by circRELN knockdown were partly repressed by miR-1290 deficiency. In addition, RORA was a target of miR-1290, and glioma cell malignant phenotypes promoted by miR-1290 restoration were partly blocked by RORA overexpression. CircRELN regulated RORA expression by targeting miR-1290. In Xenograft models, Sev inhibited tumor growth by upregulating circRELN. CONCLUSION: Sev blocked the progression of glioma by increasing circRELN expression, and circRELN played roles in glioma partly by regulating the miR-1290/RORA network.

RORα Enhances Lysosomal Acidification and Autophagic Flux in the Hepatocytes.

Lysosomes are intracellular acidic organelles with catabolic functions that contribute to the activation of autophagy. Although autophagy abnormality is associated with defects in lysosomal acidification during the progression of nonalcoholic fatty liver disease (NAFLD), the mechanisms of control of lysosomal acidification are not well understood at the molecular level. Thus, we aimed to elucidate the role of the orphan nuclear receptor retinoic acid-related orphan receptor α (RORα) in lysosomal acidification and autophagic flux, particularly in nutrition-enriched hepatocytes. First, lysosomal acidity was much lower in the hepatocytes obtained from hepatocyte-specific RORα-deleted (RORα-LKO) mice, whereas the infusion of an adenovirus encoding RORα in wild-type hepatocytes increased lysosomal acidity, as determined by LysoSensor. Second, the lysosomal translocation of the mechanistic target of rapamycin was increased and immature cathepsin D was accumulated in the liver of RORα-LKO mice. Third, the accumulation of LC3-II, p62/sequestosome 1 (SQSTM1), and neighbor of BRCA1 gene 1 (NBR1) was increased in the livers of RORα-LKO mice, indicating an impaired autophagic flux in the livers. Consistently, the number of autolysosomes containing mitochondria and lipid droplets was dramatically reduced in the RORα-deleted hepatocytes. Finally, we found that RORα induced the transcription of genes involved in lysosomal function, such as Atp6v1g1, a vacuolar H+ -ATPase (v-ATPase) subunit, which were largely down-regulated in the livers of mice with high-fat diet-induced NAFLD and patients with hepatitis. Conclusion: Targeting RORα may be a potential therapeutic strategy to restore lysosomal acidification, which inhibits the progression of NAFLD.

Integrative genetic, genomic and transcriptomic analysis of heat shock protein and nuclear hormone receptor gene associations with spontaneous preterm birth.

Heat shock proteins are involved in the response to stress including activation of the immune response. Elevated circulating heat shock proteins are associated with spontaneous preterm birth (SPTB). Intracellular heat shock proteins act as multifunctional molecular chaperones that regulate activity of nuclear hormone receptors. Since SPTB has a significant genetic predisposition, our objective was to identify genetic and transcriptomic evidence of heat shock proteins and nuclear hormone receptors that may affect risk for SPTB. We investigated all 97 genes encoding members of the heat shock protein families and all 49 genes encoding nuclear hormone receptors for their potential role in SPTB susceptibility. We used multiple genetic and genomic datasets including genome-wide association studies (GWASs), whole-exome sequencing (WES), and placental transcriptomics to identify SPTB predisposing factors from the mother, infant, and placenta. There were multiple associations of heat shock protein and nuclear hormone receptor genes with SPTB. Several orthogonal datasets supported roles for SEC63, HSPA1L, SACS, RORA, and AR in susceptibility to SPTB. We propose that suppression of specific heat shock proteins promotes maintenance of pregnancy, whereas activation of specific heat shock protein mediated signaling may disturb maternal-fetal tolerance and promote labor.

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.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.