Nuclear receptors regulate lipid metabolism and oxidative stress markers in chondrocytes.
ABSTRACT: Joint homeostasis failure can result in osteoarthritis (OA). Currently, there are no treatments to alter disease progression in OA, but targeting early changes in cellular behavior has great potential. Recent data show that nuclear receptors contribute to the pathogenesis of OA and could be viable therapeutic targets, but their molecular mechanisms in cartilage are incompletely understood. This study examines global changes in gene expression after treatment with agonists for four nuclear receptor implicated in OA (LXR, PPAR?, PPAR?, and RXR). Murine articular chondrocytes were treated with agonists for LXR, PPAR?, PPAR?, or RXR and underwent microarray, qPCR, and cellular lipid analyses to evaluate changes in gene expression and lipid profile. Immunohistochemistry was conducted to compare two differentially expressed targets (Txnip, Gsta4) in control and cartilage-specific PPAR? knockout mice subjected to surgical destabilization of the medial meniscus (DMM). Nuclear receptor agonists induced different gene expression profiles with many responses affecting lipid metabolism. LXR activation downregulated gene expression of proteases involved in OA, whereas RXR agonism decreased expression of ECM components and increased expression of Mmp13. Functional assays indicate increases in cell triglyceride accumulation after PPAR?, LXR, and RXR agonism but a decrease after PPAR? agonism. PPAR? and RXR downregulate the antioxidant Gsta4, and PPAR? upregulates Txnip. Wild-type, but not PPAR?-deficient mice, display increased staining for Txnip after DMM. Collectively, these data demonstrate that nuclear receptor activation in chondrocytes primarily affects lipid metabolism. In the case of PPAR?, this change might lead to increased oxidative stress, possibly contributing to OA-associated changes.Nuclear receptors regulate metabolic genes in chondrocytes. Nuclear receptors affect triglyceride levels. PPAR? mediates regulation of oxidative stress markers. Nuclear receptors are promising therapeutic targets for osteoarthritis.
Project description:The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPAR? binding demonstrated binding of PPAR? to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPAR?-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPAR? at the level of binding to shared genomic sites.
Project description:In this study, we used murine chondrocytes as an in vitro model and mice exhibiting destabilization of the medial meniscus (DMM) as an in vivo model to investigate the mechanisms through which S-allyl cysteine (SAC) alleviates osteoarthritis (OA). SAC significantly reduced apoptosis and senescence and maintained homeostasis of extracellular matrix (ECM) metabolism in tert-butyl hydroperoxide (TBHP)-treated chondrocytes. Molecular docking analysis showed a -CDOCKER interaction energy value of 203.76 kcal/mol for interactions between SAC and nuclear factor erythroid 2-related factor 2 (Nrf2). SAC increased the nuclear translocation of Nrf2 and activated the Nrf2/HO1 signaling pathway in TBHP-treated chondrocytes. Furthermore, Nrf2 knockdown abrogated the antiapoptotic, antisenescence, and ECM regulatory effects of SAC in TBHP-treated chondrocytes. SAC treatment also significantly reduced cartilage ossification and erosion, joint-space narrowing, synovial thickening and hypercellularity in DMM model mice. Collectively, these findings show that SAC ameliorates OA pathology in TBHP-treated chondrocytes and DMM model mice by activating the Nrf2/HO1 signaling pathway.
Project description:OBJECTIVES:We have previously shown that peroxisome proliferator-activated receptor gamma (PPAR?), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPAR? knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPAR? in OA pathophysiology. We further investigated the downstream PPAR? signalling pathway responsible for maintaining cartilage homeostasis. METHODS:Inducible cartilage-specific PPAR? KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPAR?/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPAR? signalling pathway in OA. RESULTS:Compared with control mice, PPAR? KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPAR? expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPAR? KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPAR? signalling, we created and subjected PPAR?-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPAR? KO mice (double KO) can rescue the accelerated OA phenotype observed in PPAR? KO mice. Indeed, PPAR?-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. SIGNIFICANCE:PPAR? maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.
Project description:Nuclear receptors retinoic X receptor ? (RXR?) and peroxisome proliferator activated receptor ? (PPAR?) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXR? and PPAR? is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXR? and PPAR?. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXR?:PPAR? heterodimer, instead of RXR-response element (RXRE) mediated by RXR?:RXR? homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXR? and PPAR? ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXR? L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPAR? Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXR? and PPAR? are determined. As the first report on the dual agonist targeting RXR? and PPAR? with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design.
Project description:Apolipoprotein F (ApoF) regulates cholesteryl ester transfer protein activity. We previously observed that hepatic APOF mRNA levels are decreased by high fat, cholesterol-enriched diets. Here we show in human liver C3A cells that APOF mRNA levels are reduced by agonists of LXR and PPAR? nuclear receptors. This negative regulation requires co-incubation with the RXR agonist, retinoic acid. Bioinformatic analysis of the ~2 kb sequence upstream of the APOF promoter identified one potential LXR and 4 potential PPAR? binding sites clustered between nucleotides -2007 and -1961. ChIP analysis confirmed agonist-dependent binding of LXR?, PPAR?, and RXR? to this hormone response element complex (HREc). A luciferase reporter containing the 2 kb 5' APOF sequence was negatively regulated by LXR and PPAR? ligands as seen in cells. This regulation was maintained in constructs lacking the ~1700 nucleotides between the HREc and the APOF proximal promoter. Mutations of the HREc that disrupted LXR? and PPAR? binding led to the loss of reporter construct inhibition by agonists of these nuclear receptors. siRNA knockdown studies showed that APOF gene regulation by LXR? or PPAR? agonists did not require an interaction between these two nuclear receptors. Thus, APOF is subject to negative regulation by agonist-activated LXR or PPAR? nuclear receptors binding to a regulatory element ~1900 bases 5' to the APOF promoter. High fat, cholesterol-enriched diets likely reduce APOF gene expression via these receptors interacting at this regulatory site.
Project description:Peroxisome proliferator-activated receptor ? (PPAR?) and liver X receptor ? (LXR?) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXR? and PPAR? pathways exist, including competition for the same heterodimeric partner, retinoid X receptor ? (RXR?). Although data have suggested that PPAR? and LXR? may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein-protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPAR? and LXR? interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPAR? ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPAR? and LXR? response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXR?. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPAR? and LXR? in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPAR? alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXR? overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors.
Project description:Macrophage foam cells store excess cholesterol as cholesteryl esters, which need to be hydrolyzed for cholesterol efflux. We recently reported that silencing expression of carboxylesterase 1 (CES1) in human THP-1 macrophages [CES1KD (THP-1 cells with CES1 expression knocked down) macrophages] reduced cholesterol uptake and decreased expression of CD36 and scavenger receptor-A in cells loaded with acetylated low-density lipoprotein (acLDL). Here, we report that CES1KD macrophages exhibit reduced transcription of cytochrome P45027A1 (CYP27A1) in nonloaded and acLDL-loaded cells. Moreover, levels of CYP27A1 protein and its enzymatic product, 27-hydroxycholesterol, were markedly reduced in CES1KD macrophages. Transcription of LXR? (liver X receptor ?) and ABCA1 (ATP-binding cassette transporter A1) was also decreased in acLDL-loaded CES1KD macrophages, suggesting reduced signaling through PPAR?-CYP27A1-LXR?. Consistent with this, treatment of CES1KD macrophages with agonists for PPAR?, RAR, and/or RAR/RXR partially restored transcription of CYP27A1 and LXR?, and repaired cholesterol influx. Conversely, treatment of control macrophages with antagonists for PPAR? and/or RXR decreased transcription of CYP27A1 and LXR? Pharmacologic inhibition of CES1 in both wild-type THP-1 cells and primary human macrophages also decreased CYP27A1 transcription. CES1 silencing did not affect transcript levels of PPAR? and RXR in acLDL-loaded macrophages, whereas it did reduce the catabolism of the endocannabinoid 2-arachidonoylglycerol. Finally, the gene expression profile of CES1KD macrophages was similar to that of PPAR? knockdown cells following acLDL exposures, further suggesting a mechanistic link between CES1 and PPAR?. These results are consistent with a model in which abrogation of CES1 function attenuates the CYP27A1-LXR?-ABCA1 signaling axis by depleting endogenous ligands for the nuclear receptors PPAR?, RAR, and/or RXR that regulate cholesterol homeostasis.
Project description:BACKGROUND:Epigenetic mechanisms have been reported to play key roles in osteoarthritis (OA) development. P300/CBP-associated factor (PCAF) is a member of the histone acetyltransferases, which exhibits a strong relationship with endoplasmic reticulum (ER) stress and transcription factor nuclear factor kappa B (NF-?B) signals. Salidroside, a natural histone acetylation inhibitor, showed its anti-inflammatory and anti-apoptotic effects in lipopolysaccharide (LPS)-stimulated microglia cells in our previous study. However, whether Sal has a protective effect against OA remains unknown, and its relationships to PCAF, NF-?B, and the ER stress pathway should be explored further. METHODS:We identified the role of PCAF in the pathogenesis of OA and determined the chondroprotective effect of Sal on both tumor necrosis factor alpha (TNF-?)-treated human chondrocytes and a destabilized medial meniscus (DMM) mouse OA model. FINDINGS:We found increased PCAF expression in human OA cartilage and TNF-?-driven chondrocytes. Meanwhile, silencing of PCAF attenuated nuclear p65 and C/EBP homologous protein levels in chondrocytes upon TNF-? stimulation. Furthermore, Sal was found to specifically bind to the inhibitory site of the PCAF protein structure, which subsequently reversed the TNF-?-induced activation of NF-?B signal and ER stress-related apoptosis in chondrocytes. In addition, the protective effect of Sal and its inhibitory effects on PCAF as well as inflammatory- and ER stress-related markers were also observed in the mouse DMM model. INTERPRETATION:Pharmacological blockade of PCAF by Sal ameliorates OA development via inhibition of inflammation and ER stress, which makes Sal a promising therapeutic agents for the treatment of OA.
Project description:The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice is dependent on LXR and correlates with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the role of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as PPAR signaling pathways, and subsequent ChIP-seq mapping of PPARM-NM-1 binding demonstrated binding of PPARM-NM-1 to 71-88% of the identified LXR:RXR binding sites. Sequence analysis of shared binding regions combined with sequential ChIP on selected sites indicate that LXR:RXR and PPARM-NM-1:RXR bind to degenerate response elements in a mutually exclusive manner. Together our findings suggest extensive and unexpected cross-talk between hepatic LXR and PPARM-NM-1 at the level of binding to shared genomic sites LXR, RXR, PPARalpha and RNA Polymerase II ChIP-seq on livers from female C57BL/6 wild-type and/or LXRM-NM-1/M-NM-2-deficient mice (13 weeks of age, n=1) treated by oral gavage once daily for 14 days with the RXR agonist bexarotene (100 mg/kg body weight [mpk], in 1% carboxymethylcellulose), the LXR agonist T0901317 (T09, 30 mpk) or vehicle alone.
Project description:OBJECTIVES:A role for the type II arginine methyltransferase PRMT5 in various human diseases has been identified. In this study, the potential mechanism underlying the involvement of PRMT5 in the pathological process leading to osteoarthritis (OA) was investigated. METHODS:PRMT5 expression in cartilage tissues from patients with OA and control individuals was assessed by immunohistochemical staining. The regulatory and functional roles of PRMT5 in the chondrocytes of patients with OA and control individuals were determined by western blotting and reverse transcription polymerase chain reaction. The effects of the PRMT5 inhibitor EPZ on interleukin-1?-induced inflammation were examined in the chondrocytes of patients with OA and in the destabilized medial meniscus (DMM) of a mouse model of OA. RESULTS:PRMT5 was specifically upregulated in the cartilage of patients with OA. Moreover, adenovirus-mediated overexpression of PRMT5 in human chondrocytes caused cartilage degeneration. This degeneration was induced by elevated expression levels of matrix-degrading enzymes (matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13)) in chondrocytes. The activation of the MAPK and nuclear factor ?B signaling pathways was evidenced by elevated levels of p-p65, p-p38, and p-JNK. These effects were attenuated by inhibiting the expression of PRMT5. In the mouse model, EPZ inhibited PRMT5 expression, thus protecting mouse cartilage from DMM-induced OA. CONCLUSIONS:Our results demonstrate that PRMT5 is a crucial regulator of OA pathogenesis, implying that EPZ has therapeutic value in the treatment of this cartilage-destroying disease.