The Regulatory Mechanisms Of Oxysterol Membrane Receptor(GPR183) In Osteoarthritis

Osteoarthritis(OA),the most common degenerative joint disease and one of the leading causes of chronic disability in elderly,incurs an enormous economic burden on society.OA is a disease characterized by cartilage degradation,osteophyte formation,subchondral bone sclerosis and synovial inflammation.Numerous etiologic risk factors for the development of OA such as mechanical stress,aging,obesity and metabolic syndrome were identified through preclinical research in animal models of OA and clinical studies in patients with OA.The most obvious pathology of osteoarthritis is cartilage degeneration and damage.Current therapies for OA include topical NSAIDs and total knee arthroplasty.At present,no method has been developed to relieve joint pain,effectively repair cartilage injured and improve joint function.Further research is still needed to solve this orthopedic problem in clinical practice.Epidemiological studies have suggested that there is a correlation between human serum cholesterol levels and osteoarthritis.Cholesterol and its metabolites oxysterol directly activate through the nuclear receptor ROR?axis causes OA pathogenesis by upregulating matrix-degrading.However,there are four cell membrane receptors for oxysterol except nuclear receptors.Therefore,can we find a receptor of oxysterol to help relieve joint pain,repair cartilage damage and improve joint function,to be a target of OA treatment?We examined the expression of the membrane receptors for oxysterol,we found GPR183 was the only oxysterol transmembrane receptors in OA human chondrocytes that exhibited elevated expression when stimulated by IL-1?.Subsequently,protein levels were markedly elevated in cartilage of human OA cartilage.In addition,a positive correlation was observed between GPR183 and MMP13 in human OA cartilage.To further investigate the function of Gpr183 in OA pathogenesis,we generated Gpr183-knockout(Gpr183^-/-)mice to examined the gene's effect on OA.Moreover,the DMM or HCD-associated post-traumatic OA induced manifestations,including cartilage erosion,osteophyte formation,were significantly reduced in Gpr183^-/-mice.The results also showed normal collagen organization in Gpr183^-/-articular cartilage.In addition,we verified the cartilage repair ability of Gpr183^-/-mice through cartilage injury model.The regenerated cartilage of Gpr183^-/-also stained positive for Col2a1 and ACAN,and negative for fibroblast markers such as Col1a1 and Mmp13,suggesting that stable hyaline cartilage was formed.All of the data demonstrate that GPR183 plays an essential role in regulating cartilage homeostasis.7?,25-OHC is both an oxidative product of cholesterol metabolism and an endogenous ligand of GPR183.7?,25-OHC effectively inhibit chondrogenesis in micromass cultures of primary chondrocytes and bone marrow mesenchymal stem cells of mice.Also,the expression of chondrogenic differentiation related genes COL2A,ACAN,PRG4 and COL9A1 was inhibited in human bone marrow mesenchymal stem cells after cultured with 7?,25-OHC.Under OA pathological condition,7?,25-OHC destroys the formation of extracellular matrix and accelerates the destruction of explants by inhibiting the expression of extracellular matrix genes and promoting the expression of matrix degrading enzymes genes.To further investigate whether GPR183 could be a drug target for the treatment of OA.Our results indicate,NIBR189,an inhibitor of GPR183,effectively induced chondrogenesis in micromass cultures of primary chondrocytes and bone marrow mesenchymal stem cells.In addition,under OA pathological conditions NIBR189protect collagen and glycosaminoglycan(GAG)content loss.Intra-articular injection of NIBR189 substantially inhibit the cartilage degradation,synovial inflammation,OA pain caused by DMM-induced OA model and HCD-related post-traumatic OA model.Subsequently,NIBR189 can inhibit the expression of MMP3 and MMP13,reduce GAG loss,and increase the expression of COL2A1 and ACAN in OA cartilage explants to promote the production of extracellular matrix.To further investigate the molecular mechanism of GPR183 affecting chondrocyte homeostasis,we conducted an in vitro study of Gpr183^-/-mouse cartilage chondrocytes.Our results showed that Gpr183-deficiacy restored IL-1?-induced cartilage anabolic factors expression and inhibited IL-1?-induced the expression of catabolic genes as Mmp3?Mmp12?Mmp13?Nos2 and IL-6.In addition,RNAseq analysis and verified by RT-q PCR,type II collagen(Col2a1),type IX collagen(Col9a1,Col9a2)type XI collagen(Col11a),type XVII collagen(Col27a1),proteoglycan(Acan and Ibsp),and chondrogenic factor Sox6 gene expression were up-regulated after Gpr183 knockout.And we found that the increase the content of collagen and GAG in articular cartilage of Gpr183^-/-,promote cartilage extracellular matrix generation.We found GPR183 can significantly regulate the formation of chondrocytes extracellular matrix and affect the chondrogenic differentiation ability.We used a 7?,25-OHC model to inhibit chondrogenic differentiation,and screened which signaling pathway mediates 7?,25-OHC inhibition and chondrogenic extracellular matrix formation through inhibitors of fifteen different signaling pathways.We found that MST inhibitors(YAP signaling pathway agonists)restore 7?,25-OHC-induced inhibition of extracellular matrix synthesis and differentiation.Therefore,we focus on the function of GPR183 in the regulation of YAP signaling pathway.In contrast,YAP inhibitors block the function of knockout Gpr183 to promote extracellular matrix synthesis.In addition,activation of Gpr183 or overexpression of Gpr183 significantly inhibited YAP into the nucleus,and overexpression of Gpr183 inhibited the activity of YAP-activated downstream transcription factor TEAD4,while the expression of downstream functional genes of YAP increased in Gpr183^-/-chondrocytes.In conclusion,we found that inhibiting the oxosterol membrane receptor GPR183signaling can inhibit the matrix catabolism,improve the anabolism of cartilage extracellular matrix,stabilize the network structure of extracellular matrix to reverse the damage of cartilage homeostasis and improve the regeneration ability of cartilage.The knowledge system of cholesterol signaling pathway regulating cartilage anabolism has been improved.Overall,our data demonstrated provides a novel target for potential therapeutic interventions in the treatment of osteoarthritis and cartilage injury.