Protective Effects And Mechanisms Of ADRM1 On Osteoarthritis

Background and objective:Osteoarthritis(OA)is a joint degenerative disease that seriously affects the life quality of patients,which has caused a heavy burden to patients,families and society.Because the pathogenesis of OA remains unclear,there is a lack of effective prevention and treatment methods in clinic.Therefore,the exact pathogenesis of OA deserves further study.Recent study reported the molecular programmes and lineage progression patterns controlling human OA pathogenesis using single-cell RNA sequencing,and found that adhesion regulating molecule 1(ADRM1)can participate in the occurrence and development of OA as a favorable gene.It is also found that compared with early OA cartilage,the expression of ADRM1 in late OA cartilage decreased significantly.but the exact mechanism remained unknown.ADRM1 gene is located in the long arm 13 region of human chromosome 20(20q13).ADRM1 is a ubiquitin receptor on 19 S regulatory granules,a 26 S proteasome subunit,which was newly discovered in recent years.We have previously confirmed that ADRM1 plays an important role in the maintenance of articular cartilage homeostasis and OA protection from clinical specimens,in vitro cytological experiments and in vivo animal experiments.However,so far,the specific mechanism of ADRM1 inhibiting OA remained unknown,and there is no research report.We have searched the literature and found that at the molecular level,ADRM1,as one of the ubiquitin receptors,could mediate the deubiquitination of protein substrate by stabilizing,binding and activating ubiquitin carboxy-terminal hydrolase 37(UCH37),then the activated UCH37 could stabilize the expression of activin receptor-like kinase 5(ALK5).Therefore,we predicted that ADRM1 could stabilize and activate UCH37,thereby increasing ALK5 deubiquitination and reducing ALK5 degradation,and then to maintain articular cartilage homeostasis and inhibit the progress of OA.In order to verify the above hypothesis,this project mainly intends to carry out the following studies:(1)Study on the expression and function of ADRM1 in the pathological process of OA;(2)The function and mechanism of ADRM1 in the OA pathogenesis by stabilizing,binding and activating UCH37;(3)The function and mechanism of ADRM1 stabilizing ALK5 by regulating UCH37 in the OA pathogenesis.Through this study,it is expected to clarify the function and mechanism of ADRM1 in inhibiting OA,thereby to provide new biomarkers and potential drug therapeutic targets for the diagnosis and treatment of OA.Research methods:(1)The normal cartilage requiring amputation due to trauma and deformity,and OA articular cartilage with knee replacement were collected.The cartilage of clinical specimens were evaluated and graded by HE staining and Safranin O-Fast Green staining,and then the expression level of ADRM1 in cartilage was detected by Immunohistofluorescence and q PCR.Inflammatory factor IL-1β was used to stimulated chondrocytes to simulate OA environment in vitro.Then the expression of ADRM1 and extracellular matrix related genes and proteins were detected by q PCR and Western blot.RA190 was added to inhibit ADRM1 function,and ALK5 expression level,chondrocyte phenotype and the expression of extracellular matrix related genes and proteins were detected.(2)ATDC5 cells were transfected with ADRM1 overexpression lentivirus and ADRM1 si RNA to detect the effects of ADRM1 on chondrocyte phenotype,extracellular matrix related protein and UCH37 expression.Then,immunocytofluorescence was used to detect the subcellular localization of ADRM1 and UCH37,and the effects of ADRM1 on the fluorescence expression of UCH37.Co-IP was used to verify whether there was interaction between ADRM1 and UCH37 to activate the deubiquitination activity of UCH37 and binding degree.At the same time,we detected the effect of ADRM1 on UCH37 transcription.In addition,CHX,MG132 and leupeptin were used to detect the stability mechanism of ADRM1 on UCH37 protein.(3)ATDC5 cells were transfected with UCH37 si RNA to detect the effect of UCH37 on the expression of ALK5 and extracellular matrix related proteins.Then,in the case of overexpression of ADRM1 combined with silencing UCH37,the ubiquitin binding of ALK5 and the expression of ALK5 and extracellular matrix related proteins were detected.In the case of silencing ADRM1 and UCH37,the replenishment experiment of ALK5 was carried out with MG132.Finally,RA190 and ADRM1 overexpression lentivirus were injected into the knee joint of DMM-OA mice to observe the effects of inhibiting ADRM1 and exogenous supplementation of ADRM1 on the expression of UCH37,ALK5 and extracellular matrix related protein and OA pathological process.Research results:(1)The expression levels of ADRM1 protein and m RNA were down-regulated in articular cartilage samples of OA patients.Under IL-1β stimulation,the expression level of ADRM1 m RNA decreased,while its protein level decreased in a time-dependent manner.RA190 inhibited ALK5 expression level and the synthesis of chondrocytes extracellular matrix,and promoted the degradation of chondrocytes extracellular matrix.(2)Silencing ADRM1 could down-regulate the expression of UCH37,and negatively regulate the metabolic balance of chondrocytes extracellular matrix,while the overexpression of ADRM1 could promote the expression of UCH37 and positively regulate the metabolic balance of chondrocytes extracellular matrix.Both ADRM1 and UCH37 are localized in the nucleus and cytoplasm,and ADRM1 could bind and activate UCH37.The binding degree between ADRM1 and UCH37 increased with the increase of ADRM1 expression.ADRM1 could improve the stability of UCH37.However,ADRM1 did not affect the transcriptional level of UCH37,and there was no significant change on the expression of UCH37 protein after inhibiting proteasome pathway and lysosomal pathway.(3)Silencing UCH37 could down-regulate ALK5 expression,and negatively regulate the metabolic balance of chondrocytes extracellular matrix.The overexpression of ADRM1 could up-regulate ALK5 expression with reducing the level of ALK5 ubiquitination,and positively regulate the metabolic balance of chondrocytes extracellular matrix.While the overexpression of ADRM1 combined with silencing UCH37,the level of ALK5 were down-regulated with increasing the level of ALK5 ubiquitination,and the metabolic balance of chondrocytes extracellular matrix was negatively regulated.After inhibiting the proteasome pathway,the downward trend of ALK5 caused by silencing ADRM1 and UCH37 could pick up.In vivo animal experiments,RA190 could reduce the expression levels of UCH37 and ALK5,break the homeostasis balance of cartilage extracellular matrix,and significantly increase the OARSI scores,thus aggravating the pathological process of OA in mice.However,exogenous supplementation of ADRM1 could significantly increase the expression levels of UCH37 and ALK5,maintain the extracellular matrix homeostasis of articular cartilage,and reduce the OARSI scores,thus delaying the pathological process of OA in mice.Conclusion:(1)The expression of ADRM1 is decreased in the course of OA.(2)ADRM1 could stabilize the expression of UCH37,and ADRM1 could bind and activate UCH37,which could increase the deubiquitination of ALK5 to reduce its degradation,thereby maintain the homeostasis of articular cartilage and inhibit the progress of OA.(3)Inhibiting the function of ADRM1 could aggravate the pathological process of OA in mice,while exogenous supplementation of ADRM1 could significantly delay the pathological process of OA in mice.