Function And Mechanism Study Of GREM1 In Intervertebral Disc Degeneration

Objective: The incidence of low back pain(LBP)is increasing with the aging of the population,and it has become an important cause of disability worldwide.Intervertebral disc degeneration(IDD)is the main cause of LBP.It is of great clinical significance to investigate the mechanism of IDD at the molecular biological level for preventing,delaying and even reversing the progress of IDD.Nucleus pulposus cells(NPCs)apoptosis and senescence,and NP tissues collagen loss are central events during intervertebral disc degeneration(IDD)progression.Bone morphogenetic proteins(BMPs)promote the synthesis of extracellular matrix(ECM)and help to delay the progression of IDD.Gremlin-1(GREM1)can play an important role in the development and progression of many diseases such as limb deformity and osteoarthritis by inhibiting the synthesis,secretion and signaling of BMPs;however,whether GREM1 is involved in the pathogenic mechanism of IDD remains unclear.This study intends to investigate the relationship between GREM1 and IDD and its underlying mechanism,and provide a new target for gene therapy of IDD in the future.Methods: 1.The expression of GREM1 in NP was screened by bioinformatics analysis.GREM1 levels were examined in NP tissue samples using immunohistochemical(IHC)staining and quantitative real-time PCR(q RT-PCR).2.NPCs were isolated and identified using immunofluorescent,toluidine blue staining and CD24 positive ratio.The specific effects of GREM1 overexpression/knockdown on NPCs viability,apoptosis,senescence,and extracellular matrix(ECM)production and degradation were determined using CCK-8,flow cytometry,S-Aβ-gal staining and immunoblotting.In addition,the signaling pathway regulated by GREM1 was analyzed.3.Upstream transcriptional factors of GREM1 were retrieved and analyzed by chromatin immunoprecipitation(Ch IP)and luciferase assays.Furthermore,the effects of the predicted transcription factor on the phenotype of NPCs were verified by cell function experiments.4.The IDD model was established in rats,and the specific effects of GREM1 knockdown on imaging and histopathological changes,tissue apoptosis,and IDD-associated markers were examined using MRI,hematoxylin-eosin(HE)staining,TUNEL staining,and immunoblotting respectively.Results: 1.There was a significant difference in GREM1 expression between normal and degenerative NP tissues,and GREM1 expression levels were significantly upregulated in human degenerative NP tissues.2.NPCs were isolated from normal NP tissues and identified.In vitro,GREM1 overexpression aggravated the degenerative changes in NPCs,including inhibited cell proliferation,promoted cell apoptosis and senescence,suppressed ECM production,and elevated ECM degradation.GREM1 knockdown partially eliminated the degenerative changes in NPCs.GREM1 knockdown promoted,whereas GREM1 overexpression inhibited the phosphorylation of PI3 K and Akt.GREM1 knockdown significantly promoted NPCs viability but inhibited apoptosis and senescence,which was partially reversed by LY294002.GREM1 knockdown decreased the protein levels of GREM1,MMP13,ADAMTS4,ADAMTS5,apoptosis-related proteins cleaved-caspase 3/7,and senescence-related proteins P53,P21 and P16,whereas it increased the protein levels of BMP2,collagen II,and aggrecan;LY294002partially eliminated the effects of GREM1 knockdown on markers in NPCs.3.GATA binding protein 4(GATA4)targeted GREM1 promoter region and positively regulated GREM1 expression.GATA4 knockdown ameliorated degenerative alterations of NPCs,whereas GREM1 overexpression partially abolished GATA4 knockdown effects on NPCs.4.In vivo,GREM1 knockdown improved IDD symptoms,including Pfirrmann grades,NP tissues structure loss and hyper-degradation of ECM.Conclusion: 1.GREM1 is up-regulated in human degenerative intervertebral NP tissues.2.GREM1 knockdown promotes NPCs viability,inhibits apoptosis and senescence,promotes ECM production,and inhibits ECM degradation,therefore alleviating degenerative changes of NPCs in vitro.3.GATA4 binds to GREM1 promoter,promotes GREM1 expression and inhibits the activation of PI3K/Akt signaling pathway,therefore contributing to NPCs degenerative alterations.4.Intradiscal knockdown of GREM1 in the rat IDD model significantly improves the degenerative manifestations of the intervertebral discs in terms of imaging and histology.