Knockdown Of Postn In Chondrocytes Ameliorates Cartilage Degradation Via TGF-β/SMAD3 Signaling Pathway

Background:Osteoarthritis is(OA)a most common form of chronic joint disease in elders,which maily affects hands,hips,knees,feet and spine.The symptoms of OA mainly includes stiffness,disability and pain.The epidemiology of OA is complicative and multifactorial,including epigenetic predisposition,sex,injury,aging,obesity and lifestyle.OA affects cartilage,subchondral bone,synovium and even all the joint,whose pathophysiology could be characterized by cartilage degradation,subchondral bone remodeling,osteochondral angiogenesis,formation of osteophytes and synovitis.Previous studies put forward a point that articular cartilage might be the first site to appear changes.It is possblely because the functions of chondrocytes are dysregulated after stimulation by inflammatory factors secreted by synovium and subchondral bone.Nowadays,no treatments has yet been shon to convincingly treat OA.The goals of treatment OA is to relieve pain,improve activity of the joint and decrease the erosion of joint.Patients at the end stage of OA should pay expensive fee to undergo surgery.In addition to aging of population,OA has been a heavy burden in society.With the development of molecular biology,cytobioloty and clinical medicine and permeation into each other,targeting gene therapy has been a hop topic in treatment OA.The goals of targeting gene terapy is to figure out some gene that are dysregulated by gene sequencing or microarray,and then import the normal outside gene into the targeted cells so as to change the condition that the targeted gene was loss or up-expressed,or promote the targeted cells to get new biochemical function to treat OA precisely.Our team has discovered previously that Postn gene was up-regulated in rat subchondral bone.Later,Sittisak Honsawek found that Postn was increased in the serum and synovia of OA patients.And the recent research indicates that Postn was a pivotal factor to promote hepatic sclerosis,possibly by phosphorylating of SMAD2/3 route.And in the condition of hypoxia,POSTN could inhibit phosphorylation of SMAD2/3 route.This indicates that POSTN might play an important role in regulating SMAD2/3 route.TGF-β/SMAD signaling pathway mainly regulates the metabolic activity such as cell proliferation,differentiation,apoptosis.Smad is the down stream target of TGF-(3.In chondrocyte growth,SMAD1/5/8 route was involved in cartilage degeneration while SMAD2/3 route was to maintain the homeostasis in chondrocytes.But when OA occurs,SMAD 1/5/8 route promotes cartilage degradation,while aberrant SMAD2/3 route also accelerates cartilage degradation.Some studies show that TGF-P up-regulates Mmp13 expression in OA chondrocytes.TGF-P/SMAD3 promote Mmp13 and Adamts5 expression via down-regulating miR-140 and over-expressed Smad3 could induce Mmp13 expression.Meanwhile,some reseachers discover that Smad also promotes Mmp13 expression in other tissues such as squamous carcinoma cells,breast carcinoma cells,periodontal ligament cells and osteoblast cells.Therefore,the activity of TGF-β/SMAD plays a vital roles in maintaining the metabolism and normal structure of chondrocytes.Objective:To investigate the expression of Postn in the articular cartilage of OA individuals and DMM mice.Then we determined the effect of Postn on cartilage degeneration in ATDC5 chondrogenic cell line.Afterwards,we conducted a kind of lentivirus that could silencing Postn gene and intra-articular injected the lentivirus into the knee joints of DMM mice to determined the histological and biochemical changes in OA cartilage.Finally,we investigated that whether Postn promoted MMP13 expression to promote OA progression via TGF-β/SMAD3 signaling pathway in vivro.Our aim is to explore the effects of Postn on TGF-β/SMAD3 and cartilage degeneration so as to offer a new viewpoint and theoretical foundation in treating OA.Methods:1.Human OA cartilage were obtained from OA patients.Control cartilage samples without macroscopic changes were obtained from patients undergoing knee amputation.We detected Postn mRNA expression in OA and Sham individuals by RT-qPCR.Then we purchased C57/BL6 mice to create a DMM models,we determined POSTN protein expression in 1,2,4,8 weeks postsuregery by western blot and detected its location and expression in caritilage.2.We treated ATDC5 cells with siRNA to silence Postn followed by IL-1βstimulation to induce osteoarthritic changes in vitro,and explored the effects of Postn on cartilage degeneration.3.We intraarticularly injected in the DMM knees with lentivirus to silence Postn gene in cartilage to determine the effects of Postn on histological and biochemical changes in OA pathology.4.We treated ATDC5 cells with different cell factors and examined the expression of Postn.Then we treated the cells with TGF-β1 and explored that whether Postn was regulated by TGF-β1 in a magnitude and time dependent manner.In addition,we determined the effects of silencing Postn on CollOal and Mmp13 mRNA expression induced by TGF-β1.5.We treated ATDC5 cells with Postn and siRNA-Postn to explored whether Postn promotes MMP13 to aggravate OA via TGF-β/SMAD3 signaling pathway.6.We chose SIS3 to inhibit the phosphorylation of SMAD3,then explored whether POSTN increased MMP13 expression via SMAD3.7.We treated ATDC5 cells with Postn and determined the activation levels of receptors of SMAD3 route.Results:1、Postn was up-regulated in OA individuals and DMM mice.2、Silencing Postn gene in vitro could differently down-regulated Mmp13、Co10a1、Runx2、Vegf expression,and up-regulated Col2 expression.That indicated that Postn counteracted the osteoarthritic changes induced by IL-1β to attenuate cartilage degeneration.3、After intraarticularly injection with lentivirus,the expression of POSTN was significantly down-regulated,the erosion in cartilage was relieved,the thickness of cartilage was increased,MMP13 expression was decreased while COL2 expression was increased,eventually attenuate OA progression in DMM mice.4、Postn was regulated by TGF-β in a magnitude and time dependent manner in OA pathology.Silencing Postn counteract Col10a1 and Mmp13 up-regulation induced by TGF-p.5.Postn up-regulated MMP13 production to aggravate cartilage degeneration via phosphorylation of SMAD3.6、POSTN could activate the cell surface receptors of SMAD3 route,mainly manifest as the activation of type II receptors such as Acvr2b and Tgfbr2,which assembled and activated type I receptors such as ALK5 and ALK7.Eventually,the messeage was delivered from the extracellular to the endocellular.7、Conclusion:Postn is up-regulated in OA pathology.Silencing Postn gene attenuates cartilage degeneration via decreasing MMPs production,inhibiting chondrocyte hypertrophy and angiogenesis and promote COL2 production.Silening Postn gene in chondrocyte attenuates OA progression.Postn up-regulated MMP13 production to aggravate cartilage degeneration via TGF-β/SMAD3 signaling pathway.In our study,the significance for this cause was providing a novel experimental evidence for research in OA fields and a novel therapitic target for treating OA.