Cdc42 Is Essential For Both Articular Cartilage Degeneration Ancd Subchondral Bone Deterioration In Experimental Osteoarthritis

OA(osteoarthritis)is the most common form of multifactorial joint disease characterized by articular cartilage degradation,subchondral bone sclerosis,osteophyte formation,inflammation,angiogenesis and neurogenesis.Clinical manifestations of OA include joint swelling,joint deformity,joint movement disorder and repeated episodes of pain,and the prevalence and incidence of OA exceed other two most common senile diseases.To date,no such drug has shown efficacy at slowing or reversing joint damage during OA progression in humans and drug therapy mainly aims to improve pain,necessitating joint replacement as a therapeutic option.Surgical therapy is a final choice and can't solve articular cartilage degeneration.Therefore,improving our understanding of the mechanisms driving the associated joint pathology is great beneficial to prevention,diagnosis and treatment of osteoarthritis.During the past many decades,most researchers engaged in osteoarthritis focused on articular cartilage.But the different results between clinical treatments for OA and preclinical studies suggest that targeting articular cartilage alone may not be sufficient to attenuate or perfectly halt the progression of osteoarthritis.Indeed,currently more and more researchs indicate that articular cartilage and subchondral bone form a holistic unit and mutually undertake peripheral biochemical and biomechanical stress,and OA is therefore considered as a disease of the whole joint classically accompanied by subchondral bone remodeling.Cytokines have been implicated in the metabolism of bone and cartilage,and it has been reported that synovium and cartilage can produce a variety of inflammatory factors such as IL-1? and TNF-?,which induce inhibition of proteoglycan and type ? collagen synthesis,promotion of catabolism and other inflammatory cytokines(IL-6?IL-8?PEG2 and NO et al)and finally result in cartilage matrix damage.As for subchondral bone,recent studies indicate that abberrant subchondral bone remodeling accompanies with the development of osteoarthritis.Moreover,the differentiation and function of osteoclasts is enhanced during the early stage of osteoarthritis.In addition,TGF?1 plays a fundamental role in subchondral bone sclerosis and high concentration of TGF(31 induce recruitment of MSCs clusters and formation of marrow osteoid islets accompanied by high level of angiogenesis and neurogenesis.Rho small GTPases such as Racl,Cdc42 and RhoA are molecular switches involved in regulation of multiple cell functions and play crucial roles in cartilage biology.In recent years,increasing evidences suggest that Rho small GTPases are closely associated with skeletal development.Previous studies indicate that Cdc42 is critical for chondrogenesis including mesenchymal condensation,life of chondrocyte and chondrogenic differentiation during long bone development.Moreover,Cdc42 regulates the formation and function of osteoclasts,endothelial construction and angiogenesis,and also participates in inflammatory responses especially in the case of senescence-associated inflammation.The fundamental roles of Cdc42 in inflammation,chondrogenesis,osteoclastgenesis,osteoblastogenesis and angiogenesis prompt us to speculate that Cdc42 could be implicated in the development of osteoarthritis.In the present study,we demonstrated that Cdc42 plays a fundamental role in the occurrence and development of osteoarthritis,and preliminary investigated the molecular mechanism.Firstly we detected the expression level of Cdc42 in articular cartilage and subchondral bone between Sham group and DMM group and found that expression of Cdc42 in DMM group was significantly higher than Sham group.Secondly the results of osteogenesis and chondrogenesis experiment showed that inhibition of Cdc42 activity promoted chondrogenesis and suppressed chondrocyte hypertrophy and osteogenesis.In addition,when ATDC5 and C3H10T1/2 were respectively treated with IL-1? and TGF?1,the level of Cdc42-GTP in both of them increased quite significantly,which induced high expression of MMP13 and COLX in ATDC5 cells and promoted osteoblast differentiation in mouse mesenchymal C3H10T1/2 cells.Correspondingly,inhibition of Cdc42 activity suppressed TGF?1 signaling pathway and the expression of osteogenesis-related marker genes.In animal experiments,we detected the effect of locally knock-down Cdc42 and inhibition of Cdc42 activity on osteoarthritis.Importantly,Safranin O/Fast green staining showed that locally knocking down Cdc42 and inhibition of Cdc42 activity in joint effectively attenuated cartilage damage during osteoarthritis.And then H&E staining results suggested that knock-down Cdc42 and inhibition of Cdc42 activity in DMM group decreased the calcification of articular cartilage compared with vehicle-treated DMM group.Moreover,immunostaining results showed that locally knock-down Cdc42 and inhibition of Cdc42 activity in DMM group decreased the expression of MMP13 and COLX in articular cartilage and suppressed TGF?1 signaling pathway and expression of osteogenesis-related marker genes in subchondral bone compared with vehicle-treated DMM group.In addition,trap staining similarly indicated that knock-down Cdc42 and inhibition of Cdc42 activity in DMM group inhibited the differentiation and function of osteoclasts.In parallel,through ?CT analysis of subchondral bone,we found that locally knock-down Cdc42 and inhibition of Cdc42 activity attenuated microstructural change of subchondral bone in DMM group compared with vehicle-treated DMM group.In order to know the role of Cdc42 in osteoarthritis,we then carried out many tests through ATDC5 and C3H10T1/2 cells.Western blot results showed that inhibition of Cdc42 activity and Erk phosphorylation decreased phosphorylation of Smad2/3,Smad1/5 and Erk.Co-immunoprecipitation results indicated that Erk did interact with Cdc42 forming a complex.The results above demonstrated that Cdc42 regulated condensation and osteogenesis of MSCs through Erk/Smads pathway in C3H10T1/2 cells.Similarly Western blot results showed inhibition of Cdc42 or Pak decreased phosphorylation of Jakl/2 and Stat3 induced by IL-1? in ATDC5 cells,and this result indicated that Cdc42 mediated inflammation through Jak/Stat3 signaling pathway.In concert with above results,immunostaining results suggested that locally knock-down Cdc42 and inhibition of Cdc42 activity decreased phosphorylation of Stat3 and Erk in DMM group compared with vehicle-treated DMM group.Taken together,our results indicated Cdc42 plays a fundamental role in degeneration of articular cartilage and subchondral bone sclerosis through Jak/Stat3 and TGF?1 signaling pathway respectively and knock-down Cdc42 and inhibition of Cdc42 activity attenuate the cartilage damage and subchondral bone sclerosis.