The Mechanism Of PI3K/AKT/mTORC1 Signaling In Subchondral Bone In The Pathogenesis Of OA

ObjectiveOsteoarthritis(OA)is the most common joint disease,characterized by articular cartilage degeneration and subchondral bone sclerosis.As the pathogenesis of OA is not clear,there is Iack of effective therapy methods in clinic.Increasing evidence shows aberrant subchondral bone remodeling plays an important role in development of osteoarthritis(OA).However,how subchondral bone formation is activated and the mechanism by which increased subchondral bone turnover promotes cartilage degeneration during OA remains unclear.PI3K/AKT/mTORC1 signaling is essential in regulating pathophysiology of osteoarthritis(OA).Inhibiting PI3K/AKT/mTORC1 signaling attenuates the severity of experimental OA.However,its potential modulatory role in subchondral bone remodeling during OA progression has not been investigated yet.Here,we investigate the mechanism of PI3K/AKT/mTORC1 activity in subchondral bone in the pathogenesis of OA on the molecular,cellular and animal levels.The study will provide potential target for prevention and treatment of OA.Method1.The mouse DMM-OA model was constructed and the specimens of the knee joint were collected for histological staining.The pathological degree of OA in the knee joint was evaluated by OARSI score.Micro-CT analysis and 3D reconstruction were used to evaluate the subchondral ossification.Immunohistochemistry was used to detect the PI3K/Akt activity in subchondral bone and the expression of MMP13 in cartilage.Primary preosteoblast cells were isolated from mice and PTEN siRNA was constructed in vitro to investigate the effect of PI3K/Akt activity in preosteoblasts on chondrocytes.The effect of PI3E/Akt activity in subchondral bone on the course of OA was studied by inhibitor LY294002 in vivo and vitro.2.Articular cartilage and subchondral bone specimens of normal and OA patients,and mouse ACLT-OA model were collected for histological staining.The pathological degree of OA in the knee joint was evaluated by OARSI score.Micro-CT analysis were used to evaluate the subchondral ossification.Immunohistochemistry was used to detect the mTORC1 activity and osteogenesis in subchondral bone.Inducible preosteoblast-specific Tscl(mTORC1 upstream inhibitor)and Raptor(mTORC1-specific component)knockout mice were generated to study the effect of mTORC1 activity of subchondral bone on the occurrence and development of OA.Primary preosteoblast cells were isolated from mice to investigate the mechanism of PI3K/Akt activity in preosteoblasts on chondrocytes.Results1.We revealed a significant increase in PI3K/AKT signaling activation which was associated with aberrant bone formation in tibial subchondral bone following destabilizing the medial meniscus(DMM),which was effectively prevented by treatment with PI3K/AKT signaling inhibitor LY294002.PI3K/AKT signaling inhibition attenuated articular cartilage degeneration.Serun and bone biochemical analyses revealed increased levels of MMP-13,which was found expressed mainly by preosteoblasts in subchondral bone.However,this MMP-13 induction was attenuated by LY294002 treatment.Furthermore,PI3K/AKT signaling was found to enhance preosteoblast proliferation,differentiation,and expression of MMP-13 by activating NF-κB pathway.2.we show that the mechanistic target of rapamycin complex 1(mTORC1)pathway is activated in subchondral bone preosteoblasts(Osterix+)from OA patients and mice.Constitutive activation of mTORC1 in preosteoblasts by deletion of the mTORC1 upstream inhibitor,tuberous sclerosis 1(TSC1)induced aberrant subchondral bone formation and sclerosis,resulting in an OA-like phenotype and aggravated destabilized OA in mice.In contrast,inhibition of mTORC1 in preosteoblasts by disruption of Raptor(mTORC1-specific component)reduced subchondral bone formation and cartilage degeneration,and attenuated destabilized OA in mice.Mechanistically,mechanical loading reduced primary cilia and activated mTORC1 in preosteoblasts.mTORC1 activation promoted preosteoblast expansion and Cxcl12 secretion,which induced cartilage degeneration during OA.A Cxcl12 neutralizing antibody reduced cartilage degeneration and alleviated OA in mice.ConclusionTogether,these findings demonstrate that PI3K/AKT/mTORC1 activation in subchondral preosteoblast is important in OA development,acting by inducing aberrant subchondral bone formation and secretion of MMP13,Cxcl12 to promote cartilage degeneration.Pharmaceutical inhibition of the pathway represents a promising therapeutic approach for OA treatment.