Objectives To investigate the roles and regulatory mechanisms of synovial macrophages and their polarisation in the development of osteoarthritis (OA). Methods Synovial tissues from normal patients and patients with OA were collected. M1 or M2-polarised macrophages in synovial tissues of patients with OA and OA mice were analysed by immunofluorescence and immunohistochemical staining. Mice with tuberous sclerosis complex 1 (TSC1) or Rheb deletion specifically in the myeloid lineage were generated and subjected to intra-articular injection of collagenase (collagenase-induced osteoarthritis, CIOA) and destabilisation of the medial meniscus (DMM) surgery to induce OA. Cartilage damage and osteophyte size were measured by Osteoarthritis Research Society International score and micro-CT, respectively. mRNA sequencing was performed in M1 and control macrophages. Mice and ATDC5 cells were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in OA. Results M1 but not M2-polarised macrophages accumulated in human and mouse OA synovial tissue. TSC1 deletion in the myeloid lineage constitutively activated mechanistic target of rapamycin complex 1 (mTORC1), increased M1 polarisation in synovial macrophages and exacerbated experimental OA in both CIOA and DMM models, while Rheb deletion inhibited mTORC1, enhanced M2 polarisation and alleviated CIOA in mice. The results show that promoting the macrophage M1 polarisation leads to exacerbation of experimental OA partially through secretion of Rspo2 and activation of β-catenin signalling in chondrocytes. Conclusions Synovial macrophage M1 polarisation exacerbates experimental CIOA partially through Rspo2. M1 macrophages and Rspo2 are potential therapeutic targets for OA treatment.

The mammalian target of rapamycin, mTOR, is a central regulator of cell growth. Its activity is regulated by Rheb, a Ras-like small guanosine triphosphatase (GTPase), in response to growth factor stimulation and nutrient availability. We show that Rheb regulates mTOR through FKBP38, a member of the FK506-binding protein (FKBP) family that is structurally related to FKBP12. FKBP38 binds to mTOR and inhibits its activity in a manner similar to that of the FKBP12-rapamycin complex. Rheb interacts directly with FKBP38 and prevents its association with mTOR in a guanosine 5′-triphosphate (GTP)–dependent manner. Our findings suggest that FKBP38 is an endogenous inhibitor of mTOR, whose inhibitory activity is antagonized by Rheb in response to growth factor stimulation and nutrient availability.

It has been established that reactive oxygen species (ROS) such as H₂O₂ or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-κB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H₂O₂ or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early stage both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes. At late stage, MTORC1-p-EIF2AK3-mediated ERS apoptosis were predominant, while p-ERN1 and autophagic flux were inhibited. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis, and reduced TMJ cartilage loss induced by UAC. In contrast, MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1, an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA. Abbreviations : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A₁; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4ʹ,6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.