Excessive mechanical loading promotes osteoarthritis development by upregulating Rcn2.

Exposure of articular cartilage to excessive mechanical loading is closely related to the pathogenesis of osteoarthritis (OA). However, the exact molecular mechanism by which excessive mechanical loading drives OA remains unclear. In vitro, primary chondrocytes were exposed to cyclic tensile strain at 0.5 Hz and 10 % elongation for 30 min to simulate excessive mechanical loading in OA. In vivo experiments involved mice undergoing anterior cruciate ligament transection (ACLT) to model OA, followed by interventions on Rcn2 expression through adeno-associated virus (AAV) injection and tamoxifen-induced gene deletion. 10 µL AAV2/5 containing AAV-Rcn2 or AAV-shRcn2 was administered to the mice by articular injection at 1 week post ACLT surgery, and Col2a1-creERT: Rcn2flox/flox mice were injected with tamoxifen intraperitoneally to obtain Rcn2-conditional knockout mice. Finally, we explored the mechanism of Rcn2 affecting OA. Here, we identified reticulocalbin-2 (Rcn2) as a mechanosensitive factor in chondrocytes, which was significantly elevated in chondrocytes under mechanical overloading. PIEZO type mechanosensitive ion channel component 1 (Piezo1) is a critical mechanosensitive ion channel, which mediates the effect of mechanical loading on chondrocytes, and we found that increased Rcn2 could be suppressed through knocking down Piezo1 under excessive mechanical loading. Furthermore, chondrocyte-specific deletion of Rcn2 in adult mice alleviated OA progression in the mice receiving the surgery of ACLT. On the contrary, articular injection of Rcn2-expressing adeno-associated virus (AAV) accelerated the progression of ACLT-induced OA in mice. Mechanistically, Rcn2 accelerated the progression of OA through promoting the phosphorylation and nuclear translocation of signal transducer and activator of transcription 3 (Stat3).

Inhibition of cyclooxygenase-2 activity in subchondral bone modifies a subtype of osteoarthritis.

Osteoarthritis (OA) causes the destruction of joints. Its pathogenesis is still under investigation, and there is no effective disease-modifying therapy. Here, we report that elevated cyclooxygenase-2 (COX-2) expression in the osteocytes of subchondral bone causes both spontaneous OA and rheumatoid arthritis (RA). The knockout of COX-2 in osteocytes or treatment with a COX-2 inhibitor effectively rescues the structure of subchondral bone and attenuates cartilage degeneration in spontaneous OA (STR/Ort) mice and tumor necrosis factor-α transgenic RA mice. Thus, elevated COX-2 expression in subchondral bone induces both OA-associated and RA-associated joint cartilage degeneration. The inhibition of COX-2 expression can potentially modify joint destruction in patients with arthritis.

Inhibition of overactive TGF-ß attenuates progression of heterotopic ossification in mice.

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-ß initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-ß that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-ß in tendon induces spontaneous HO, whereas systemic injection of a TGF-ß neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-ß type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-ß as inducers and promoters of ectopic bone formation, and suggest that TGF-ß might be a therapeutic target in HO.

MiR-497∼195 cluster regulates angiogenesis during coupling with osteogenesis by maintaining endothelial Notch and HIF-1α activity.

A specific bone vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), is identified as coupling angiogenesis and osteogenesis. The abundance of type CD31hiEmcnhi vessels decrease during ageing. Here we show that expression of the miR-497∼195 cluster is high in CD31hiEmcnhi endothelium but gradually decreases during ageing. Mice with depletion of miR-497∼195 in endothelial cells show fewer CD31hiEmcnhi vessels and lower bone mass. Conversely, transgenic overexpression of miR-497∼195 in murine endothelium alleviates age-related reduction of type CD31hiEmcnhi vessels and bone loss. miR-497∼195 cluster maintains the endothelial Notch activity and HIF-1α stability via targeting F-box and WD-40 domain protein (Fbxw7) and Prolyl 4-hydroxylase possessing a transmembrane domain (P4HTM) respectively. Notably, endothelialium-specific activation of miR-195 by intravenous injection of aptamer-agomiR-195 stimulates CD31hiEmcnhi vessel and bone formation in aged mice. Together, our study indicates that miR-497∼195 regulates angiogenesis coupled with osteogenesis and may represent a potential therapeutic target for age-related osteoporosis.

MiR-142-5p promotes bone repair by maintaining osteoblast activity.

MicroRNAs play important roles in regulating bone regeneration and remodeling. However, the pathophysiological roles of microRNAs in bone repair remain unclear. Here we identify a significant upregulation of miR-142-5p correlated with active osteoblastogenesis during the bone healing process. In vitro, miR-142-5p promoted osteoblast activity and matrix mineralization by targeting the gene encoding WW-domain-containing E3 ubiquitin protein ligase 1. We also found that the expression of miR-142-5p in the callus of aged mice was lower than that in the callus of young mice and directly correlated with the age-related delay in bone healing. Furthermore, treatment with agomir-142-5p in the fracture areas stimulated osteoblast activity which repaired the bone fractures in aged mice. Thus, our study revealed that miR-142-5p plays a crucial role in healing fractures by maintaining osteoblast activity, and provided a new molecular target therapeutic strategy for bone healing.

GDF11 Inhibits Bone Formation by Activating Smad2/3 in Bone Marrow Mesenchymal Stem Cells.

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß superfamily. Recent studies confirmed that GDF11 plays an important role in regulating the regeneration of brain, skeletal muscle, and heart during aging; however, its role in bone metabolism remains unclear. Thus, the aim of this study was to determine the effects of GDF11 on bone metabolism, including bone formation and bone resorption, both in vitro and in vivo. Our results showed that GDF11 inhibited osteoblastic differentiation of bone marrow mesenchymal stem cells in vitro. Mechanistically, GDF11 repressed Runx2 expression by inducing SMAD2/3 phosphorylation during osteoblast differentiation. Moreover, intraperitoneal injection of GDF11 inhibited bone formation and accelerated age-related bone loss in mice. Our results also showed that GDF11 had no effect on osteoclast differentiation or bone resorption both in vitro and in vivo. These results provide a further rationale for the therapeutic targeting of GDF11 for the treatment of age-related osteoporosis.