Mechanical forces are fundamental regulators of cell behaviors. However, molecular regulation of mechanotransduction remain poorly understood. Here, we identified the mechanosensitive channels Piezo1 and Piezo2 as key force sensors required for bone development and osteoblast differentiation. Loss of Piezo1, or more severely Piezo1/2, in mesenchymal or osteoblast progenitor cells, led to multiple spontaneous bone fractures in newborn mice due to inhibition of osteoblast differentiation and increased bone resorption. In addition, loss of Piezo1/2 rendered resistant to further bone loss caused by unloading in both bone development and homeostasis. Mechanistically, Piezo1/2 relayed fluid shear stress and extracellular matrix stiffness signals to activate Ca2+ influx to stimulate Calcineurin, which promotes concerted activation of NFATc1, YAP1 and ß-catenin transcription factors by inducing their dephosphorylation as well as NFAT/YAP1/ß-catenin complex formation. Yap1 and ß-catenin activities were reduced in the Piezo1 and Piezo1/2 mutant bones and such defects were partially rescued by enhanced ß-catenin activities.

BackgroundOsteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted.MethodsHuman bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs.ResultsThe current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling.ConclusionThe current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multif...

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gαs that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia (FD) or progressive osseous heteroplasia (POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gαs regulates Hh signaling in a ligand-independent manner. We further show that Gαs controls intramembranous ossification by regulating both Hh and Wnt/β-catenin signaling. In addition, Gαs activation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

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Adolescent idiopathic scoliosis (AIS) is the most common type of spinal deformity and has a significant genetic background. Genome-wide association studies (GWASs) identified several susceptibility loci associated with AIS. Among them is a locus on chromosome 6q24.1 that we identified by a GWAS in a Japanese cohort. The locus is represented by rs6570507 located within GPR126. To ensure the association of rs6570507 with AIS, we conducted a meta-analysis using eight cohorts from East Asia, Northern Europe and USA. The analysis included a total of 6,873 cases and 38,916 controls and yielded significant association (combined P = 2.95 × 10−20; odds ratio = 1.22), providing convincing evidence of the worldwide association between rs6570507 and AIS susceptibility. In silico analyses strongly suggested that GPR126 is a susceptibility gene at this locus.

Heterotopic ossification (HO) occurs as a common complication after injury or in genetic disorders. The mechanisms underlying HO remain incompletely understood, and there are no approved prophylactic or secondary treatments available. Here, we identify a self-amplifying, self-propagating loop of Yes-associated protein (YAP)–Sonic hedgehog (SHH) as a core molecular mechanism underlying diverse forms of HO. In mouse models of progressive osseous heteroplasia (POH), a disease caused by null mutations in GNAS, we found that Gnas−/− mesenchymal cells secreted SHH, which induced osteoblast differentiation of the surrounding wild-type cells. We further showed that loss of Gnas led to activation of YAP transcription activity, which directly drove Shh expression. Secreted SHH further induced YAP activation, Shh expression, and osteoblast differentiation in surrounding wild-type cells. This self-propagating positive feedback loop was both necessary and sufficient for HO expansion and could act independently of Gnas in fibrodysplasia ossificans progressiva (FOP), another genetic HO, and nonhereditary HO mouse models. Genetic or pharmacological inhibition of YAP or SHH abolished HO in POH and FOP and acquired HO mouse models without affecting normal bone homeostasis, providing a previously unrecognized therapeutic rationale to prevent, reduce, and shrink HO.

Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder characterized by three-dimensional spinal curvatures, affecting 2%-3% of school age children, yet the causes underlying AIS are not well understood. Here, we first conducted a whole-exome sequencing and linkage analysis on a three-generation Chinese family with autosomal-dominant (AD) AIS, and then performed targeted sequencing in a discovery cohort comprising 20 AD AIS families and 86 simplex patients, and finally identified three disease-associated missense variants (c.886G> A, c.1943C> T, and c.1760C> T) in the MAPK7 gene (encoding mitogen-activated protein kinase 7). Genotyping of the three rare variants in a Chinese replication cohort comprising 1,038 simplex patients and 1,841 controls showed that their combined allele frequency was significantly over-represented in patients as compared with controls (2.0% [41/2,076] vs. 0.7% [27/3,682]; odds ratio = 2.7; P = 2.8 × 10 ). In vitro, we demonstrated that the three MAPK7 mutants disrupted nuclear translocation in cellular models, which is necessary for the normal function of MAPK7. In vivo, we also conducted CRISPR/Cas9-mediated deletion of mapk7 in zebrafish recapitulating the characteristic phenotype of idiopathic scoliosis. Taken together, our findings suggest that rare coding variants in MAPK7 predispose to AIS, providing clues to understanding the mechanisms of AIS.

Heparan sulfate (HS) interacts with growth factors and has been implicated in regulating chondrogenesis. However, the effect of HS on TGF-β-mediated mesenchymal stem cell (MSC) chondrogenesis and molecular mechanisms remains unknown. In this study, we explored the effects of exogenous HS alone and in combination with TGF-β3 on chondrogenic differentiation of human MSCs and possible signal mechanisms. The results indicated that HS alone had no obvious effects on chondrogenic differentiation of human MSCs and TGF-β/Smad2/3 signal pathways. However, the combined TGF-β3/HS treatment resulted in a significant increase in GAG synthesis, cartilage matrix protein secretion, and cartilage-specific gene expression compared to cells treated with TGF-β3 alone. Furthermore, HS inhibited type III TGF-β receptors (TβRIII) expression and increased TGF-β3-mediated ratio of the type II (TβRII) to the type I (TβRI) TGF-β receptors and phosphorylation levels of Smad2/3. The inhibitor of the TGF-β/Smad signal, SB431542, not only completely inhibited HS-stimulated TGF-β3-mediated Smad2/3 phosphorylation but also completely inhibited the effects of HS on TGF-β3-induced chondrogenic differentiation. These results demonstrate exogenous HS enhances TGF-β3-induced chondrogenic differentiation of human MSCs by activating TGF-β/Smad2/3 signaling.