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Local injection of tumor necrosis factor-alpha (TNF-α) at bone fracture sites during the early stage of the inflammatory response is reported to improve fracture repair in a murine model. However, the underlying mechanism is unclear. Endochondral bone formation, a process that is highly related to fracture repair, requires a certain amount of chondrocyte hypertrophy. This study aimed to investigate the effect of TNF-α on the differentiation of murine chondrogenic ATDC5 cells and the underlying mechanism. In this study, improved chondrogenic differentiation of ATDC5 cells was achieved by brief TNF-α stimulation. Moreover, the expression of Yes-associated protein 1 (YAP1) was suppressed after brief TNF-α stimulation. The expressions of inflammatory mediators and chondrogenic and hypertrophic-associated genes in ATDC5 cells triggered by TNF-α were suppressed in the YAP1 overexpression group but enhanced in the YAP1 knockdown group. Mechanistically, TNF-α-induced activation of the 5' AMP-activated protein kinase (AMPK) signaling pathway was regulated by YAP1, as revealed by the phosphorylated-AMPK/AMPK change ratios in the YAP1 overexpression and knockdown groups, respectively. Moreover, the potential for TNF-α to enhance chondrogenic differentiation could be partially reversed with an AMPK inhibitor. Taken together, we demonstrate, for the first time, that YAP1 modulates the ability of TNF-α to enhance chondrocyte differentiation partly through AMPK signaling.

Yes-associated protein 1 (YAP1) transcriptional coactivator has recently been identified to regulate skeletal lineage cell differentiation and bone development. However, the role and molecular mechanisms of YAP1 in the regulation of osteoblastic differentiation remains to be elucidated. In this study, we demonstrated that YAP1 expression was increased during osteogenic differentiation of rat bone mesenchymal stem cells and MC3T3-E1. YAP1 overexpression MC3T3-E1 showed increased expression of osteogenesis markers, such as runt-related transcription factor 2, osteocalcin, and osteopontin, as well as alkaline phosphatase and alizarin red staining. Conversely, YAP1 knockdown significantly suppressed MC3T3-E1 osteoblastic differentiation. Mechanistically, we found that YAP1 overexpression upregulated the mRNA and protein expression of the inhibitor of differentiation/DNA binding 1 (ID1), which was contrary to the results of YAP1-knockdown group. Moreover, the early osteogenic differentiation of MC3T3-E1 cells was enhanced by ID1 overexpression. Furthermore, transient transfection with exogenous ID1 overexpression plasmid completely recaptured the decreased effects of YAP1 knockdown on MC3T3-E1 cell differentiation. In addition, β-catenin and AMP-activated protein kinase signaling pathways participated in YAP1 regulation processes. Taken together, our study suggests that YAP1 is a crucial modulator of osteoblast differentiation in vitro, and provides insight into the mechanism by which YAP1 regulates osteoblast differentiation.

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.

Yes-associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1-knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad-dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP-stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad-dependent BMP and Erk1/2 signaling pathways.

Yes-associated protein 1 (YAP1), the core downstream effector of the Hippo signaling cascade, was involved in the regulation of osteoblast and osteoclast differentiation and in bone metabolism. However, the regulatory effects and mechanisms of YAP1 on bone-remodeling molecules in osteoblasts under inflammation remain unknown. In this study, YAP1 expression level was downregulated after treatment with inflammatory cytokine tumor necrosis factor-α (TNF-α) in MC3T3-E1 cells. The key osteoclastogenic molecules induced by TNF-α, namely, interleukin-6 and receptor activator of nuclear factor-κB (NF-κB) ligand, were suppressed after lentivirus-induced YAP1 overexpression, which dramatically increased the expression level of osteoprotegerin. Conversely, the expression levels of the above factors showed opposite trends in the YAP1 small interfering RNA and YAP1 inhibitor (verteporfin) group. Mechanistically, YAP1 attenuated the TNF-α-induced activation of the NF-κB signaling pathway as revealed by the reduced expression of phosphorylated-p65 and NF-κB reporter activity and the nuclear translocation of p65. Moreover, the expression level of YAP1 suppressed by TNF-α was reversed by berberine in concentration-dependent manner. Taken together, our study suggests that YAP1 plays a critical role in the regulation of bone metabolism and is a potential therapeutic target for treating inflammatory bone resorption.

DDIT3 is of great importance in endoplasmic reticulum stress and is involved in many inflammatory diseases and mineralization processes. The cementum protects teeth from periodontitis and provides attachment for Sharpey's fibers of the periodontal ligament. However, the effect of DDIT3 on cementoblast differentiation remains largely unknown. In this study, we found that DDIT3 was suppressed during cementoblast differentiation. Knockdown of DDIT3 increased the messenger RNA (mRNA) and protein levels of several key osteogenic markers in vitro, including alkaline phosphatase, runt-related transcription factor 2, and osteocalcin (OCN). In addition, isocitrate dehydrogenase 1 (IDH1) was increased during cementoblast differentiation, and knockdown of DDIT3 increased the protein and mRNA levels of IDH1. Furthermore, inhibition of IDH1 could partially reduce the effect of DDIT3 on cementoblast differentiation. The DDIT3 knockdown activated nuclear factor-κB (NF-κB) transcriptional activity and upregulated the expression of p-p65 and p-IκBα. The increased osteogenic differentiation ability and IDH1 expression, as induced by the DDIT3 knockdown, could be partially turned over by the addition of NF-κB inhibitor BAY 11-7082. Overall, our data clarified that DDIT3 suppresses cementoblast differentiation through IDH1, via the NF-κB pathway.

YAP1 (Yes-associated protein 1) transcriptional coactivator is a downstream gene of the Hippo signaling pathway, which controls cell proliferation and differentiation. YAP1 plays a significant role in the regulation of cartilage and bone development. However, the molecular mechanism by which YAP1 regulates chondrocyte differentiation remains to be elucidated. Immunofluorescent staining was used to visualize the localization of YAP1 expression in the mouse chondroprogenitor ATDC5 cell line. ATDC5 cells with lentivirus-vector-mediated YAP1 overexpression and knockdown were established. The differentiation abilities were examined by real-time quantitative PCR and two staining methods The expression levels of sex-determining region Y-type high mobility group box protein (SOX9) and key proteins in the Wnt/β-catenin pathway were analyzed by Western blot. The Dickkopf-1 (Dkk1) and small interfering RNA (siRNA) of β-catenin were used for further study. The YAP1 protein was mainly expressed in the nucleus of ATDC5 cells. YAP1 overexpression enhanced chondrocyte proliferation but inhibited chondrocyte differentiation, which were contrary to the findings of the YAP1-knockdown group. Moreover, YAP1 overexpression activated Wnt/β-catenin signaling pathway. Treatment with exogenous DKK1 and β-catenin siRNA partially recaptured the effects of YAP1 overexpression on ATDC5 cell differentiation. Taken together, our study suggested that YAP1 attenuated ATDC5 cell chondrogenic and hypertrophic differentiation. We also demonstrated that YAP1 exerted its effect on the chondrocyte differentiation by activating the Wnt/β-catenin signaling pathway.