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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. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs ( Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system. [ABSTRACT FROM AUTHOR]

Sirtuin 6 (SIRT6) is a NAD-dependent deacetylase involved in lifespan regulation. To evaluate the effect of SIRT6 on osteogenesis, rat bone marrow mesenchymal stem cells (rBMSCs) with enhanced or reduced SIRT6 function were developed. We observed that SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin, while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor-κB (NF-κB) transcriptional activity and upregulated the expression of acetyl-NF-κB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF-κB inhibitor BAY 11-7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of collagen/chitosan/hydroxyapatite scaffold could significantly boost new bone formation in rat cranial critical-sized defects, as determined by microcomputed tomography and histological examination. These data confirm that SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF-κB signaling. S tem C ells 2014;32:1943-1955 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DNA damage-inducible transcript 3 (DDIT3), a member of the CCAAT/enhancer-binding protein (C/EBP) family, is involved in cellular apoptosis and differentiation. DDIT3 participates in the regulation of adipogenesis and osteogenesis in vitro and in vivo. However, the role of DDIT3 in osteoclastogenesis is not yet known. In this study, the involvement of DDIT3 in osteoclast differentiation and function was reported for the first time. CRISPR/Cas9-mediated DDIT3 knockout (KO) mice were generated for functional assessment. Tartrate-resistant acid phosphatase (TRAP) staining of distal femurs showed increased positive cells in DDIT3 KO mice. DDIT3 expression was downregulated during the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation of bone marrow-derived macrophages (BMMs). The loss of DDIT3 increased the expression of osteoclast-specific markers, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K (CTSK), and dendritic cell-specific transmembrane protein (DC-STAMP) and promoted the formation of TRAP-positive multinucleated osteoclasts. The actin ring number and resorption area of bone slices were also increased in DDIT3 KO BMMs. Lentivirus-mediated DDIT3 overexpression significantly inhibited the osteoclast differentiation of RAW264.7 cells. In the tumor necrosis factor-α-induced osteolysis model, DDIT3 deficiency enhanced osteoclast formation and aggravated bone resorption. DDIT3 inhibited osteoclast differentiation by regulating the C/EBPα-CTSK axis. Furthermore, DDIT3 KO intensified the RANKL-triggered activation of the MAPKs and Akt signaling pathways. Taken together, the results revealed the essential role of DDIT3 in osteoclastogenesis in vitro and in vivo and its close relationship with osteoclast-associated transcription factors and pathways. • DDIT3 deficiency enhanced osteoclastogenesis in vitro and in vivo. • DDIT3 regulated C/EBPα-CTSK axis and MAPKs and Akt pathways. • DDIT3 influenced bone mass by regulating osteoclast-mediated bone resorption. [ABSTRACT FROM AUTHOR]