Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.     

阻断ERK1/2激酶可增强骨形态发生蛋白9诱导的间充质干细胞成骨分化

骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有很强的诱导间充质干细胞定向成骨分化的能力.但对于其所涉及的相关分子机理了解并不深入.利用BMP9重组腺病毒感染间充质干细胞,Western blot检测ERK1/2激酶的磷酸化,ERK1/2的特异性抑制剂PD98059阻断ERK1/2活性,或以RNA干扰抑制ERK1/2表达,通过体外细胞实验和体内动物实验,初步分析和揭示ERK1/2对于BMP9诱导的间充质干细胞成骨分化的调控作用及其可能机制.结果发现:BMP9可以促进ERK1/2激酶的磷酸化,ERK1/2抑制剂PD98059可增强由BMP9诱导的碱性磷酸酶(alkaline phosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,并促进由BMP9诱导的Runx2基因的表达和转录活性,以及Smad经典途径的活化;而RNA干扰导致ERK1/2基因沉默同样也可进一步促进BMP9诱导的ALP活性和钙盐沉积,并促进BMP9诱导的间充质干细胞在裸鼠皮下异位成骨.因此,BMP9可以促进ERK1/2蛋白激酶的活化,而阻断ERK1/2蛋白激酶可进一步增强BMP9诱导的成骨分化,ERK1/2极可能对于BMP9诱导的间充质干细胞成骨分化起着负向调控作用.     

Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins

Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1(-/-) mice. Vertebral bone volume and strength of TNF-Tg/Smurf1(-/-) mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1(-/-) mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.