肥大软骨细胞自噬调控Notch1/β-catenin在骨形成和骨重塑中的作用机制研究

Chondrocyte hypertrophy is the central step of endochondral ossification, which is essential for physiological skeletal development. Hypertrophic chondrocytes not only produce factors to regulate osteoclastogenesis at the chondroosseous front, but also transdifferentiate into osteoblasts involved in endochondral bone formation. There is growing awareness that dysregulation of the autophagic response is involved in the pathogenesis of skeletal disorders, yet the role and underlying mechanisms of autophagy in hypertrophic chondrocytes remain unclear. Of note, our previous study indicated that Atg7fl/fl; Col2a1-CreERT2 mice exhibited growth retardation associated with reduced chondrocyte proliferation and hypertrophic differentiation. We also found that suppression of autophagy in hypertrophic chondrocytes resulted in an increased Notch1 level but a decreased β-catenin level. Based on the evidence, we proposed that autophagy in hypertrophic chondrocytes may regulate both the early bone formation and the later bone remodeling through Notch1/β-catenin dependent manner. To test this hypothesis, we generated conditional deletion of Atg7 in hypertrophic chondrocytes by a Col10a1-Cre line to investigate the direct role of autophagy during developmental and remodeling stages using microCT、histological stainings as well as immunohistochemistry. Especially, we will clarify the underlying molecular mechanisms utilizing double-knockout mice as well as multiple cell models. The study will contribute to a better understanding of the functional mechanism of autophagy in endochondral ossification, and may provide new insights into the treatment of skeletal disorders, especially osteoporosis.

肥大软骨细胞不仅是软骨内成骨的核心，它还可分泌多种细胞因子调控破骨细胞生成，或转分化为成骨细胞参与骨形成。研究证实骨细胞自噬调节异常参与多种骨骼疾病，而自噬在肥大软骨细胞中的具体作用和机制不明。申请人前期发现：使用Col2a1-Cre鼠敲除自噬基因Atg7显著抑制软骨内成骨；抑制肥大软骨细胞自噬可上调Notch1同时下调β-catenin的表达。据此我们推测：肥大软骨细胞自噬可能通过Notch1/β-catenin调控骨形成及骨重塑。本项目拟利用肥大软骨细胞特异性Atg7敲除鼠，采用Micro-CT、形态学染色、免疫组化等方法，明确抑制肥大软骨细胞自噬对骨形成及骨重塑的作用；运用抑制/激活自噬、干扰表达/过表达及细胞共培养模型、双敲除小鼠模型阐明肥大软骨细胞自噬调控骨形成及骨重塑的分子机制。本项目有助于全面理解自噬在软骨内成骨中的作用机制，为骨骼疾病尤其是骨质疏松的治疗提供新思路。

肥大软骨细胞不仅是软骨内成骨的核心，它还可分泌多种细胞因子调控破骨细胞生成，或转分化为成骨细胞参与骨形成。研究证实骨细胞自噬调节异常参与多种骨骼疾病，而自噬在肥大软骨细胞中的具体作用和机制不明。本项目利用肥大软骨细胞特异性Atg7敲除鼠，采用Micro-CT、形态学染色、免疫组化等方法，明确抑制肥大软骨细胞自噬对骨形成及骨重塑的作用；运用抑制/激活自噬、干扰表达/过表达及细胞共培养模型、体内注射腺相关病毒载体等小鼠模型阐明肥大软骨细胞自噬调控骨形成及骨重塑的分子机制。我们的结果表明，在肥大软骨细胞中敲除自噬可以抑制生长板软骨细胞的肥大分化，进而抑制肥大软骨细胞转分化为成骨细胞，抑制骨形成及骨发育过程。在机制上，自噬缺陷导致Notch1蛋白降解减少，进而抑制β-catenin的表达及活性，抑制软细胞肥大分化及转分化。本项目有助于全面理解自噬在骨发育中的作用机制，为骨发育不良相关疾病的创新分子药物研发及临床转化奠定基础。