From the Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,; the Department of Physiology, School of Basic Medical Science, Wuhan University, Wuhan 430072, Hubei Province, China.
From the Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
J Biol Chem. 2013 Nov 8;288(45):32229-32240. doi: 10.1074/jbc.M113.463554. Epub 2013 Sep 18.
The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.
表皮生长因子受体（EGFR）是胚胎期和出生后多个器官发育过程中的重要参与者。为了了解其在骺板软骨发育中的作用，我们生成了条件性敲除软骨细胞 EGFR 的转基因小鼠。出生后，这些小鼠在软骨膜处正常启动软骨管的形成，但这些软骨管向软骨内的挖掘受到强烈抑制，导致次级骨化中心（SOC）的形成延迟。这种延迟伴随着正常的软骨细胞肥大，但肥大软骨细胞的矿化和凋亡减少，骨髓腔边界处的破骨细胞数量减少。免疫组织化学分析表明，软骨细胞特异性 EGFR 信号的失活减少了靠近骨髓腔的肥大软骨细胞中基质金属蛋白酶（MMP9、-13 和 -14）和 RANKL（核因子-κB 配体受体激活剂）的含量，并减少了 SOC 中的软骨基质降解。对初级骺板软骨细胞中 EGFR 下游信号通路的分析表明，EGFR 信号通过经典的 Wnt/β-catenin 通路部分介导 MMP9 和 RANKL 的上调，而 EGFR 增强的 MMP13 表达不受其介导。进一步的生化研究表明，EGFR 信号刺激 LRP6 的磷酸化，增加活性 β-catenin 水平，并诱导其核转位。与这些体外研究一致，软骨细胞特异性 EGFR 活性的缺失减少了体内肥大软骨细胞中β-catenin 的含量。总之，我们的工作表明，软骨细胞特异性 EGFR 信号是 SOC 形成和骺板软骨发育过程中软骨基质降解的重要调节因子，其作用部分通过激活β-catenin 通路来介导。
