Elucidation of molecular mechanism of endochondral ossification targeting novel therapeutic strategy for skeletal dysplasia

利钠肽家族由ANP、BNP和CNP三种内源性配体及其膜上的鸟苷酸环化酶受体GC-A和GC-B组成。结果表明，CNP-GC-B系统对软骨内骨生长有很强的刺激作用，激活CNP-GC-B系统可显著改善FGFR3激活的软骨再生模型小鼠软骨内骨生长障碍。至于机制，CNP/GC-B/cGMP通过抑制成纤维细胞生长因子信号的MAPK通路的激活来恢复减少的软骨基质合成。在本项目中，我们进一步研究了CNP-GC-B系统和成纤维细胞生长因子系统之间的细胞内信号相互作用。首先，我们证明了成纤维细胞生长因子系统同时刺激MAPK和STAT1通路，而CNP-GC-B系统不干扰STAT1通路。我们还发现，成纤维细胞生长因子信号刺激PI3/PKB通路，而CNP-GC-B系统干扰PI3/PKB通路，而PI3/PKB系统在肝素的作用下受到抑制。另一方面，激活成纤维细胞生长因子途径可剂量依赖性地减少CNP-GC-B系统第二信使cGMP的产生。这些结果揭示了软骨中CNP-GC-B系统和成纤维细胞生长因子系统的相互调节。接下来，我们利用胎鼠胫骨器官培养系统研究了软骨局部钠尿肽清除系统。利钠肽清除受体选择性激动剂C-ANF(4-23)呈剂量依赖性延长培养的胎儿胫骨，因此，我们认为局部利钠肽清除系统是软骨中CNP-GC-B系统的调节之一。

Natriuretic peptide family consists of three endogenous ligands, ANP, BNP and CNP, and their membranous guanylyl cyclase receptors, GC-A and GC-B. We have shown that CNP-GC-B system is a strong stimulator of endochondral bone growth and that disturbed endochondral bone growth in mice model of achondroplastic with an activated FGFR3 in cartilage is greatly improved by activation of CNP-GC-B system. As for the mechanism, CNP/GC-B/cGMP restores the decreased cartilaginous matrix synthesis by inhibiting activation of MAPK pathway of FGF signaling.In this project, further we investigated the intracellular signaling interaction between CNP-GC-B system and FGF system in cartilage. First we showed that FGF system stimulates both MAPK and STAT1 pathway and that CNP-GC-B system does not interfere to STAT1 pathway. We also showed that FGF signaling stimulates PI3/PKB pathway to which CNP-GC-B system interferes, and that PI3/PKB system is suppressed under the condition with heparin. On the other hand, activation of FGF pathway decreased the production of cGMP, the second messenger of CNP-GC-B system, dose-dependently. These results exhibit the reciprocal regulation of CNP-GC-B system and FGF system in cartilage.Next we investigated local natriuretic peptide clearance system in cartilage by using fetal mice tibial organ culture system. Selective agonist for natriuretic peptide clearance receptor, C-ANF (4-23), elongated cultured fetal tibiae dose-dependently, and accordingly, we concluded that local natriuretic peptide clearance system works as one of the regulator of CNP-GC-B system in cartilage.