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

利钠肽家族由三种内源性配体ANP、BNP和CNP及其膜上鸟苷酸环化酶受体GC-A和GC-B组成。我们已经表明，CNP-GC-B系统是软骨内骨生长的强刺激剂，并且通过激活CNP-GC-B系统，在软骨中具有活化的FGFR 3的软骨发育不全小鼠模型中，紊乱的软骨内骨生长得到极大改善。CNP/GC-B/cGMP通过抑制FGF信号通路中MAPK通路的激活，恢复了软骨基质合成的下降。本课题进一步研究了软骨中CNP-GC-B系统与FGF系统的胞内信号相互作用。我们首先发现FGF系统同时刺激MAPK和STAT 1通路，而CNP-GC-B系统不干扰STAT 1通路。我们还发现FGF信号刺激了PI 3/PK B通路，而CNP-GC-B系统干扰了该通路，并且在肝素的条件下，PI 3/PK B系统被抑制。另一方面，FGF途径的激活可剂量依赖性地减少CNP-GC-B系统第二信使cGMP的产生。这些结果表明软骨中CNP-GC-B系统和FGF系统的相互调节作用。选择性利钠肽清除受体激动剂C-ANF（4-23）可剂量依赖性地延长培养的胎鼠软骨细胞，因此，我们认为局部利钠肽清除系统是软骨中CNP-GC-B系统的调节者之一。

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.