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In vivo role of the GDNF/RET signaling pathway in morphogenesis and pathogenesis

GDNF/RET 信号通路在形态发生和发病机制中的体内作用

基本信息

批准号：
15209014
负责人：
TAKAHASHI Masahide
金额：
$ 32.2万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

The RET receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in RET represents a binding site for the PTB domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, PI3K/AKT, and JNK pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice were generated in which tyrosine 1062 in the Ret gene was replaced with phenylalanine. Although homozygous knock-in mice were born normally, they died by day 27 after birth with growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract as observed in Ret-deficient mice. The rest of the mutant mice exhibited the development of enteric neurons in the intestine to varying extent, although the size and number of ganglion cells were … More significantly reduced. Unlike Ret-deficient mice, the small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.In addition, we demonstrated that the Rac1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of Ret in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the Ret signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not Erk, Akt and Src activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of Ret function regulates JNK signaling responsible for proper migration of the ENCCs in the developing gut. Less

RET受体酪氨酸激酶在肠神经系统和肾脏的发育中起着至关重要的作用。RET中的酪氨酸1062代表了几种衔接子和效应蛋白的PTB结构域的结合位点，这些衔接子和效应蛋白对于细胞内信号传导途径（例如RAS/ERK、PI 3 K/AKT和JNK途径）的活化是重要的。为了研究酪氨酸1062对体内器官发生的重要性，产生了Ret基因中的酪氨酸1062被苯丙氨酸取代的敲入小鼠。虽然纯合子基因敲入小鼠出生正常，但它们在出生后第27天死亡，生长迟缓。在纯合突变小鼠中肠神经系统的发育严重受损，其中约40%的小鼠在整个肠道中缺乏肠神经元，如在RET缺陷小鼠中观察到的。其余的突变小鼠表现出不同程度的肠神经元在肠中的发育，尽管神经节细胞的大小和数量与正常小鼠相比有所不同。 ...更多信息大幅减少。与RET缺陷小鼠不同，所有基因敲入小鼠均发育出小肾脏，并伴有轻微的组织学变化。肾脏缩小是由于胚胎发育过程中输尿管芽分支减少所致。因此，这些结果表明，通过酪氨酸1062信号在肠神经系统和肾发生的组织发生中起着重要作用，此外，我们证明，Rac 1/JNK途径是由丝氨酸磷酸化的Ret的质膜区域在cAMP依赖的方式进行调节。为了确定cAMP依赖性修饰的Ret信号在体内的重要性，我们产生了突变小鼠，其中丝氨酸残基697，一个假定的蛋白激酶A（PKA）磷酸化位点，被替换为丙氨酸（指定S697 A小鼠）。纯合子S697 A突变小鼠由于肠神经嵴细胞（ENCC）迁移缺陷，导致远端结肠缺乏ENS。体外器官培养显示突变ENCC对GDNF的趋化反应受损。GDNF对JNK的激活作用在突变小鼠的神经元中显著降低，而Erk、Akt和Src的激活作用则没有显著降低。JNK抑制剂SP 600125和PKA抑制剂KT 5720抑制来自野生型小鼠的培养肠道中的ENCC的迁移至可比较的程度。因此，这些发现表明，cAMP依赖性的Ret功能修饰调节JNK信号传导，负责ENCC在发育中的肠道中的适当迁移。少