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

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代表了几种衔接蛋白和效应蛋白的PTB结构域的结合位点，这些衔接蛋白和效应蛋白对细胞内信号通路的激活很重要，如RAS/ERK、PI3K/AKT和JNK通路。为了研究酪氨酸1062在体内器官发生中的重要性，我们用苯丙氨酸代替Ret基因中的酪氨酸1062来产生敲入小鼠。虽然纯合子敲入小鼠正常出生，但它们在出生后第27天死亡，生长迟缓。纯合突变小鼠的肠神经系统发育严重受损，在ret缺乏小鼠中观察到，约40%的小鼠在整个肠道中缺乏肠神经元。其余的突变小鼠在不同程度上表现出肠内神经元的发育，但神经节细胞的大小和数量明显减少。与ret缺乏小鼠不同的是，所有敲入小鼠的肾脏都很小，并伴有轻微的组织学改变。肾脏大小的减小是由于胚胎发生时输尿管芽分支减少所致。因此，这些发现表明，通过酪氨酸1062的信号在肠神经系统的组织发生和肾发生中起重要作用。此外，我们证明了Rac1/JNK通路以camp依赖的方式受到Ret近膜区域丝氨酸磷酸化的调节。为了确定camp依赖性Ret信号在体内修饰的重要性，我们产生了一种突变小鼠，其中丝氨酸残基697（假定的蛋白激酶a （PKA）磷酸化位点）被丙氨酸取代（指定为S697A小鼠）。纯合子S697A突变小鼠在远端结肠缺乏ENS，这是由于肠神经嵴细胞（ENCCs）迁移缺陷造成的。体外器官培养显示突变ENCCs对GDNF的化学引诱反应受损。在突变小鼠的神经元中，GDNF对JNK的激活而不是Erk、Akt和Src的激活显著降低。JNK抑制剂SP600125和PKA抑制剂KT5720在相当程度上抑制野生型小鼠培养肠道中ENCCs的迁移。因此，这些研究结果表明，camp依赖性的Ret功能修饰调节了JNK信号，这些信号负责发育中的肠道中ENCCs的适当迁移。少

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