Tissue regeneration and homeostasis through functional Met/HGF receptor modification associated with tissue injury.

通过与组织损伤相关的功能性 Met/HGF 受体修饰来实现组织再生和稳态。

基本信息

批准号：
13470037
负责人：
MATSUMOTO Kunio
金额：
$ 6.72万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

HGF is involved in regeneration of various tissues, including the liver. HGF exerts its biological activities in injured tissue-specific manner, whereas mechanisms involved in the injured tissue-specific responsiveness/activation of the c-Met remain unknown. Elucidation of injured tissue-specific c-Met activation is expected to lead to understanding of fundamental mechanisms of tissue regeneration and homeostasis. This research focused to elucidate regulatory mechanisms for c-Met receptor function in signal transduction and their significance in tissue regeneration and homeostasis.1.Regulation of cellular responsiveness associated with c-Met Ser985 phosphorylation :We found that 1) c-Met receptor tyrosine phosphorylation/activation upon HGF-stimulus is regulated with a reciprocal manner to c-Met Ser985 phosphorylation, 2) c-Met Ser985 phosphorylation is bidirectionally regulated by PKC and PP2A (protein phosphatase-2A), i.e., it is phosphorylated by PKC instead dephosphorylated by PP2A … More , and 3) c-Met Ser985 phosphorylation is induced oxidative stress of cells these cells wherein c-Met Ser985 is phosphorylated are incapable of responding to extracellular HGF-stimulus. In addition, c-Met Ser985 phosphorylation is regulated through cell-cell adhesiveness in hepatocytes in primary culture, suggesting that change in cell-cell adhesiveness in response to tissue and cellular injury may affect Ser985 phosphorylation, thereby regulating cellular responsiveness to HGF.2.Analysis of physiological significance of c-Met receptor juxtamembrane region including Ser985 through transgenic mice :The juxtamembrane region of the c-Met receptor contains Ser985 and is considered to be negative regulatory domain for HGF-dependent tyrosine phosphorylation. To elucidate physiological significance of the negative regulation of c-Met receptor function, we planned to prepare genetically modified mice (knock-in mice) wherein mutated c-Met receptor deleted with the juxtamembrane (Δjxt-c-Met) is expressed, instead of full-length c-Met. ES cells having knock-in constract were selected and chimeric mice were obtained.3.In vivo injured tissue-specific activation of c-Met and its relationship to Ser985 phosphorylation:c-Met receptor tyrosine phosphorylation was induced in the injured liver 24-48 hr after CC14-administration in mice, whereas c-Met receptor was not activated in intact organs. Instead, c-Met Ser985 was phosphorylated in intact liver, whereas it was dephosphorylated 24-48 hr after liver injury. Thus, c-Met Ser985 and c-Met tyrosine phosphorylation is reciprocally regulated in response to tissue injury, suggesting that Ser985 phosphorylation is a mechanism responsible for injured tissue-specific modification of the c-Met receptor function.4.Discussion :Ser985 phosphorylation mediated by activation of PKC and PP2A is likely to play a role in the regulation of c-Met receptor activation and cellular responsiveness to HGF, which depend on the extracellular environment and stimuli. Further studies on regulatory mechanisms responsible for c-Met receptor activation through juxtamembrane domain and Ser985 phosphorylation will provide even further understanding of the HGF-c-Met system in development, tissue regeneration, and tumorigenesis. Less

HGF 参与多种组织的再生，包括肝脏，以受损组织特异性方式发挥其生物活性，而参与受损组织特异性反应/c-Met 激活的机制仍不清楚。特异性 c-Met 激活有望促进对组织再生和稳态的基本机制的理解。这项研究的重点是阐明 c-Met 受体功能在信号转导中的调节机制及其在组织再生和稳态中的意义。 1. 与 c-Met Ser985 磷酸化相关的细胞反应性调节：我们发现 1) HGF 刺激后的 c-Met 受体酪氨酸磷酸化/激活以与 c-Met Ser985 磷酸化相反的方式进行调节，2) c- Met Ser985 磷酸化受 PKC 和 PP2A（蛋白质磷酸酶-2A），即它被 PKC 磷酸化，而不是被 PP2A 去磷酸化……更多，以及 3）c-Met Ser985 磷酸化会诱导细胞的氧化应激，因此这些细胞中的 c-Met Ser985 磷酸化无法对细胞外 HGF- 做出反应此外，c-Met Ser985 磷酸化是通过细胞间调节的。原代培养物中肝细胞的粘附性，表明响应组织和细胞损伤的细胞间粘附性的变化可能影响 Ser985 磷酸化，从而调节细胞对 HGF 的反应性。 2. 通过分析 c-Met 受体近膜区（包括 Ser985）的生理意义转基因小鼠：c-Met受体的近膜区含有Ser985，被认为是HGF依赖性酪氨酸的负调节结构域为了阐明c-Met受体功能负调节的生理意义，我们计划制备表达缺失近膜的突变c-Met受体（Δjxt-c-Met）的基因改造小鼠（敲入小鼠），选择具有敲入构建体的ES细胞代替全长c-Met，获得嵌合小鼠。3.c-Met体内损伤组织特异性激活及其与体内损伤的关系。 Ser985 磷酸化：给予小鼠 CC14 后 24-48 小时，在受损肝脏中诱导 c-Met 受体酪氨酸磷酸化，而在完整器官中 c-Met 受体未激活，相反，c-Met Ser985 在完整肝脏中被磷酸化。而在肝损伤后 24-48 小时，c-Met Ser985 和 c-Met 酪氨酸被去磷酸化。磷酸化在组织损伤时相互调节，表明 Ser985 磷酸化是一种负责损伤组织特异性修饰 c-Met 受体功能的机制。 4.讨论：PKC 和 PP2A 激活介导的 Ser985 磷酸化很可能发挥重要作用。 c-Met 受体激活和细胞对 HGF 反应的调节作用，这取决于细胞外环境和刺激的调节机制的进一步研究。通过近膜结构域和 Ser985 磷酸化激活 c-Met 受体将进一步了解 HGF-c-Met 系统在发育、组织再生和肿瘤发生中的作用。