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

HGF 参与多种组织的再生，包括肝脏。 HGF 以受损组织特异性方式发挥其生物活性，而涉及受损组织特异性 c-Met 反应/激活的机制仍然未知。阐明受损组织特异性 c-Met 激活有望有助于理解组织再生和稳态的基本机制。本研究重点阐明c-Met受体在信号转导中的功能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刺激下的c-Met受体酪氨酸磷酸化/激活以与c-Met Ser985磷酸化相反的方式调节，2）c-Met Ser985磷酸化磷酸化受 PKC 和 PP2A（蛋白磷酸酶-2A）双向调节，即，它被 PKC 磷酸化，而不是被 PP2A 去磷酸化……更多，3) c-Met Ser985 磷酸化诱导细胞氧化应激，其中 c-Met Ser985 磷酸化的这些细胞无法对细胞外 HGF 刺激做出反应。此外，原代培养的肝细胞中c-Met Ser985磷酸化是通过细胞间粘附性来调节的，提示响应组织和细胞损伤时细胞间粘附性的变化可能会影响Ser985磷酸化，从而调节细胞对HGF的反应性。2.通过转基因小鼠分析c-Met受体近膜区（包括Ser985）的生理意义：c-Met受体的近膜区含有Ser985并被认为是 HGF 依赖性酪氨酸磷酸化的负调控域。为了阐明c-Met受体功能负调节的生理意义，我们计划制备转基因小鼠（敲入小鼠），其中表达缺失近膜的突变c-Met受体（Δjxt-c-Met），而不是全长c-Met。选择具有敲入契约的ES细胞，获得嵌合小鼠。 3.体内损伤组织特异性c-Met激活及其与Ser985磷酸化的关系：给予小鼠CC14后24-48小时，损伤肝脏中c-Met受体酪氨酸磷酸化，而完整器官中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 小时则去磷酸化。因此，c-Met Ser985 和 c-Met 酪氨酸磷酸化在组织损伤时相互调节，表明 Ser985 磷酸化是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 磷酸化很可能在调节 c-Met 受体激活和细胞对 HGF 的反应性中发挥作用，这取决于与细胞外环境和刺激有关。对通过近膜结构域和 Ser985 磷酸化负责 c-Met 受体激活的调节机制的进一步研究将有助于进一步了解 HGF-c-Met 系统在发育、组织再生和肿瘤发生中的作用。较少的

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