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

HGF参与各种组织的再生，包括肝脏。HGF以损伤组织特异性方式发挥其生物学活性，而涉及损伤组织特异性应答/c-Met激活的机制仍不清楚。阐明损伤组织特异性c-Met激活有望导致对组织再生和稳态的基本机制的理解。本研究旨在阐明c-Met受体在信号转导中的调控机制及其在组织再生和体内平衡中的意义。1. c-Met Ser 985磷酸化相关的细胞反应性调节：我们发现：1）HGF刺激后c-Met受体酪氨酸磷酸化/激活与c-Met Ser 985磷酸化以相互作用的方式调节; 2）c-Met Ser 985磷酸化受PKC和PP 2A（蛋白磷酸酶-2A）双向调节，即：它被PKC磷酸化，而不是被PP 2A去磷酸化 ...更多信息 和3）c-Met Ser 985磷酸化诱导细胞的氧化应激，其中c-Met Ser 985被磷酸化的这些细胞不能响应细胞外HGF刺激。此外，原代培养的肝细胞中c-Met Ser 985的磷酸化通过细胞-细胞间的相互作用来调节，提示组织和细胞损伤时细胞-细胞间相互作用的变化可能影响Ser 985的磷酸化，从而调节细胞对HGF的反应性。2.通过转基因小鼠分析含Ser 985的c-Met受体跨膜区的生理意义：c-Met受体的跨膜区含有Ser 985，被认为是HGF依赖的酪氨酸磷酸化的负调控结构域。为了阐明c-Met受体功能负调控的生理学意义，我们计划制备基因修饰小鼠（敲入小鼠），其中表达了缺失质膜的突变c-Met受体（Δjxt-c-Met），而不是全长c-Met。3.体内损伤组织特异性c-Met激活及其与Ser 985磷酸化的关系：CCl 4处理后24-48小时，小鼠损伤肝脏c-Met受体酪氨酸磷酸化被诱导，而完整器官c-Met受体未被激活。相反，c-Met Ser 985在完整肝脏中磷酸化，而在肝损伤后24-48小时去磷酸化。因此，c-Met Ser 985和c-Met酪氨酸磷酸化在组织损伤中受到双向调节，提示Ser 985磷酸化是c-Met受体功能受损的组织特异性修饰的机制。4.讨论：Ser 985磷酸化可能通过激活PKC和PP 2A介导，在c-Met受体激活和细胞对HGF反应性的调节中发挥作用，这依赖于细胞外环境和刺激。对c-Met受体通过质膜结构域和Ser 985磷酸化激活的调控机制的进一步研究将提供对HGF-c-Met系统在发育、组织再生和肿瘤发生中的进一步理解。少

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