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以损伤组织特异性的方式发挥其生物活性，但c-Met的损伤组织特异性反应/激活机制尚不清楚。阐明受损组织特异性c-Met的激活有望有助于理解组织再生和动态平衡的基本机制。本研究旨在阐明c-Met受体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的调节：我们发现：1)c-Met受体酪氨酸在肝细胞生长因子刺激下的磷酸化/激活与c-Met Ser985的磷酸化是相互调节的；2)c-Met Ser985的磷酸化是由PKC和PP2A(蛋白磷酸酶-2A)双向调节的，即它被PKC磷酸化，而不是被PP2A…去磷酸化C-Met Ser985磷酸化诱导细胞氧化应激这些c-Met Ser985磷酸化的细胞不能对细胞外HGF刺激作出反应。此外，c-Met Ser985的磷酸化是通过原代培养的肝细胞的细胞-细胞粘附性来调节的，提示在组织和细胞损伤时细胞-细胞粘附性的改变可能会影响Ser985的磷酸化，从而调节细胞对HGF的反应。2.通过转基因小鼠分析包括Ser985在内的c-Met受体近膜区域的生理意义：c-Met受体的近膜区域含有Ser985，被认为是HGF依赖的酪氨酸磷酸化的负调节域。为了阐明c-Met受体功能负调控的生理意义，我们计划制备表达突变的c-Met受体的转基因小鼠(Δjxt-c-Met)，而不是全长c-Met。3.体内c-Met损伤组织特异性激活及其与Ser985磷酸化的关系：给予CC14后24-48小时，损伤小鼠肝脏c-Met受体酪氨酸磷酸化，而完整器官c-Met受体不激活。相反，c-Met Ser985在完整肝脏中被磷酸化，而它在肝损伤后24-48小时被去磷酸化。因此，c-Met Ser985和c-Met酪氨酸磷酸化在组织损伤反应中是相互调节的，提示Ser985磷酸化是导致c-Met受体功能损伤的组织特异性修饰的一个机制。4.讨论：通过激活PKC和PP2A介导的Ser985磷酸化可能在调节c-Met受体激活和细胞对HGF的反应性中发挥作用，这依赖于细胞外环境和刺激。进一步研究通过膜旁结构域和Ser985磷酸化激活c-Met受体的调控机制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

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