Molecular Mechanisms for injury-dependent activation of c-Met, HGF receptor

在这个项目中，我们试图阐明组织识别其损伤或稳态状态的分子机制。我们特别关注了HGF受体c-Met激活状态的生理变化，得到了如下1 ~ 4.1所示的结果，(1)HGF在低细胞密度培养时容易诱导原代肝细胞的细胞增殖，但在融合培养中，HGF没有表现出这种效果。这被认为是模仿损伤肝脏实质肝细胞的反应。在这个系统中，我们发现即使在hgf刺激后，c-Met酪氨酸自磷酸化在融合细胞中也被下调。(2)氧化应激导致细胞增殖停滞，我们发现过氧化氢诱导的应激剥夺了细胞对HGF的有丝分裂反应，这是通过对c-Met的ser985磷酸化导致c-Met活化下调。(3)原代肝细胞在无脯氨酸的培养基中即使经过hgf刺激也不能增殖，而c-Met和相关的信号转导细胞很容易被激活。2、我们成功地鉴定出负责1，(1)~(3)的分子如下。(1)酪氨酸磷酸酶LAR以细胞密度依赖的方式对c-Met进行去磷酸化；(2)PKC增强了Ser985的磷酸化，过氧化氢处理抑制了靶向磷酸化Ser985的PP2A；(3)在无pro的培养基中抑制了触发细胞增殖的CDK4的表达。3、建立ES细胞构建携带Ser985缺失c-Met的转基因小鼠。

In this project, we attempted to clarify the molecular mechanisms by which the tissues recognize their injured- or homeostatic states. We especially focused on the physiological changes of activation states of c-Met, the receptor for HGF and obtained the results as descried bellow 1〜4.1, (1) HGF readily induces cellular proliferation of primary hepatocytes when they are cultured at low cellular density, but in confluent cultures, HGF failed to show the effect. which was thought to mimic the response of parenchymal) hepatocytes in injured liver. In this system, we found that c-Met tyrosine autophosphorylation was down regulated in the confluent cells even after HGF-stimulation. (2) Oxidant stress causes arrest of cellular proliferation, and we found that the stress induced by hydrogen peroxide deprives cells of mitotic response to HGF, which was caused down regulation of c-Met activation through Ser985-phosphorylation on c-Met. (3) Primary hepatocytes could not proliferate in proline-free culture medium even after HGF-stimulation, while the c-Met and the associated signal transducers were readily activated.2, We successfully identified molecules responsible for 1,(1)〜 (3) as follows. (1) A tyrosine phosphatase LAR, which dephosphorylates c-Met in a cell density dependent way, (2) PKC enhances Ser985 phosphorylation and PP2A which targets phosphorylated Ser985 was suppressed by hydrogen peroxide-treatment, (3) Expression of CDK4 that trigger the cellular proliferation was suppressed under Pro-free medium.3, We established ES cells for construction of transgenic mouse carrying Ser985 deleted c-Met.