Machide Mitsuru, Hashigasako Atsuko, Matsumoto Kunio, Nakamura Toshikazu
Division of Molecular Regenerative Medicine, Department of Biochemistry and Molecular Biology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2006 Mar 31;281(13):8765-72. doi: 10.1074/jbc.M512298200. Epub 2006 Jan 16.
Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, we investigated the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF), focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at a sparse cell density, HGF stimulation induced prolonged c-Met tyrosine phosphorylation for over 5 h and a marked mitogenic response. In contrast, HGF stimulation induced transient c-Met tyrosine phosphorylation in <3 h and failed to induce mitogenic response in hepatocytes cultured at a confluent cell density. Treatment of the confluent cells with HGF plus orthovanadate, a broad spectrum protein-tyrosine phosphatase inhibitor, however, prolonged c-Met tyrosine phosphorylation for over 5 h and permitted the subsequent mitogenic response. The mitogenic response to HGF was associated with the duration of c-Met tyrosine phosphorylation even in the sparse cells. We found that the activity and expression of the protein-tyrosine phosphatase LAR increased following HGF stimulation specifically in confluent hepatocytes and not in sparse hepatocytes. LAR and c-Met were associated, and purified LAR dephosphorylated tyrosine-phosphorylated c-Met in in vitro phosphatase reactions. Furthermore, antisense oligonucleotides specific for LAR mRNA suppressed the expression of LAR, allowed prolonged c-Met tyrosine phosphorylation, and led to acquisition of a mitogenic response in hepatocytes even under the confluent condition. Thus functional association of LAR and c-Met underlies the inhibition of c-Met-mediated mitogenic signaling through the dephosphorylation of c-Met, which specifically occurs under the confluent condition.
接触抑制，即细胞间紧密接触对细胞增殖的抑制，是正常细胞的一个基本特征。我们使用原代培养的肝细胞，研究了接触抑制降低肝细胞生长因子（HGF）促有丝分裂活性的机制，重点关注c-Met/HGF受体激活的调节。在低密度培养的肝细胞中，HGF刺激诱导c-Met酪氨酸磷酸化持续超过5小时，并引发显著的有丝分裂反应。相比之下，在汇合细胞密度培养的肝细胞中，HGF刺激诱导c-Met酪氨酸磷酸化在3小时内短暂出现，且未能诱导有丝分裂反应。然而，用HGF加原钒酸钠（一种广谱蛋白酪氨酸磷酸酶抑制剂）处理汇合细胞，可使c-Met酪氨酸磷酸化延长超过5小时，并允许随后的有丝分裂反应。即使在稀疏细胞中，对HGF的有丝分裂反应也与c-Met酪氨酸磷酸化的持续时间相关。我们发现，蛋白酪氨酸磷酸酶LAR的活性和表达在HGF刺激后特异性地在汇合肝细胞中增加，而在稀疏肝细胞中未增加。LAR与c-Met相关，并且纯化的LAR在体外磷酸酶反应中使酪氨酸磷酸化的c-Met去磷酸化。此外，针对LAR mRNA的反义寡核苷酸抑制了LAR的表达，使c-Met酪氨酸磷酸化延长，并导致肝细胞即使在汇合条件下也获得有丝分裂反应。因此，LAR和c-Met的功能关联通过c-Met的去磷酸化构成了对c-Met介导的有丝分裂信号传导的抑制，这种去磷酸化特异性地发生在汇合条件下。
