Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
Biochem Biophys Res Commun. 2013 May 24;435(1):120-5. doi: 10.1016/j.bbrc.2013.04.052. Epub 2013 Apr 22.
Hepatocyte growth factor (HGF) is a key ligand that elicits G1/S progression of epithelial cells, including hepatocytes. Proline is also required for DNA synthesis that is induced by growth factors in primary culture of hepatocytes. However, it remains unknown how proline contributes to the G1/S progression of hepatocytes. The primary culture of rat hepatocytes using HGF plus proline can be a conceptual model for elucidating the molecular linkage of amino acids and growth factors during G1/S progression. Using this in vitro model, we provide evidence that not only induction of cyclin-D1 by HGF but also up-regulation of cyclin-E1 by proline is required for hepatocytes to enter the S-phase. Proline-enhanced cyclin-E1 induction, without changing its mRNA level, is associated with the activation of mammalian target of rapamycin (mTOR)-dependent pathways. Indeed, proline enhanced the ribosomal protein S6 phosphorylations (i.e., mTOR target), concomitantly with an increase in cyclin-E1. Inversely, mTOR-inhibitor, rapamycin suppressed the proline-mediated induction of cyclin-E1. As a result, DNA synthesis of hepatocytes, which was induced by HGF in the presence of proline, was largely abolished by mTOR-inhibitor treatment. Such a co-mitogenic effect of proline was also dependent on collagen synthesis: collagen synthesis inhibitors, such as cis-OH-proline, diminished the proline-induced cyclin-E1, and then the G1/S progression of hepatocytes was also suppressed. Overall, proline-mediated mTOR activation and collagen synthesis were found critical for HGF-induced DNA synthesis, partly via the sufficient accumulation of cyclin-E1. This is the first report to demonstrate the molecular bridge between amino acids and growth factors that drive mitogenic outcomes.
肝细胞生长因子 (HGF) 是一种关键的配体，可引发包括肝细胞在内的上皮细胞的 G1/S 期进展。脯氨酸也是生长因子诱导原代培养肝细胞 DNA 合成所必需的。然而，脯氨酸如何促进肝细胞的 G1/S 期进展仍不清楚。使用 HGF 加脯氨酸的大鼠原代肝细胞培养可以作为阐明氨基酸和生长因子在 G1/S 期进展过程中的分子联系的概念模型。使用这种体外模型，我们提供的证据表明，HGF 不仅诱导 cyclin-D1，而且脯氨酸上调 cyclin-E1 对于肝细胞进入 S 期也是必需的。脯氨酸增强 cyclin-E1 的诱导，而不改变其 mRNA 水平，与哺乳动物雷帕霉素靶蛋白 (mTOR) 依赖性途径的激活有关。事实上，脯氨酸增强核糖体蛋白 S6 的磷酸化（即 mTOR 靶标），同时 cyclin-E1 增加。相反，mTOR 抑制剂雷帕霉素抑制脯氨酸介导的 cyclin-E1 诱导。结果，在存在脯氨酸的情况下由 HGF 诱导的肝细胞 DNA 合成，被 mTOR 抑制剂处理大大消除。脯氨酸的这种共促效应对胶原合成也是依赖的：胶原合成抑制剂，如 cis-OH-proline，可减少脯氨酸诱导的 cyclin-E1，然后肝细胞的 G1/S 期进展也受到抑制。总的来说，发现脯氨酸介导的 mTOR 激活和胶原合成对于 HGF 诱导的 DNA 合成至关重要，部分原因是 cyclin-E1 的充分积累。这是第一个证明驱动有丝分裂结果的氨基酸和生长因子之间的分子桥梁的报告。
