Up-regulation of cyclin-E(1) via proline-mTOR pathway is responsible for HGF-mediated G(1)/S progression in the primary culture of rat hepatocytes

Abstract

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.