Id1 Is Down-Regulated by Hepatocyte Growth Factor via ERK-Dependent and ERK-Independent Signaling Pathways, Leading to Increased Expression of p16 in Hepatoma Cells

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16 through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88) Introduction Cell proliferation is tightly regulated by extracellular factors such as growth factors and cytokines. To maintain tissues and organs and to avoid tumorigenesis, the extracellular factors regulate both stimulation and inhibition of cell proliferation. Hepatocyte growth factor (HGF) was originally described as a mesenchymal cell-derived mitogenic protein for primary hepatocytes (1, 2). HGF is a heparin-binding glycoprotein produced by stroma cells in a wide variety of tissues and plays a key role in tumor-stroma interactions. HGF is unique because it has opposing effects on the regulation of cell proliferation: whereas treatment with HGF stimulates the proliferation of some tumor cell lines, it inhibits the proliferation of several others (3-5). Elucidation of the cause of the differences in the effects of HGF on cell proliferation is essential for applying HGF to cancer therapy. Because the HGF signal is transduced through the receptor tyrosine kinase c-Met, the product of the c-met protooncogene, the opposing effects of HGF on cell proliferation are thought to be derived from differences in downstream signaling pathways (6, 7). The association of HGF with c-Met induces phosphorylation of some tyrosine residues on c-Met followed by the recruitment of various signal transducers and adaptors such as Grb2/Sos, Shc, and Gab1 (6, 8, 9), which leads to the activation of two major signaling pathways, the extracellular signal-regulated kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3K) pathway. We reported previously that HGF inhibits the proliferation of a hepatoma cell line, HepG2, by arresting the cell cycle at G1 (10, 11). The inhibition was suppressed by a low concentration of MEK inhibitor, which causes the high-intensity ERK activity to become weak; thus, the G1 arrest is dependent on the high-intensity ERK activity induced by HGF (10, 12). We also showed that the strong activation of ERK is required for up-regulation of a cyclin-dependent kinase (Cdk) inhibitor, p16 (13), which is also known as a tumor suppressor. p16 preferentially associates with Cdk4 and Cdk6 and inhibits cell proliferation by preventing binding to the regulatory subunit, cyclin D (14, 15). We showed that up-regulation of p16 with HGF treatment of the cells increases the association of p16 with Cdk4, leading to the redistribution of other Cdk inhibitors, p21 and p27, from Cdk4 to Cdk2. Because Cdk2 activity is responsible for progression of the cell cycle though the G1 phase in HepG2 cells, the repression of Cdk2 activity by the Cdk inhibitors results in G1 arrest (13). Therefore, up-regulation of p16 is crucial for the inhibitory effect of HGF on the proliferation of HepG2 cells. Received 6/19/08; revised 3/19/09; accepted 4/13/09; published OnlineFirst 6/30/09. Grant support: Grant-in-Aid for Scientific Research 19570124 from Japan Society for the Promotion of Science (T. Tanaka) and Grant-in-Aid for Cancer Research 17014032 from the Ministry of Education, Science, Sports and Culture of Japan (N. Kitamura). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Toshiaki Tanaka, Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Phone: 81-45-924-5702; Fax: 81-45-924-5771. E-mail: [email protected] Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-08-0289 Mol Cancer Res 2009;7(7). July 2009 1179 on July 6, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst June 30, 2009; DOI: 10.1158/1541-7786.MCR-08-0289