Id1 Is Down-Regulated by Hepatocyte Growth Factor via ERK-Dependent and ERK-Independent Signaling Pathways, Leading to Increased Expression of p16INK4a in Hepatoma Cells

Abstract

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 G 1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a 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.

ERK-Dependent Downregulation of Skp2 Reduces Myc Activity with HGF, Leading to Inhibition of Cell Proliferation through a Decrease in Id1 Expression

“…HepG2 cells and HuH7 cells were provided by Dr. S. Taketani and Dr. N. Kitamura, respectively, and have been described previously (7)(8)(9)(10). Cells were cultured as previously described (9).…”

“…cDNA synthesis was as described previously (10,23). PCR was conducted with pairs of specific oligonucleotide primers (Skp2: 5 0 -CCTG-TCTGTGCCTCCCTG-3 0 and 5 0 -CTGAGTGATAGGT-GTTGG-3 0 , p27 Kip1 : 5 0 -ATGTCAAACGTGCGAGTG-TC-3 0 and 5 0 -ACGTTTGACGTCTTCTGAGG-3 0 ).…”

“…Cell lysates were prepared as described previously (10). Equal amounts of protein in the precleared cell lysates (20-70 mg total protein) were resolved by SDS-PAGE on a 12% gel after heat denaturation.…”

“…The strong activation of ERK by HGF upregulated expression of a Cdk inhibitor, p16 Ink4a , which led to a redistribution of other Cdk inhibitors, p21 and p27, from Cdk4 to Cdk2, resulting in low phosphorylation of pRb and G 1 arrest (8,9). Because the upregulation of p16 expression is crucial to the G 1 arrest, we studied the mechanism responsible for it, and found that transcription factor Ets upregulates p16 through downregulation of a repressor protein for Ets, Id1 (10). The expression of Id1 is shown to be regulated at the transcription level (10), but the regulatory mechanism remains to be elucidated.…”

“…Because the upregulation of p16 expression is crucial to the G 1 arrest, we studied the mechanism responsible for it, and found that transcription factor Ets upregulates p16 through downregulation of a repressor protein for Ets, Id1 (10). The expression of Id1 is shown to be regulated at the transcription level (10), but the regulatory mechanism remains to be elucidated.…”