JOURNAL OF CELLULAR PHYSIOLOGY 210:766–773 (2007) Effect of Hepatocyte Growth Factor on Methionine Adenosyltransferase Genes and Growth Is Cell Density-Dependent in HepG2 Cells HEPING YANG, 1 NATHANIEL MAGILNICK, 1 MAZEN NOUREDDIN, 1 JOSE ´ M. MATO, 2 AND SHELLY C. LU 1 * 1 Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine USC, Los Angeles, California 2 CIC Biogune, Center for Cooperative Research in Biosciences, Parque Tecnolo ´ gico de Bizkaia, Derio, Bizkaia Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen but its effect in liver cancer is conflicting. Methionine adenosyltransferase (MAT) is an essential enzyme encoded by two genes (MAT1A and MAT2A), while a third gene (MAT2b) encodes for a subunit that regulates the MAT2A-encoded isoenzyme. MAT1A is silenced while MAT2A and MAT2b are induced in hepatocellular carcinoma (HCC). The current work examined expression of HGF/c-met in HCC and whether HGF regulates MAT genes and growth in HepG2 cells. We found the mRNA levels of HGF and c-met are markedly increased in HCC. To study the influence of cell density, HepG2 cells were plated under high-density (HD) or low-density (LD) and treated with HGF (10 ng/ml). Cell density had a dramatic effect on MAT1A expression, being nearly undetectable at LD to a ninefold induction under HD. Cell density also determined the effect of HGF. At HD, HGF increased the mRNA levels of p21 and p27, while lowering the levels of MAT genes, cyclin A, and c-met. At LD, HGF increased the mRNA levels of cyclin A, MAT2A, MAT2b, and c-met. Consistently, HGF inhibits growth under HD but stimulates growth under LD. HGF induced sustained high ERK activation under HD as compared to LD. In summary, HGF induces genes favoring growth and is mitogenic when HepG2 cells are plated under LD; however, the opposite occurs under HD. This involves cell density-dependent differences in HGF-induced ERK activation. This may explain why HGF is mitogenic only when there is loss of cell-cell contact in vivo. J. Cell. Physiol. 210: 766 – 773, 2007. ß 2006 Wiley-Liss, Inc. Hepatocyte growth factor (HGF), also known as scatter factor, is a potent mitogen for a variety of cells through activation of its receptor c-Met (Zarnegar and Michalopoulos, 1995). HGF acts as a hepatotropic factor in vivo after partial hepatectomy and was initially isolated as a potent mitogen for hepatocytes in primary culture (Zarnegar and Michalopoulos, 1995). However, HGF’s mitogenic effect in primary cultures of rat hepatocytes depends on the cell density of the culture. It induced growth at low cell density whereas it induced albumin synthesis at high cell density (Takehara et al., 1992). Recently, Machide et al. (2006) reported that lack of HGF’s mitogenic effect in confluent primary cultures of rat hepatocytes is due to activation of LAR (leukocyte common antigen-related protein-tyrosine phosphatase) which shortened the duration of c-Met activation and activation of its downstream targets such as extracel- lular signal-regulated kinases 1 and 2 (ERK1/2). Even though HGF is a potent mitogen for hepatocytes, the role of HGF in hepatocellular carcinoma (HCC) is highly controversial. Full-length HGF transgenic mice under the control of the albumin promoter had lower HCC risk (Santoni-Rugui et al., 1996), whereas HGF transgenic mice under the control of the metallothionein promoter had higher HCC risk in older animals (Sakata et al., 1996). Adding to this controversy, transgenic mice overexpressing a five amino acid-deleted variant of HGF in the liver by an albumin expression vector also had higher HCC risk (Bell et al., 1999). Opposite results have been reported on the effect of HGF administration on DNA synthesis in diethylnitrosamine-induced rat liver tumors in vivo (Liu et al., 1995; Yaono et al., 1995). Conflicting results regarding HGF’s effect in HepG2 cells have also been published ranging from inducing a mitogenic effect (Lee et al., 1998) and increased inva- siveness (Jiang et al., 2001; Wang et al., 2004), to inhibiting growth and inducing apoptosis (Matteucci et al., 2001; Tsukada et al., 2001, 2004; Han et al., 2005). These studies used different culture conditions and the influence of cell density was not examined in any of them. Methionine adenosyltransferase (MAT) is an essen- tial cellular enzyme that catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor and the ultimate source of the propyla- mine moiety used in polyamine biosynthesis (Mato et al., 2002). Its importance is due to the fact that SAMe is the principal biological methyl donor, the precursor of aminopropyl groups utilized in polyamine biosynthesis, and in the liver, a precursor of glutathione (GSH) (Lu, 2000). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, a1 and a2; whereas a third gene MAT2b, encodes for a regulatory subunit b that regulates MAT2A-encoded isoenzyme (Kotb et al., 1997; Halim et al., 1999). MAT1A is expressed mostly in liver and is a ß 2006 WILEY-LISS, INC. Contract grant sponsor: NIH; Contract grant numbers: DK51719, AT1576; Contract grant sponsor: Ministerio de Educacio ´n y Ciencia; Contract grant sponsor: USC Research Center for Liver Diseases; Contract grant number: DK48522. *Correspondence to: Shelly C. Lu, MD, Division of Gastrointest- inal and Liver Diseases, HMR Bldg., 415, Department of Medicine, Keck School of Medicine USC, 2011 Zonal Ave., Los Angeles, CA, 90033. E-mail: shellylu@usc.edu Received 27 July 2006; Accepted 31 August 2006 DOI: 10.1002/jcp.20891