Junhong Han, Yu-ichi Tsukada, Eiji Hara, Naomi Kitamura, and Toshiaki Tanaka

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 36, Issue of September 9, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hepatocyte Growth Factor Induces Redistribution of p21 CIP1 and p27 KIP1 through ERK-dependent p16 INK4a Up-regulation, Leading to Cell Cycle Arrest at G 1 in HepG2 Hepatoma Cells* Received for publication, March 29, 2005, and in revised form, June 14, 2005 Published, JBC Papers in Press, July 13, 2005, DOI /jbc.M Junhong Han, Yu-ichi Tsukada, Eiji Hara, Naomi Kitamura, and Toshiaki Tanaka From the Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama , Japan and the Division of Protein Information, Institute for Genome Research, University of Tokushima, Tokushima , Japan Hepatocyte growth factor (HGF) has an anti-proliferative effect on many types of tumor cell lines and tumors in vivo. We found previously that inhibition of HGFinduced proliferation in HepG2 hepatoma cells is caused by cell cycle arrest at G 1 through a high intensity ERK signal, which represses Cdk2 activity. To examine further the mechanisms of G 1 arrest by HGF, we analyzed the Cdk inhibitor p16 INK4a, which has an antiproliferative function through cell cycle arrest at G 1.We found that HGF treatment drastically increased endogenous p16 levels. Knockdown of p16 with small interfering RNA reversed the arrest, indicating that the induction of p16 is required for G 1 arrest by HGF. Analysis of the promoter of the human p16 gene identified the proximal Ets-binding site as a responsive element for HGF, and this responded to the high intensity ERK signal. HGF treatment of the cells led to a redistribution of p21 CIP1 and p27 KIP1 from Cdk4 to Cdk2. The redistribution was blocked by the knockdown of p16 with small interfering RNA, which restored the Cdk2 activity repressed by HGF, demonstrating the requirement of p16 induction for the redistribution and eventual repression of Cdk2 activity. Our results reveal a signaling pathway for G 1 arrest induced by HGF. Hepatocyte growth factor (HGF) 1 was originally described as a mesenchymal cell-derived mitogenic protein for hepatocytes (1 3). At present, HGF is recognized as a pleiotropic growth factor that acts as a potent mitogen, morphogen, motility factor, and angiogenic factor for various types of cells (4 10). HGF and its high affinity receptor c-met are widely expressed in normal tissues and are thought to play a key role in the interaction between mesenchymal (the site of HGF expression) and epithelial (the site of c-met expression) cells (11 13); thus, the * This work was supported by Grant-in-aid for Cancer Research (to N. K.) from the Ministry of Education, Science, Sports, and Culture of Japan. 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. Present address: Dept. of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC To whom correspondence should be addressed. Tel.: ; Fax: ; ttanaka@bio.titech.ac.jp. 1 The abbreviations used are: HGF, hepatocyte growth factor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Cdk, cyclindependent kinase; CdkI, cyclin-dependent kinase inhibitor; sirna, small interfering RNA; BrdUrd, bromodeoxyuridine; ChIP, chromatin immunoprecipitation various effects of HGF seem to be important for regulation of the formation and functions of normal tissues and organs. Among the pleiotropic effects, HGF has opposing effects on cell growth: treatment with HGF inhibits the proliferation of a number of tumor cell lines (14 16), whereas it stimulates the proliferation of other tumor cell lines (17 22). The anti-proliferative effect of HGF is especially important in that it is observed not only in cultured tumor cells, but also in tumors in vivo: administration of HGF decreases the synthesis of DNA in diethylnitrosamine-induced rat liver tumors (23), and c-mycinduced hepatocarcinogenesis is inhibited by HGF in a transgenic mouse model coexpressing c-myc and HGF (24). The anti-proliferative effect of HGF thus seems to be concerned with the control of tumorigenesis, but the mechanism of the effect remains to be elucidated. Because all of the pleiotropic effects of HGF are transduced through the activation of the c-met receptor, which is known as the c-met proto-oncogene product (25 29), the opposing effects of HGF on cell growth are thought to be derived from differences in the downstream signaling pathways involving multiple effector molecules (30, 31). We have investigated the molecular mechanisms of the antiproliferative effect of HGF and found previously that HGF induces cell cycle arrest at G 1 in the human hepatocellular carcinoma cell line HepG2. This G 1 arrest was shown to be induced through a high intensity ERK signal because the antiproliferative effect of HGF was suppressed by a low concentration of MEK inhibitor, which reduces the strong activation of ERK to weak activation (32). The high intensity ERK signal leads to the repression of Cdk2 (cyclin-dependent kinase-2) kinase activity for prb, which mainly regulates the proliferation of HepG2 cells (33). Cdk2 generally controls the transition of the cell cycle from G 1 to S phase, and its activity is regulated by phosphorylation, association with cyclins A and E, and association with the CIP/KIP family of Cdk inhibitors (CdkIs) (34). HGF down-regulates the expression of cyclin A protein and up-regulates that of p21 CIP1 protein in HepG2 cells, suggesting that these changes result in the repression of Cdk2 activity and induction of G 1 arrest (33). It has also been reported that p21/p27 KIP1 proteins are expressed upon treatment of HepG2 cells with HGF and that the induction is accompanied by cell cycle arrest (3, 35). However, the molecular mechanism of how CdkIs are involved in cell cycle arrest at G 1 by HGF has not been uncovered. In this study, we focused on p16 INK4a, which is included in another group of CdkIs, the INK4 family, because p16 has been implicated in cell cycle arrest at G 1 in various types of cells. p16 specifically associates with Cdk4 and Cdk6 (36, 37) and keeps them from their binding partners, D-type cyclins. The association of p16 with Cdk4 and Cdk6 thus results in inactivation of This paper is available on line at

2 Redistribution of p21 and p27 by HGF-induced p the Cdk proteins, accumulation of hypophosphorylated prb, and eventual G 1 arrest of the cell cycle (34, 37). Here, we found that treatment of HepG2 cells with HGF up-regulated p16 mrna and protein. Overexpression of p16 increased the HepG2 cell population in G 1 phase, and small interfering RNA (sirna)-mediated knockdown of p16 reversed G 1 arrest induced by HGF, suggesting that the induction of p16 by HGF is responsible for the arrest in G 1 by HGF. Analysis of the p16 promoter identified the proximal Ets-binding site as a HGFresponsive element. Although p16 is incapable of directly associating with Cdk2, the induction of p16 by HGF was necessary for the repression of Cdk2 activity because sirna-mediated knockdown of p16 restored the Cdk2 activity repressed by HGF. The HGF treatment led to a redistribution of p21 and p27 from Cdk4 to Cdk2. sirna-mediated p16 knockdown blocked the redistribution, demonstrating that the induction of p16 is required for the redistribution, which causes inactivation of Cdk2 and leads to G 1 arrest. MATERIALS AND METHODS Cell Culture and Transient Transfection HepG2 cells were cultured in Dulbecco s modified Eagle s medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 C as described previously (32, 33). For transient transfection, the cells were seeded at a density of in 6-well plates and cultured for 1 day. They were treated with 4 g of plasmid mixed with 11.2 l of jetpei (Polyplus-transfection) for 24 h, and the medium was replaced with fresh medium. The cells were cultured further in the absence or presence of HGF (50 ng/ml) and were either harvested after trypsinization for flow cytometry or extracted for lysate production. To measure DNA synthesis, at 24 h after transfection, cells were trypsinized and reseeded. Quantitative Reverse Transcription-PCR of p16 mrna Total RNA was purified with ISOGEN (Nippon Gene Co., Ltd.) according to the manufacturer s instruction, and 1 g of RNA was used for cdna synthesis using the 1st Strand cdna synthesis kit with oligo(dt) primers (Invitrogen). PCR for p16 was performed in a total volume of 50 l containing 1 l of the cdna and 10 pmol of forward primer 5 -AGCAC- CGGAGGAAGAAAGAGGAG-3, 10 pmol of reverse primer 5 -AGTT- GTGGCCCTGTAGGACCTTC-3, 50mM KCl, 10 mm Tris-HCl (ph 8.3), 200 mm dntps, 2 mm MgCl 2, 10% dimethyl sulfoxide, and 2.5 units of LA Taq polymerase (Takara) with 28 cycles of denaturing (94 C, 1 min), annealing (60 C, 1 min), and extension (72 C, 1 min). PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. Experiments conducted using various amounts of input template ensured that the assay was quantitative under these conditions. Construction of a p16 Expression Plasmid PCR amplification to construct a p16 expression plasmid was performed with a pair of p16- specific oligonucleotide primers (5 -CCAAGCTTAGCATGGAGCCG- GCGGC-3 and 5 -CGGGATCCTCAATCGGGGATGTCTGAGGG-3 ) and cdna as described above with 32 cycles of denaturing (94 C, 1 min), annealing (60 C, 1 min), and extension (72 C, 1 min). The amplified cdna was cloned into the vector pt7blue (Novagen), and its sequence was confirmed. The entire coding sequence of p16 was inserted into the BamHI and Hind III sites of pcdna3. Cell Proliferation Assay Following replacement of the medium after transfection as described above, the cells were cultured and harvested at specific time points after trypsinization. The number of cells was counted with a hemocytometer. Cell Cycle Analysis by Flow Cytometry Cells were trypsinized and harvested at specific time points after HGF treatment and washed twice with ice-cold phosphate-buffered saline. They were incubated in flow reagent (0.1% sodium citrate, 0.1% Triton X-100, 50 g/ml propidium iodide, and 1 g/ml DNase-free RNase (Roche Applied Science)) for an appropriate time period at room temperature. After filtration with a nylon cell strainer (pore size of 70 m), cell cycle distribution was monitored with a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). RNA Interference p16-targeting sirna was synthesized according to published data (38). Its sense sequence (5 -CGCACCGCCTAGT- TACGGT-3 ) corresponds to nucleotides of the human p16 coding sequence, which is in exon 1 of the INK4a locus. A DNA oligonucleotide containing the sense target sequence, a hairpin loop, and the antisense target sequence was synthesized, annealed, and inserted into a polymerase III-mediated sirna expression plasmid vector, psilencer1.0-u6 (Ambion Inc.). The sirna expression plasmid was transfected as described above. Measuring DNA Synthesis DNA synthesis was assayed by measuring the incorporation of bromodeoxyuridine (BrdUrd) into the genome. Cells were transfected as described above. At 24 h after transfection, the cells were trypsinized and seeded at in 96-well plates in the absence or presence of HGF. The cells were incubated with 10 M BrdUrd for 2 h before being harvested, and DNA synthesis was measured with the Biotrak cell proliferation enzyme-linked immunosorbent assay system (Amersham Biosciences). Peroxidase-conjugated anti-brdurd antibody was used at 1:100 dilution, and the resultant immune complexes were quantified by measurements of absorbance after the subsequent reaction with substrate using a microtiter plate spectrophotometer (Wallac Oy, Turku, Finland). Luciferase Reporter Assays A series of p16 promoter constructs containing 5 -truncated promoter regions of the p16 gene (bases 1216 to 1 from the transcription initiation site) fused to the luciferase gene were produced as described previously (39). Each promoter construct was cotransfected along with a standard amount of the psv- -galactosidase control plasmid (Promega). Transfected cells were stimulated with HGF (50 ng/ml) for 24 h, and cell extracts were prepared with reporter lysis buffer (Promega). The luciferase and -galactosidase assays were performed in 20 l of cell extract with the luciferase assay system kit (Promega) and the -galactosidase enzyme assay system (Promega), respectively, on a microtiter plate multi-label counter (Wallac Oy). Each luciferase activity was normalized to the corresponding -galactosidase activity. Site-directed Mutagenesis of the Ets-binding Sites in the p16 Promoter Mutations in the p16 promoter were designed based on published data (40). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) with the following mutant primers: M1, 5 -GAGGGGGCTCTTCCGCCAGCACCCCA- TTAAGAAAG-3 ; M2, 5 -GAGGGGGCTCTTAAGCCAGCACCGGAG- GAAGAAAG-3 ; and M3, 5 -GAGGGGGCTCTTAAGCCAGCACCCCA- TTAAGAAAG-3. Ets-binding motifs are shown in boldface, and point mutations are underlined. Chromatin Immunoprecipitation (ChIP) Cells ( ) were washed twice with phosphate-buffered saline and then treated with 1% formaldehyde for 10 min at room temperature for cross-linking. The reaction was stopped by addition of 0.1 volume of 1.25 M glycine. Cells were scraped and collected in a 1.5-ml tube. They were washed twice with ice-cold phosphate-buffered saline, lysed with ChIP lysis buffer (50 mm Tris-Cl (ph 8.1), 1% Triton X-100, 0.1% deoxycholate, 150 mm NaCl, 5mM EDTA, 5 g/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 5 g/ml aprotinin), and sonicated using a Branson Model 250 sonifier to shear the chromatin. After clarification by centrifugation, the lysate was precleared by incubation for 2 h at 4 C with protein A- Sepharose (Amersham Biosciences), which had been blocked previously with 1 mg/ml herring sperm DNA and 1 mg/ml bovine serum albumin in ChIP lysis buffer. ChIP lysis buffer was added to the precleared lysate up to 1 ml, and 100 l was used for control PCR. The aliquot of the rest of the lysate was incubated with 10 g of anti-ets1/2 antibody (C-275, Santa Cruz Biotechnology, Inc.) or normal rabbit IgG (Upstate Biotechnology, Inc.) for 16 h at 4 C. After a 1-h incubation with blocked protein A-Sepharose, immunoprecipitates were collected by centrifugation; washed once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris (ph 8.1), and 150 mm NaCl), once with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris (ph 8.1), and 500 mm NaCl), once with LiCl wash buffer (0.25 M LiCl, 0.1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, and 10 mm Tris (ph 8.0)), and twice with TE buffer (10 mm Tris (ph 8.0) and 1 mm EDTA); and incubated for 2hat55 Cwith 20 g of proteinase K. The precipitates were eluted three times with 80 l of elution buffer (1% SDS, 0.1 M NaHCO 3, and 0.01 mg/ml herring sperm DNA) and combined. The eluted samples were incubated for 5 h at 65 C to reverse cross-links and extracted with phenol/chloroform, followed by precipitation of the DNA in ethanol. The resultant DNA was resuspended in 100 l of TE buffer, and 2 l was used as a template for PCR with a pair of specific primers flanking the Ets site (5 -TGCTCGGGATTAATAG- CACC-3 and 5 -CTCCATGCTGCCCCGCCG-3 ) (40). PCR was performed with denaturing for 5 min at 95 C; 34 cycles of denaturing (94 C, 1 min), annealing (60 C, 30 s), and amplification (72 C, 1 min); and extension (72 C, 10 min). PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. Preparation of Cell Extracts Cells were washed with ice-cold phosphate-buffered saline and lysed with 200 l of ice-cold cell lysis buffer (50 mm Tris-HCl (ph 7.4), 150 mm NaCl, 1 mm EDTA, 0.1% Tween 20,

3 31550 Redistribution of p21 and p27 by HGF-induced p16 30 mm tetrasodium pyrophosphate, 50 mm NaF, 1 mm Na 3 VO 4,5 g/ml leupeptin, 1 g/ml pepstatin A, and 1 mm phenylmethylsulfonyl fluoride) per 100-mm plate. The cell lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4 C, and the protein concentration of the precleared cell extract was measured with the BCA protein assay reagent (Pierce). In Vitro Kinase Assays Kinase assays were carried out as described by Matsushime et al. (41) with slight modifications. Equal amounts of protein in the precleared cell extracts ( g of total protein) were immunoprecipitated with 2 g of anti-cdk2 antibody. Precipitated immune complexes were washed twice with cell lysis buffer and twice with kinase buffer (20 mm HEPES (ph 7.5), 1 mm dithiothreitol, 10 M Na 3 VO 4,2mM -glycerophosphate, 100 M EGTA, 100 M phenylmethylsulfonyl fluoride, 0.1 g/ml leupeptin, and 12 mm MgCl 2 ). Kinase reactions were performed in 30 l of kinase buffer containing 0.2 g of full-length prb (QED Bioscience Inc.), 24 M ATP, and 0.22 MBq of [ - 32 P]ATP at 30 C for 30 min. Reactions were stopped by addition of 30 lof2 Laemmli sample buffer (125 mm Tris-HCl (ph 6.8), 4% SDS, 20% glycerol, 0.002% bromphenol blue, and 10% 2-mercaptoethanol), and samples were resolved by SDS-PAGE on a 10% gel after heat denaturation. Phosphorylation of the substrate was visualized by autoradiography. The band intensity on the autoradiogram was quantitated by NIH Image-J software. Antibodies, Immunoprecipitation, and Immunoblotting Antibodies used for immunoblotting and immunoprecipitation were obtained as follows: anti-p16 (H-156), anti-p21 (C-19), anti-p27 (C-19), anti-cdk2 (M-2), anti-cdk4 (H-22), and anti-cyclin D 2 (C-17) antibodies from Santa Cruz Biotechnology, Inc.; -tubulin antibody (T 9026) from Sigma; and horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins from Amersham Biosciences. Immunoprecipitation and immunoblotting were performed as described previously (33). RESULTS HGF Treatment Induces the Expression of p16 INK4a We have reported previously that HGF treatment induces cell cycle arrest at G 1 in HepG2 cells and that this arrest first appears at 48 h after HGF treatment and reaches a maximum by 72 h, at which time 70% of the cells are in G 1. Pretreatment with a low concentration (10 M) of the MEK inhibitor PD98059 prevents cell cycle arrest by HGF, indicating that the effect of HGF is mediated by an intensive activation of the ERK signal (32, 33). To examine the expression pattern of a Cdk inhibitor, p16 INK4a, we performed a quantitative reverse transcription- PCR analysis with total RNA extracted from HepG2 cells treated with HGF. The level of the p16 transcript increased at 4 h and peaked at 72 h after HGF treatment (Fig. 1A, upper panels). This increase was completely blocked by pretreatment with the MEK inhibitor PD98059 (10 M) (Fig. 1A, lower panels). Immunoblotting showed that the level of p16 protein increased at 12 h of HGF treatment and that addition of the MEK inhibitor blocked the accumulation of p16 (Fig. 1B). Thus, the increase in p16 mrna was followed by an increase in the protein. Altered stability of p16 mrna is unlikely to account for the increase in p16 mrna because it has been reported that p16 mrna is stable, with a half-life of 24 h (39). Thus, p16 expression is regulated at the transcriptional level through the ERK signal. Increased Expression of p16 Is Responsible for G 1 Arrest in HepG2 Cells It was previously shown that down-regulation of Cdk2 activity primarily contributes to cell cycle arrest by HGF at G 1 in HepG2 cells (33). Because p16 does not bind to Cdk2 and does not directly inhibit Cdk2 activity, we examined whether the increase in p16 expression leads to cell cycle arrest at G 1 in HepG2 cells. We transfected a p16 expression plasmid into HepG2 cells and examined cell proliferation and cell cycle distribution. The expression of exogenous p16 peaked at 24 h after transfection, and its level remained essentially unchanged after this point (data not shown). The exogenous p16 level was almost the same as the endogenous p16 level induced by HGF (Fig. 2A). At 24 and 48 h after transfection, the number of HepG2 cells exogenously expressing p16 was smaller than FIG. 1.Effect of HGF on the expression of p16 in HepG2 cells. Cells were seeded at a density of /100-mm dish. After 2 days of culture, the medium was replaced with fresh medium; and following preincubation with or without PD98059 (10 M) for 1 h, HGF (50 ng/ml) was added. Cells were then cultured. A, expression of p16 mrna. Total RNA was purified at the indicated times and subjected to quantitative reverse transcription-pcr. The time course of p16 mrna expression (upper panels) and the effect of the MEK inhibitor PD98059 on expression (lower panels) are shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mrna was used as an internal control. B, expression of p16 protein. Cell lysates were prepared at the indicated times and subjected to immunoblotting using anti-p16 and anti- -tubulin antibodies. Tubulin was used as a loading control. The results are representative of at least three independent experiments. that of untransfected cells (Fig. 2B). Analysis of cell cycle distribution by flow cytometry showed that the elevated p16 expression increased cell numbers in G 1 phase and reduced cell numbers in S phase compared with untransfected controls (Fig. 2C). These results suggest that the increased expression of p16 contributes to cell cycle arrest at G 1 in HepG2 cells. Next, to examine whether the endogenous expression of p16 induced by HGF is actually required for G 1 arrest of HepG2 cells induced by HGF, we used the sirna method. p16 is known to be encoded at the INK4a/ARF locus of the genome, and this locus also encodes another tumor suppressor gene, p14 ARF (36, 42 46), which is also responsible for cell cycle arrest through the regulation of a different pathway (47 49). Each of their first exons is present in the genome as exons 1 and 1, but p16 and p14 share common exons, 2 and 3, in which their reading frames are different (36). The sirna for exon 1 was synthesized according to published data (38), and it was confirmed previously that the sirna eliminates the expression of p16 specifically with no effect on p14, which is encoded by an alternative exon, 1 (38). Introduction of the sirna reduced the expression of p16 at 48 and 72 h after HGF treatment (Fig. 3A). This sirna retained the level of -tubulin protein (Fig. 3A), and introduction of the empty vector or sirna targeting another gene did not affect the p16 level (data not shown), confirming that this repression of p16 was specific and was not the result of any cell damage by transfection or expression of sirna itself. This knockdown of p16 alleviated the HGF-induced repression of BrdUrd uptake (Fig. 3B). The cell cycle distribution analysis showed that G 1 arrest induced by HGF was partially rescued by the knockdown of p16 (Fig. 3C). These results suggest that the HGF-induced expression of p16 is required for G 1 arrest by HGF in HepG2 cells. The Proximal Ets-binding Site in the p16 Promoter Mediates Up-regulation of p16 Induced by HGF HGF-induced up-regulation of p16 expression appears to contribute to cell cycle arrest at G 1 in HepG2 cells; thus, it is important to elucidate the regulatory mechanism of the induction by HGF. Because

4 Redistribution of p21 and p27 by HGF-induced p FIG. 2.Cell proliferation and cell cycle distribution of HepG2 cells overexpressing p16. Cells were transiently transfected with a vector encoding p16 or an empty vector (Mock). At 24 h after transfection, the medium was replaced with fresh medium, and cells were further cultured. A, comparison of the expression levels of endogenous and exogenous p16 proteins. To detect exogenous p16, cell lysates were prepared at 48 h after transfection. The exogenous p16 levels remained essentially constant after 24 h of transfection (data not shown). For HGF treatment, cells were seeded at a density of /100-mm dish. After 2 days of culture, the medium was replaced with fresh medium with or without HGF (50 ng/ml). Cell lysates were prepared at 72 h after HGF treatment. Each cell lysate was subjected to immunoblotting. B, cell proliferation. Cell numbers were counted at 0, 24, and 48 h. Each value represents the mean S.D. of triplicate determinations from a representative experiment. *, p 0.01 by Student s t test. C, cell cycle distribution. Cells were collected at 48 h, stained with propidium iodide, and analyzed by flow cytometry. The percentages of cells in G 1, S, and G 2 /M phases were calculated from a sample of 20,000 cells using the program CellQuest. Experiments were done twice with similar results, and representative data are shown (A and C). the up-regulation occurred at the transcriptional level, we examined the activity of a series of 5 -truncated promoters of the p16 gene, each of which was placed upstream of the luciferase gene (39). The basal activity of the p16 promoters was influenced by the series of truncations, but responsiveness to HGF was observed for all truncated promoters, including the shortest one (Fig. 4A). Thus, the HGF-responsive element seemed to be located within the shortest 247-bp promoter. The ERK signal was involved in the HGF-induced up-regulation of the p16 promoter (Fig. 1A); thus, the transcription factor responsible for the expression of p16 should be regulated by the ERK signal. Most transcription factors of the Ets family, which play important roles in cell proliferation, are phosphorylated and activated through ERK activity and thus are thought to be downstream nuclear targets of the Ras-ERK signal (50). Ohtani et al. (40) have reported that the shortest p16 promoter has two consensus sequences for binding Ets around 120 bp upstream from the transcription initiation site and that this region mediates senescence-induced up-regulation of p16. To examine whether these Ets-binding sites are involved in the HGF-induced expression of p16, we analyzed the shortest 247-bp promoter with point mutations within each or both of the Ets-binding sites described by Ohtani et al. (Fig. 4B). The mutant promoter M1, with a replacement of nucleotides at the proximal Ets-binding site, completely lost HGF-induced promoter activity, whereas the mutant promoter M2, in which two nucleotides were changed at the distal Etsbinding site, showed little reduction in promoter activity in response to HGF (Fig. 4C). The M3 promoter, which had all of FIG. 3. Effect of p16 sirna on DNA synthesis and cell cycle distribution of HepG2 cells treated with HGF. Cells were transiently transfected with a vector encoding p16 sirna or an empty vector (Mock). At 24 h after transfection, the cells were reseeded at the proper density (B); the medium was replaced with fresh medium with or without HGF (50 ng/ml), and the cells were cultured (A C). A, expression levels of p16 protein. Cell lysates were prepared at 24, 48, and 72 h after HGF treatment and subjected to immunoblotting. B, DNA synthesis. At 0, 24, 48, and 72 h after HGF treatment, cells were incubated with 10 M BrdUrd for 2 h before being harvested, and DNA synthesis was measured. Each value represents the mean S.D. of triplicate determinations from a representative experiment. *, p C, cell cycle distribution. Cells were collected at 72 h, stained with propidium iodide, and analyzed by flow cytometry. The percentages of cells in each phase were calculated from a sample of 20,000 cells using the program CellQuest. Experiments were done twice with similar results, and representative data are shown (A and C). the mutations in both Ets-binding sites, lost all responsiveness to HGF. These results suggest that the proximal Ets-binding site is required for HGF-induced p16 expression. Addition of a low concentration of MEK inhibitor reduced the activity of the 247-bp promoter almost to the basal level (Fig. 4C), indicating that the induction of p16 by HGF through the proximal Ets-binding site is mediated by the high intensity of ERK activity in HepG2 cells. ChIP assay showed that the transcription factor Ets physically bound to the Ets-binding site in the p16 promoter in HGF-treated HepG2 cells (Fig. 4D), suggesting that Ets is involved in the HGF-induced expression of p16. Up-regulation of p16 Expression by HGF Contributes to Suppression of Cdk2 Activity To determine the relationship between the induction of p16 and the activity of Cdk2, we examined the time course of Cdk2 activity in HGF-treated HepG2

5 31552 Redistribution of p21 and p27 by HGF-induced p16 FIG. 4. Determination of the HGF-responsive element in the p16 promoter. A, cells were transiently transfected with vectors encoding a series of 5 -truncated promoter regions of the p16 gene fused to the luciferase gene or an empty vector (Mock). At 24 h after transfection, the medium was replaced with fresh medium, and cells were cultured in the absence ( ) or presence ( ) of HGF (50 ng/ml). At 24 h after HGF treatment, cell lysates were prepared and subjected to the luciferase assay. Luciferase activities were normalized as described under Materials and Methods. The average -fold increase compared with the mock transfection is indicated. Each value represents the mean S.D. of triplicate determinations from a representative experiment. Each difference between the values in the absence or presence of HGF was statistically significant (p 0.01). B, shown are the sequences of Ets-binding motifs (underlined) inthe 247-bp region of the p16 promoter. The positions of point mutations in the Ets-binding motifs are indicated. C, cells were transiently transfected with vectors encoding the wild-type (WT) 247-bp region or its mutants (M1 M3) fused to the luciferase gene or an empty vector (Mock). At 24 h after transfection, the medium was replaced with fresh medium; and following preincubation with ( ) or without ( ) PD98059 (10 M) for 1 h, HGF (50 ng/ml) was added. Cells were then cultured in the absence ( ) or presence ( ) of HGF. At 24 h after HGF treatment, cell lysates were prepared and subjected to the luciferase assay. Luciferase activities were normalized as described under Materials and Methods. The average -fold increase compared with the mock transfection is indicated. Each value represents the mean S.D. of triplicate determinations from a representative experiment. *, p D, cells were seeded at a density of /100-mm dish. After 2 days of culture, the medium was replaced with fresh medium, and cells were further cultured in the presence of HGF (50 ng/ml) for 48 h. Chromatin immunoprecipitates from the cell lysate using anti-ets1/2 or control antibody (normal rabbit serum (NRS)) were analyzed by PCR with a pair of specific primers flanking the Ets site. The arrow indicates the specifically amplified DNA fragments. cells and compared it with the time course of p16 induction by HGF. Although Cdk2 was active for 12 h after HGF treatment, its activity started to decline at 24 h. Cdk2 activity was drastically reduced at 36 h after HGF treatment and remained weak after this point (Fig. 5A). The time course of the reduction of Cdk2 activity correlated well with that of the induction of FIG. 5. Kinase activity of Cdk2 in HepG2 cells treated with HGF. A, effect of HGF on the kinase activity of Cdk2. Cells were seeded at a density of /100-mm dish. After 2 days of culture, the medium was replaced with fresh medium, and cells were further cultured in the presence of HGF (50 ng/ml). Cell lysates were prepared at the indicated times and immunoprecipitated (IP) with anti-cdk2 antibody. Immune complex kinase assays were performed using prb as a substrate (upper panel). The amounts of Cdk2 in the immunoprecipitates were analyzed by immunoblotting (IB) (lower panel). B, effect of PD98059 on the HGF-reduced kinase activity of Cdk2. Cells were cultured as described for A. After replacement of the medium, cells were preincubated with ( ) or without ( ) PD98059 for 1 h, and HGF (50 ng/ml) was added. Cells were then cultured in the absence ( ) or presence ( ) of HGF. At 48 h after HGF treatment, immune complex kinase assays were performed as described for A. C, effect of p16 sirna on the HGF-reduced kinase activity of Cdk2. Cells were transiently transfected with a vector encoding p16 sirna ( ) or an empty vector ( ). At 24 h after transfection, the medium was replaced with fresh medium, and cells were further cultured in the absence ( ) or presence ( ) of HGF (50 ng/ml). At 48 h after HGF treatment, immune complex kinase assays were performed as described for A. The band intensity of the upper panel was quantitated using NIH Image-J software and graphed. The amounts of Cdk2 in the immunoprecipitates were analyzed by immunoblotting (lower panel). Experiments were done twice with similar results, and representative data are shown. p16 expression (Fig. 1B), suggesting that induction participates in G 1 arrest by mediating the down-regulation of Cdk2 activity. Addition of PD98059 resulted in an alleviation of the decrease in Cdk2 activity caused by HGF (Fig. 5B), confirming that the activity was regulated by p16 induction because the expression of p16 was also repressed by addition of PD98059, as shown in Fig. 1. To examine whether the expression of p16 induced by HGF is necessary for the suppression of Cdk2 activity, we performed sirna-mediated p16 knockdown and analyzed Cdk2 activity at 48 h after treatment of the cells with HGF. The level of Cdk2 activity was very high in the absence of HGF, and HGF treatment reduced the activity to 30%. Knockdown of p16 in HGF-treated HepG2 cells using sirna restored the Cdk2 activity to 55% (Fig. 5C). It is worth noting that the extent of

6 Redistribution of p21 and p27 by HGF-induced p FIG. 6.Effect of HGF on expression of Cdk2, Cdk4, and cyclin D 2 and formation of the cyclin-cdk-cdki complex. Cells were seeded at a density of /100-mm dish. After 2 days of culture, the medium was replaced with fresh medium, and cells were further cultured in the presence of HGF (50 ng/ml). A, expression of Cdk2, Cdk4, and cyclin D 2. Cell lysates were prepared at the indicated times and subjected to immunoblotting. B, formation of the cyclin-cdk-cdki complex. Cell lysates were prepared at the indicated times and immunoprecipitated (IP) with anti-cdk4, anti-p21, or anti-p27 antibody. The immunoprecipitates were subjected to immunoblotting using anti-cdk, anti-cyclin D 2, or anti-cdki antibody. C, relative ratio of the amount of Cdk2 associated with p21/p27 to the amount of Cdk4 or cyclin D 2 (cycd2) associated with p21/p27 before and after up-regulation of p16 (12 and 36 h after HGF treatment, respectively). The band intensity of the data in B was quantitated with NIH Image-J software. Each result is presented as -fold induction relative to the ratio before p16 induction (12 h). The results are representative of two or three independent experiments (A and B). the recovery of Cdk2 activity with sirna resembled that of the recovery of DNA synthesis with the sirna shown in Fig. 3C. These results suggest that the induction of p16 expression by HGF is required for the suppression of Cdk2 activity, which primarily regulates the proliferation of HepG2 cells. p21 and p27 Are Redistributed by p16 Up-regulation Induced by HGF It has been reported that overexpression of INK4 family proteins leads to cell cycle arrest in other types of cells through a redistribution of p21 and p27 from Cdk4 to Cdk2 (51 53). Thus, it is possible that the suppression of Cdk2 activity by the expression of p16 is mediated through the same mechanism. To test this possibility, we analyzed the time course of the physical association of p16, p21, and p27 with Cdk2, Cdk4, and cyclin D 2 by immunoprecipitation, followed by immunoblotting. In addition, the total amounts of Cdk2, Cdk4, and cyclin D 2 were analyzed by immunoblotting of the total lysate. The total amounts of p21 and p27 were essentially the same as those shown in the immunoprecipitation/immunoblot experiments in Fig. 6B (33) (data not shown). The amount of p16 associated with Cdk4 was increased by 24 h after HGF treatment (Fig. 6B), and the increase correlated well with the expression of p16. The amount of Cdk4 remained unchanged during this period (Fig. 6A). On the other hand, the amounts of Cdk4 and cyclin D 2 associated with p21 were increased by 4 h and abruptly declined after 24 h. A loss of the cyclin D 2 -Cdk4- p21 complex was detected following p16 up-regulation by HGF. The amount of Cdk2 associated with p21 was almost constant (Fig. 6B), and the total amount of Cdk2 protein diminished after 24 h (Fig. 6A); thus, the ratio of Cdk2 associated with p21 to the total amount of Cdk2 was increased after 24 h. The relative ratio of the Cdk2-p21 complex to the Cdk4-p21 or cyclin D 2 -p21 complex before and after the induction of p16 (12 and 36 h after HGF treatment, respectively) was calculated from the band intensity in Fig. 6B, and the result showed that the ratio was increased after the induction of p16 (Fig. 6C, left panel). These results suggest that the redistribution of p21 from Cdk4 to Cdk2 occurs along with the expression of p16 after 24 h of HGF treatment. In addition, the amount of Cdk4 and cyclin D 2 associated with p27 declined, and the amount of Cdk2 associated with p27 increased, resulting in an increased ratio of the Cdk2-p27 complex to the Cdk4-p27 or cyclin D 2 -p27 complex after the time when the expression of p16 was upregulated by HGF treatment (Fig. 6, B and C, right panel). Therefore, these results suggest that, not only p21, but also p27 is redistributed from Cdk4 to Cdk2 along with the expression of p16 induced by HGF. To examine whether the up-regulation of p16 expression is necessary for the redistribution of p21 and p27, we reduced p16 levels in HepG2 cells using the specific sirna and analyzed the formation of the cyclin-cdk-cdki complex. The total amounts of Cdk2, Cdk4, and cyclin D 2 (Fig. 7A) and p21 and p27 (Fig. 7B) were unchanged by the introduction of the sirna, but the sirna repressed the increase in Cdk4 associated with p16 (Fig. 7B). The decline in the amount of the cyclin D 2 -Cdk4-p21 complex was alleviated with p16 sirna after 24 h, and the amount of the complex simply paralleled the expression level of p21 protein. The amount of Cdk2 associated with p21 also paralleled the expression of p21 protein, suggesting no redistribution of p21 from Cdk4 to Cdk2 in the presence of p16 sirna. No change in the amount of the cyclin D 2 -Cdk4-p27 complex was observed in the presence of p16 sirna, indicating that the redistribution of p27 is suppressed by the sirna. The amount of Cdk2 associated with p27 increased even in the presence of p16 sirna (Fig. 7B), but it was less than the amount of the Cdk2-p27 complex in the absence of the sirna. In addition, the relative ratio of the Cdk2-p27 complex to the Cdk4-p27 or cyclin D 2 -p27 complex was unchanged before and after the time of up-regulation of p16 (Fig. 7C). These results suggest that the induction of p16 expression by HGF is necessary for the redistribution of p21 and p27 from Cdk4 to Cdk2, which leads to the suppression of Cdk2 activity. DISCUSSION In this study, we have shown that treatment with HGF up-regulated the expression of the Cdk inhibitor p16 through the proximal Ets-binding site in the p16 promoter (Fig. 4). The

7 31554 Redistribution of p21 and p27 by HGF-induced p16 FIG. 7.Effect of p16 sirna on formation of the cyclin-cdk-cdki complex. Cells were transiently transfected with a vector encoding p16 sirna. At 24 h after transfection, the medium was replaced with fresh medium, and cells were cultured in the presence of HGF (50 ng/ml). A, expression of Cdk2, Cdk4, cyclin D 2, and p16 in cells transfected with p16 sirna. Cell lysates were prepared at the indicated times and subjected to immunoblotting. B, formation of the cyclin-cdk-cdki complex. Cell lysates were prepared at the indicated times and analyzed as described in the legend to Fig. 6B. IP, immunoprecipitate. C, relative ratio of the amount of Cdk2 associated with p21/p27 to the amount of Cdk4 or cyclin D 2 (cycd2) associated with p21/p27 before and after up-regulation of p16 (12 and 36 h after HGF treatment, respectively) in cells transfected with p16 sirna. The band intensity of the data in B was quantitated with NIH Image-J software. Each result is presented as -fold induction relative to the ratio before p16 induction (12 h). The results are representative of two or three independent experiments (A and B). expression of exogenous p16 resulted in cell cycle arrest at G 1 in HepG2 cells (Fig. 2), and sirna-mediated p16 knockdown alleviated the inhibitory effect of HGF (Fig. 3), suggesting that the up-regulation of p16 is responsible for G 1 arrest induced by HGF. Treatment of HepG2 cells with the proper concentration of the MEK inhibitor PD98059, which represses the anti-proliferative effect of HGF (32, 33), prevented the induction of p16 by HGF (Fig. 1B), suggesting that a high intensity ERK signal is required for the up-regulation of p16 expression. These results indicate that p16 is a key regulator that links the high intensity ERK signal to cell cycle arrest at G 1 induced by HGF. Upon analysis of progressively deleted and point mutant p16 promoters, we found that the proximal Ets-binding site in the p16 promoter was required for the induction of p16 expression by HGF (Fig. 4C), suggesting that a member of the Ets family of transcription factors regulates the induction. Our previous experiment with a reporter construct of the Ras-responsive element including Ets-binding sites showed that HGF treatment induces activation of Ets by approximately 3-fold in HepG2 cells (32). Also, we have shown that Ets protein actually associated with the p16 promoter site in HGF-treated HepG2 cells (Fig. 4D). These facts confirm that Ets is involved in p16 induction. p16 has been reported to accumulate in response not only to pathological abnormalities such as the exogenous expression of oncogenic Ras and inactivation of prb (54, 55), but also to cellular senescence under physiological conditions. Ohtani et al. (40) reported that the up-regulation of p16 expression in senescent cells is mediated by Ets1 protein, the level of which is specifically elevated in such cells, through the proximal Ets-binding site in the p16 promoter and that this leads to the slower progression of the cell cycle in senescent cells than in young cells. Ets1 has also been suggested to be involved in the age-associated increase in p16 in vivo (56). Our preliminary study showed that the treatment of HepG2 cells with HGF increased the amount of Ets1 protein before the induction of p16 (data not shown); thus, it is possible that the up-regulation of p16 expression caused by HGF is mediated by the increased expression of Ets1. We found that the expression of p16 was up-regulated through strong activation of ERK in HepG2 cells treated with HGF. On the other hand, the induction of p16 expression is not mediated by the ERK signal in senescent cells (40). Thus, the increase in Ets1 protein appears to be mediated in each type of cell by different mechanisms. Ohtani et al. (40) also reported that another transcription factor, Ets2, is upregulated and binds to the proximal Ets-binding site in the p16 promoter in young cells, although p16 is not up-regulated in such cells because of the concomitant expression of a negative regulator of Ets, Id1. This up-regulation of Ets2 is mediated by the ERK signal; thus, it is possible that the up-regulation of p16 by HGF is mediated by Ets2. ChIP assay indicated that at least Ets1 and/or Ets2 was physically associated with the Etsbinding site in HGF-treated HepG2 cells (Fig. 4D), but it was impossible to distinguish which Ets protein bound to the site because of the specificity of the antibody used. Further analysis is required to uncover which member of the Ets family is responsible for activation of the p16 promoter in HGF-treated HepG2 cells. Besides the amount of the Ets family proteins, their activities are regulated by phosphorylation (57 59); thus, it is also possible that phosphorylation of the Ets family proteins may be involved in the regulation of p16 expression. We reported previously that the proliferation of HepG2 cells is mainly regulated by Cdk2 activity and that Cdk4 activity is much weaker in HepG2 cells. Furthermore, HGF treatment reduces the level of Cdk2 activity (33). In this study, sirnamediated knockdown of p16 indicated that the induction of p16 by HGF contributed to the repression of Cdk2 activity (Fig. 5C) in spite of the fact that p16 did not bind to Cdk2 and did not directly repress Cdk2 activity. We found that decreases in p21- and p27-associated cyclin D 2 /Cdk4 and increases in p21- and p27-associated Cdk2 correlated well with the increase in p16- associated Cdk4. Moreover, sirna-mediated knockdown of p16 reversed these changes (Fig. 7B). The relative ratio of Cdk2 associated with p21 or p27 to Cdk4 or of cyclin D 2 associated with each of them at 36 h clearly indicates that p16 sirna suppressed the redistribution of p21 and p27 from cyclin D 2 / Cdk4 to Cdk2 (Figs. 6C and 7C). These results suggest that the redistribution of p21 and p27 from Cdk4 to Cdk2 is induced by the increase in p16 expression, which causes the inactivation of

8 Redistribution of p21 and p27 by HGF-induced p Cdk2 and the eventual G 1 arrest of HepG2 cells. It has been reported that the redistribution of p21 and/or p27 by INK4 family proteins is induced by the exogenous expression of regulators such as p16, p15 INK4b, and p18 INK4c (51, 60 62). Furthermore, it has also been reported that physiological factors (transforming growth factor- and progesterone) induce the expression of endogenous p15 INK4b and p18 INK4c, respectively, which may be involved in the redistribution of p27 from Cdk4 and Cdk6 to Cdk2 (53, 63, 64). However, in these reports, the redistribution of p27 was shown by overexpression of p15 or p18; thus, the contribution of the induction of endogenous p15 and p18 by transforming growth factor- and progesterone to the redistribution was not directly proven. In this study, we have demonstrated, using the sirna method, that up-regulation of endogenous p16 expression by HGF led to the redistribution of p21 and p27, which is direct proof that stimulation by a physiological factor induces the redistribution of p21 and p27 by induction of an INK4 family protein. We have shown previously that treatment of HepG2 cells with HGF induces p21 expression in an ERK-dependent manner and suggested that this induction may be a cause of the G 1 arrest of the cell (33). It has also been reported that strong and/or sustained activation of the ERK pathways induces growth arrest of other cells through induction of p21 (65 68). However, our time course analysis showed that the amount of p21 protein increases at 3 h after HGF treatment and gradually decreases to the basal level before 72 h (33), indicating that the up-regulation of p21 precedes cell cycle arrest induced by HGF. In this study, we suggested that the redistribution of p21 induced by p16 is involved in cell cycle arrest at G 1 ; thus, it is possible that the p21 protein produced in response to HGF before cell cycle arrest accumulates as a complex with cyclin D-Cdk4, which acts as a low affinity reservoir for p21 and is used for the redistribution (69). However, exogenously expressed p16 induced cell cycle arrest at G 1 in HepG2 cells (Fig. 2C), indicating that the amount of endogenous p21 (and p27) protein in untreated HepG2 cells is sufficient to arrest the cell cycle. Thus, the significance and function of the up-regulation of p21 expression induced by HGF are still to be elucidated. The expression of p27 was also increased in HGF-treated HepG2 cells, but the time course of the increase was different from that of p21. The level of p27 increased gradually and peaked at 36 h after HGF treatment (Fig. 6B). The high level of p27 correlated well with the large amount of the Cdk2-p27 complex, suggesting that, in addition to the redistribution of p27 from Cdk4 to Cdk2 upon induction of p16, the increased p27 may contribute to the formation of the Cdk2-p27 complex, which leads to the repression of Cdk2 activity. It may be noteworthy that Cdk2 activity increased at4hand abruptly declined after 24 h (Fig. 5A). The amounts of Cdk2 and its main partner in HepG2 cells (cyclin A) gradually decreased during the 24 h after HGF treatment (Fig. 6A) (33); thus, the increase in Cdk2 activity is regulated through a mechanism other than the expression of these proteins. At the same time points, a transient increase in the cyclin D 2 -Cdk4- p21 complex was shown (Fig. 6B). This increased complex may indicate the reverse redistribution of p21 associated with the cyclin-cdk2 complex to cyclin D 2 -Cdk4 in addition to an increased amount of the cyclin D 2 -Cdk4 complex associated with the newly synthesized p21, which acts as a reservoir of p21 as described above. This may result in the transient activation of Cdk2 and may cause the delay of HGF effects on cell cycle arrest in HepG2 cells. The experiment using sirna showed that the knockdown of p16 restored Cdk2 activity (which was repressed to 30% by HGF treatment) to 55% (Fig. 5C). Because the efficiency of FIG. 8. Schematic model of the signaling pathway involving p16 for HGF-induced cell cycle arrest at G 1 in HepG2 cells. The binding of HGF to its specific receptor c-met elicits phosphorylation of the receptor; and subsequently, the phosphorylated c-met receptor recruits adapter proteins Grb2 and SOS, which activate the Ras-ERK signaling pathway. Intensive ERK activity results in activation of an Ets family transcription factor(s). The activated Ets protein binds to the p16 promoter at the proximal Ets-binding motif and facilitates expression of p16. Treatment of the cells with the proper concentration of PD98059 represses the high intensity ERK signal; thus, Ets retains an inactive form, leading to cancellation of p16 induction. Expression of p16 increases the association of p16 with Cdk4, leading to a redistribution of p21 and p27 from Cdk4 to Cdk2. The association of p21 and p27 with Cdk2 represses Cdk2 activity, resulting in hypophosphorylation of prb and repression of transcription by E2F. Eventually, the cell cycle of HepG2 cells is arrested in G 1. cycd, cyclin D; cyca, cyclin A. transient transfection of the plasmid expressing p16 sirna was 50%, the 25% restoration of Cdk2 activity by sirna would rise to 50% if the transfection efficiency were 100%. However, this is not enough for the full restoration of Cdk2 activity repressed by HGF. Thus, in addition to the redistribution of p21 and p27 from Cdk4 to Cdk2 upon the induction of p16 expression, other mechanisms would be involved in the repression of Cdk2 activity by HGF. As described above, a direct association of p27 with Cdk2 may be one of the mechanisms. Moreover, we have shown previously that the expression of cyclin A, a regulatory subunit for Cdk2 in HepG2 cells, is significantly decreased at 24 h after HGF treatment and suggested that the decrease contributes to the repression of Cdk2 activity by HGF (33). Thus, the decrease in cyclin A levels may be another mechanism for the repression of Cdk2 activity. In summary, the data from this study have delineated one of the molecular pathways that link the high intensity ERK signal to cell cycle arrest at G 1 in HepG2 cells stimulated by HGF (Fig. 8). The binding of HGF to its specific receptor c-met highly activates the ERK signal, leading to the activation of a member of the Ets family of transcription factors. The activated transcription factor up-regulates p16 expression. p16 forms a complex with Cdk4, leading to the redistribution of p21 and p27 from Cdk4 to Cdk2. The association of p21 and p27 with Cdk2 represses Cdk2 activity, resulting in hypophosphorylation of prb (33). The hypophosphorylated prb eventually causes cell cycle arrest at G 1. In addition to the expression of p16, the high intensity ERK signal induced by HGF changes the expression of other regulatory molecules, including p21, cyclin A, and