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Phosphorylated Heat Shock Protein 27 Represses Growth of Hepatocellular Carcinoma via Inhibition of Extracellular Signal-regulated Kinase

2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês

10.1074/jbc.m801301200

1083-351X

Rie Matsushima‐Nishiwaki, Shinji Takai, Seiji Adachi, Chiho Minamitani, Eisuke Yasuda, Takahiro Noda, Kanefusa Kato, Hidenori Toyoda, Yuji Kaneoka, Akihiro Yamaguchi, Takashi Kumada, Osamu Kozawa,

Enzyme Structure and Function

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-α, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma. Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-α, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma. The mammalian small stress protein, heat shock protein (HSP) 2The abbreviations used are: HSP, heat shock protein; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; HCC, hepatocellular carcinoma; IKK, IκB kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein kinase; MEK, MAPK/ERK kinase; MKK, mitogen-activated protein kinase kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; TNFα, tumor necrosis factor-α; WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: HSP, heat shock protein; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; HCC, hepatocellular carcinoma; IKK, IκB kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein kinase; MEK, MAPK/ERK kinase; MKK, mitogen-activated protein kinase kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; TNFα, tumor necrosis factor-α; WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 27, is a widely expressed 27-kDa protein, and it is one of 10 members of the human low molecular weight HSP family. HSPs are classified into high molecular weight HSPs such as HSP70 and HSP90, and low molecular weight HSPs with molecular masses from 10 to 30 kDa based on their apparent molecular sizes. Low molecular weight HSPs have significant similarities in terms of amino acid sequences, known as the α-crystallin domain and WDPF motif (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar, 2Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The high molecular weight HSPs act as molecular chaperones in protein folding, oligomerization, and translocation (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar). Although the functions of low molecular weight HSPs are not as well characterized as those of the high molecular weight HSPs, it is recognized that they may have chaperone activities (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar). The functions of HSP27 are regulated by post-translational modifications such as phosphorylation (3Benjamin I.J. McMillan D.R. Circ. Res. 1998; 83: 117-132Crossref PubMed Scopus (783) Google Scholar, 4Welch W.J. J. Biol. Chem. 1985; 260: 3058-3062Abstract Full Text PDF PubMed Google Scholar). Mouse HSP27 is phosphorylated at two sites (Ser-15 and Ser-82), whereas human HSP27 is phosphorylated at three sites (Ser-15, Ser-78, and Ser-82) (3Benjamin I.J. McMillan D.R. Circ. Res. 1998; 83: 117-132Crossref PubMed Scopus (783) Google Scholar). Ser-78 and Ser-82 of HSP27 are adjacent to the amino-terminal sequence of the α-crystallin domain, whereas Ser-15 is on the amino terminus of the WDPF motif. HSP27 can form oligomers up to 1000 kDa and interfere with cell death induced by several stimuli (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar, 5Beere H.M. J. Clin. Invest. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar). The oligomerization is regulated by phosphorylation of Ser-78 and/or Ser-82 and the WDPF motif, although phosphorylation of Ser-15 is unrelated to oligomerization (2Lambert H. Charette S.J. Bernier A.F. Guimond A. Landry J. J. Biol. Chem. 1999; 274: 9378-9385Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). HSP27 is reportedly phosphorylated through the following activation of the p38 mitogen-activated protein kinase (MAPK) pathway by the MAPK-activated protein kinase (MAPKAP) 2 and 3 (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar). Phosphorylated HSP27 forms a dimer, and the chaperone function is diminished (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar). However, the role of phosphorylated HSP27 has not yet been precisely elucidated. Proinflammatory stimuli, such as tumor necrosis factor-α (TNFα), are involved in the pathophysiology of viral hepatitis, alcoholic liver disease, and nonalcoholic fatty liver disease (6Wullaert A. van Loo G. Heyninck K. Beyaert R. Endocr. Rev. 2007; 28: 365-386Crossref PubMed Scopus (173) Google Scholar). TNFα plays a dichotomous role in the liver, where it not only acts as a mediator of cell death but also induces hepatocyte proliferation and liver regeneration. HSP27 was reported to be able to suppress TNFα-induced apoptosis and enhance NF-κB activity via promotion of the proteasome-dependent degradation of IκB in a human leukemic cell line (7Parcellier A. Schmitt E. Gurbuxani S. Seigneurin-Berny D. Pance A. Chantôme A. Plenchette S. Khochbin S. Solary E. Garrido C. Mol. Cell. Biol. 2003; 23: 5790-5802Crossref PubMed Scopus (278) Google Scholar). Otherwise, TNFα activates MAPK that enhances phosphorylation of HSP27 (8Haddad J.J. Land S.C. Br. J. Pharmacol. 2002; 135: 520-536Crossref PubMed Scopus (133) Google Scholar). Phosphorylated HSP27 inhibits IκB kinase (IKK) and reduces IκB degradation, thus resulting in the suppression of the NF-κB activation in HeLa cells (9Park K.J. Gaynor R.B. Kwak Y.T. J. Biol. Chem. 2003; 278: 35272-35278Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Accumulating evidence indicates that the phosphorylation of HSP27 holds the key to the TNFα related liver diseases. Hepatocellular carcinoma (HCC) commonly arises in the liver with chronic inflammation and ranks fifth in frequency on a worldwide basis, thus causing more than 1 million deaths annually (10Kojima S. Okuno M. Matsushima-Nishiwaki R. Friedman S.L. Moriwaki H. Int. J. Oncol. 2004; 24: 797-805PubMed Google Scholar). The overall survival of patients with HCC even after resection is still unsatisfactory because of frequent recurrence. The recurrence rate at 5 years after the curative treatment may exceed 70% (10Kojima S. Okuno M. Matsushima-Nishiwaki R. Friedman S.L. Moriwaki H. Int. J. Oncol. 2004; 24: 797-805PubMed Google Scholar). This high recurrence rate is not because of local recurrence or metastasis from the original lesion but rather from second primary lesions (10Kojima S. Okuno M. Matsushima-Nishiwaki R. Friedman S.L. Moriwaki H. Int. J. Oncol. 2004; 24: 797-805PubMed Google Scholar). However, the most suitable prognostic factor that suggests patients with HCC are at high risk for early recurrence has not yet been identified. MAPKs are essential components of intracellular signal transduction and are activated by phosphorylation in response to various extracellular stimuli, including growth factors, cytokines, and environmental stress. Among the MAPK family, extracellular signal-regulated kinase (ERK) is a key molecule that transfers signals into the nuclei to induce proliferation and differentiation (11Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar). In HCC, the ERK are activated, and they up-regulate cyclin D1 expression, which thus stimulates progression (12Ito Y. Sasaki Y. Horimoto M. Wada S. Tanaka Y. Kasahara A. Ueki T. Hirano T. Yamamoto H. Fujimoto J. Okamoto E. Hayashi N. Hori M. Hepatology. 1998; 27: 951-958Crossref PubMed Scopus (337) Google Scholar). Conversely, p38 MAPK negatively regulates cyclin D1 and antagonizes the c-Jun NH2-terminal kinase (JNK)-c-Jun pathway to suppress HCC development (13Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 14Hui L. Bakiri L. Mairhorfer A. Schweifer N. Haslinger C. Kenner L. Komnenovic V. Scheuch H. Beug H. Wagner E.F. Nat. Genet. 2007; 39: 741-749Crossref PubMed Scopus (302) Google Scholar). Previous studies showed that the level of phosphorylated HSP27 in human HCC tissues inversely correlates with the tumor size and the TNM stage of HCC (15Yasuda E. Kumada T. Takai S. Ishisaki A. Noda T. Matsushima-Nishiwaki R. Yoshimi N. Kato K. Toyoda H. Kaneoka Y. Yamaguchi A. Kozawa O. Biochem. Biophys. Res. Commun. 2005; 337: 337-342Crossref PubMed Scopus (54) Google Scholar). In addition, a proapoptotic, tumor-suppressive molecule protein kinase Cδ regulates HSP27 phosphorylation at a point upstream of p38 MAPK in the human HCC-derived cell line, HuH7 cells (16Takai S. Matsushima-Nishiwaki R. Tokuda H. Yasuda E. Toyoda H. Kaneoka Y. Yamaguchi A. Kumada T. Kozawa O. Life Sci. 2007; 81: 585-591Crossref PubMed Scopus (21) Google Scholar). However, the exact role and regulatory mechanism of HSP27 in human HCC remain to be clarified. This study aimed to clarify the role of phosphorylated HSP27 in HCC and to analyze the proliferation of the HCC cells transfected with unphosphorylatable or phospho-mimic mutants of human HSP27. The results showed that phosphorylated HSP27 repressed the HCC cell proliferation in the presence of proinflammatory cytokine, TNFα, via inhibition of the Raf-ERK kinase (MEK)-ERK signaling pathway and the activation of p38 MAPK-MAPK phosphatase-1 (MKP-1) pathway. Plasmids—Wild-type (WT) and mutant human HSP27s subcloned into pcDNA3.1 mammalian expression vector were kindly provided by Dr. C. Schäfer (Klinikum Grosshadern, Ludwig-Maximilians University Munich, Munich, Germany). For mutant HSP27 vectors, the cDNA of HSP27 had been mutated at serine residues 15, 78, and 82 to aspartate (3D) to imitate the phosphorylated HSP27 form or mutated at the same residues to alanine (3A) to prevent phosphorylation of HSP27 (17Kubisch C. Dimagno M.J. Tietz A.B. Welsh M.J. Ernst S.A. Brandt-Nedelev B. Diebold J. Wagner A.C.C. Göke B. Williams J.A. Schäfer C. Gastroenterology. 2004; 127: 275-286Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A constitutively active MEK1 cDNA was the generous gift from Dr. N. G. Ahn (Howard Hughes Medical Institute, University of Colorado, Boulder) (18Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn M.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1257) Google Scholar). Antibodies and Chemicals—HSP27 antibodies, phosphorylated HSP27 (Ser-15) antibodies, and phosphorylated HSP27 (Ser-78) antibodies were purchased from StressGen Biotechnologies Corp. (Atlanta, GA). Phosphorylated HSP27 (Ser-82) antibodies were obtained form Biomol (Plymouth Meeting, PA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma, respectively. Caspase 9 antibodies, ERK (p44/p42 MAPK) antibodies, phospho-ERK antibodies, MEK antibodies, phospho-MEK antibodies, phospho-c-Raf antibodies, cyclin D1 antibodies, p38 MAPK antibodies, phospho-p38 MAPK antibodies, and phospho-MKP-1 (Ser-359) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Recombinant human TNFα was a kind gift from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). Caspase-9 inhibitor I (benzyloxycarbonyl-LEHD-fluoromethyl ketone), a cell-permeable and irreversible inhibitor of caspase 9, was purchased from Merck. Cell Culture and Stable Transfections—Human HCC-derived HuH7 cells, which originated from well differentiated HCC tissues, were obtained from the Japanese Cancer Research Resources Bank. HuH7 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 1% fetal calf serum. For stable transfections, 4 × 105 HuH7 cells were cultured in 6-well dishes and then transfected with 2 μg of the WT or mutant HSP27 plasmids that expresses geneticin (G418) resistance using 12 μl of UniFECTOR transfection reagent (B-Bridge International, Mountain View, CA) in 1 ml of RPMI 1640 medium without fetal calf serum per well. One ml/well medium with 10% fetal calf serum was added 5 h after transfection. The cells were subcultured and grown in the presence of 1 mg/ml of G418 (EMD Chemicals, Inc., San Diego) 2 days later. After about 2 weeks, single G418-resistant colonies were obtained by serial dilution in 96-well dishes. The colonies then were maintained and analyzed individually for the expression of HSP27s. Cell Growth Assay—Empty vector-transfected, WT, or mutant HSP27s stably expressing HuH7 cells were plated on 96-well dishes (1 × 103 cells/well). Twenty four h after seeding, the cells were treated with or without 1 nm TNFα for the indicated time, and cell numbers were counted using the trypan blue dye exclusion method or using WST-1 reagent (Roche Diagnostics) according to the manufacturer's instructions. To investigate the influence of caspase 9 on the cell growth, WT, or the 3D HSP27, stably expressed HuH7 cells were treated with caspase-9 inhibitor I simultaneously with or without 1 nm TNFα for 6 days. Western Blotting—The cultured cells, which overexpressed WT or mutant HSP27s, were stimulated with or without TNFα for the indicated time. The cells or the snap-frozen human HCC samples were lysed, homogenized, and sonicated in lysis buffer containing 62.5 mm Tris/HCl (pH 6.8), 2% SDS, 50 mm dithiothreitol, and 10% glycerol. A Western blot analysis was performed as described previously (16Takai S. Matsushima-Nishiwaki R. Tokuda H. Yasuda E. Toyoda H. Kaneoka Y. Yamaguchi A. Kumada T. Kozawa O. Life Sci. 2007; 81: 585-591Crossref PubMed Scopus (21) Google Scholar, 19Noda T. Kumada T. Takai S. Matsushima-Nishiwaki R. Yoshimi N. Yasuda E. Kato K. Toyoda H. Kaneoka Y. Yamaguchi A. Kozawa O. Oncol. Rep. 2007; 17: 1309-1314PubMed Google Scholar). Band intensities were visualized on x-ray film with the ECL Western blotting detection system (GE Healthcare). The protein band intensities were determined by integrating the optical density over the band area (band volume) using NIH image software. The samples from the cell cultures to be quantitatively compared by Western blots were run in the same gel. Values represent the amount of phospho-ERK or phospho-MEK divided by those of total ERK or total MEK, respectively. The values represent the amount of full-length and cleaved caspase 9, phospho-c-Raf, cyclin D1, phospho-p38 MAPK, and phospho-MKP-1 divided by those of GAPDH. To quantify the protein from the HCC tissue extracts, 0.25 μl of MagicMark XP Western protein standard (Invitrogen), the marker protein, was run in every gel. Based on the intensity of the marker protein band on x-ray film, the proteins of the tissue samples were quantitatively compared. After being normalized by the intensity of the marker protein, values represent the amount of phospho- and total HSP27s or phospho-ERK divided by those of β-actin or total-ERK, respectively. The data of the normalized values of the protein bands were statistically analyzed as described under “Statistics.” Luciferase Reporter Assay—A reporter plasmid, activator protein-1 (AP-1)-Luc was kindly provided by Dr. S. Kojima (RIKEN, Wako, Japan). The cells were stimulated with or without 1 nm TNFα for 48 h before transfection. At 5 h after transfection, another 24 h of stimulation of TNFα was performed. Transient transfection with the AP-1-Luc reporter (1 μg/35-mm dish) and measurement of luciferase activity of cell lysates were performed using UniFECTOR transfection reagent and a dual luciferase reporter assay system (Promega Corp., Madison, WI) as described previously (20Matsushima-Nishiwaki R. Okuno M. Adachi S. Sano T. Akita K. Moriwaki H. Friedman S.L. Kojima S. Cancer Res. 2001; 61: 7675-7682PubMed Google Scholar). The cells were cotransfected with pRL-CMV (Renilla luciferase; 100 ng/35-mm dish) as an internal standard to normalize transfection efficiency. To examine the involvement of MEK-ERK system in AP-1-mediated transactivation activity within the 3D HSP27 mutant overexpressed cells, active MEK1 was cotransfected with the reporter plasmid. Tissue Specimens—HCC tissues were obtained by surgical resection from 44 patients infected with hepatitis viruses B (10 cases) or C (31 cases) and 3 patients with alcoholic cirrhosis at the Department of Surgery, Ogaki Municipal Hospital. No patient had previously undergone chemotherapy. The resected tissues were snap-frozen in liquid nitrogen and then stored at -80 °C until used for the Western blot analysis. The resected HCC specimens were obtained according to protocol approved by the Committee for the Conduct of Human Research at Ogaki Municipal Hospital. Informed consent was obtained from all patients. Statistics—Data are expressed as the means ± S.D. Statistical significance of the data from the cell cultures was analyzed using one-way analysis of variance, followed by Dunnett's test, and the patient clinical data were analyzed using the Pearson correlation coefficient (r). All p values were derived from two-tailed tests, and p < 0.05 was accepted as statistically significant. A Pearson correlation coefficient of |r| > 0.400 was accepted as a positive correlation. Expression of HSP27 in Wild-type, Unphosphorylated Type, or Phospho-mimic Type HSP27-transfected HuH7 Cells—To investigate the effect of phosphorylated HSP27 on HCC cell growth, human HCC-derived HuH7 cells were stably transfected with cDNAs of mutant HSP27s with alanine 15, alanine 78, and alanine 82 (3A) that mimicked the unphosphorylated type or with aspartate 15, aspartate 78, and aspartate 82 (3D) that mimicked the phosphorylated type. For comparison purposes, HuH7 cells were also transfected with wild-type (WT) HSP27 cDNA or an empty pcDNA3.1 vector (empty). A Western blot analysis demonstrated that HSP27 was overexpressing in WT, 3A, or 3D HSP27 cDNA-transfected HuH7 cells (Fig. 1). The empty vector-transfected cells only expressed intact HSP27 proteins. Anti-phospho-Ser-15 HSP27 antibodies and anti-phospho-Ser-82 HSP27 antibodies reacted with the HSP27 protein that was overexpressed in both WT and 3D HSP27 vector-transfected cells (Fig. 1). The phosphorylated HSP27 protein level in WT HSP27 cDNA-transfected HuH7 cells was almost the same as that in 3D HSP27 cDNA-transfected cells. The antibodies for human-specific phospho-Ser-78 HSP27 also reacted with the HSP27 in WT or 3D HSP27 cDNA-transfected cells as the antibodies for other phosphorylated forms (data not shown). On the other hand, the overexpressed HSP27 protein in the 3A HSP27 cDNA-transfected cells did not react with the phospho-HSP27 antibodies (Fig. 1). Phosphorylated HSP27 Retarded the HCC Cell Growth in the Presence of TNFα—To clarify the relationship between phosphorylation of HSP27 and HCC cell growth, we first studied whether the cell growth of phosphorylated HSP27-overexpressed HuH7 cells was suppressed compared with that of unphosphorylated HSP27-overexpressed cells. HCC commonly arises in the liver with chronic inflammation (10Kojima S. Okuno M. Matsushima-Nishiwaki R. Friedman S.L. Moriwaki H. Int. J. Oncol. 2004; 24: 797-805PubMed Google Scholar, 21Maeda S. Kamata H. Luo J.L. Leffert H. Karin M. Cell. 2005; 121: 977-990Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar). In the liver, the levels of TNFα, a proinflammatory stimuli, in patients with cirrhosis and HCC have been reported to be significantly higher than those in normal individuals (22Ataseven H. Bahcecioglu I.H. Kuzu N. Yalniz M. Celebi S. Erensoy A. Ustundag B. Mediators Inflamm. 2006; 2006: 1-6Crossref Scopus (89) Google Scholar). Therefore, the cell growth of phosphorylated HSP27-overexpressed HuH7 cells was examined both in the presence and in the absence of TNFα. In the absence of TNFα, all WT, 3A, or 3D HSP27-overexpressed cell lines and the empty vector-transfected cell line showed almost the same growth curve (Fig. 2A, curves 1–4). Even in the presence of 1 nm TNFα, the empty vector or 3A HSP27 vector-transfected HuH7 cells also exhibited almost similar growth rate as in the absence of TNFα (Fig. 2A, curves 5 and 7). However, the cell growth of WT and 3D HSP27-overexpressed HuH7 cells was remarkably delayed in comparison with that of 3A HSP27-overexpressed HuH7 cells in the presence of TNFα 6 days after incubation (Fig. 2A, curves 6 and 8). Phosphorylated HSP27 therefore seems to inhibit the cell growth of HCC under inflammatory conditions. Nonphosphorylated HSP27 is an inhibitor for caspase-dependent apoptosis (1Garrido C. Brunet M. Didelot C. Zermati Y. Schmitt E. Kroemer G. Cell Cycle. 2006; 5: 2592-2601Crossref PubMed Scopus (546) Google Scholar, 5Beere H.M. J. Clin. Invest. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar). It inhibits the interaction of cytochrome c, which is released from the permeabilized mitochondria, and pro-caspase 9. To study the relationship of apoptosis and the growth retardation of phosphorylated HSP27-overexpressed HuH7 cells, the activities of caspase 9 were examined in WT, 3A, or 3D HSP27-transfected HuH7 cells in the presence of TNFα. Regardless of the presence or the absence of TNFα, the amount of the full-length caspase 9 increased in 3D HSP27 cDNA-transfected HuH7 cells (Fig. 2B, lanes 7 and 8). The cleaved and activated caspase 9 significantly increased in WT HSP27 cDNA-transfected HuH7 cells after 2 h of stimulation of TNFα (Fig. 2C, lane 4). However, the increased activation of caspase 9 was shown also in the 3D HSP27-overexpressed cells in the absence of TNFα (Fig. 2C, lane 7). The cleavage of caspase 9 in the 3D HSP27-overexpressed cells in the presence of TNFα was even weaker than in the absence of TNFα (Fig. 2C, lanes 7 and 8). The similar tendency of the caspase 9 activities in the 2-h TNFα-stimulated cells was also shown in the cells after 72 h of stimulation of TNFα (data not shown). To confirm the cell growth retardation of WT and 3D HSP27-overexpressed HuH7 cells in the presence of TNFα is not related to caspase 9, we investigated whether caspase-9 inhibitor I, an irreversible inhibitor of caspase 9, restored the cell growth of WT and 3D HSP27 in the presence of TNFα. As shown in Fig. 2D, caspase-9 inhibitor I, which alone had little effect on the cell number, did not affect the cell growth of both WT and 3D HSP27-overexpressed HuH7 cells treated with 1 nm TNFα for 6 days (columns 4 and 8, in comparison with columns 3 and 7, respectively). Caspase-9 inhibitor I at a dose of 50 μm was toxic (data not shown). Therefore, it seems unlikely that caspase-dependent apoptosis caused the growth retardation of the phosphorylated HSP27-overexpressed cells in the presence of TNFα. Phosphorylated HSP27 Inhibited Prolonged Activation of ERK Signal Transduction in the HCC Cells—ERK has been reported to act as a potent proliferative factor of HCC and be constitutively activated in the human HCC cells and tissues (12Ito Y. Sasaki Y. Horimoto M. Wada S. Tanaka Y. Kasahara A. Ueki T. Hirano T. Yamamoto H. Fujimoto J. Okamoto E. Hayashi N. Hori M. Hepatology. 1998; 27: 951-958Crossref PubMed Scopus (337) Google Scholar, 23Adachi S. Okuno M. Matsushima-Nishiwaki R. Takano Y. Kojima S. Friedman S.L. Moriwaki H. Okano Y. Hepatology. 2002; 35: 332-340Crossref PubMed Scopus (62) Google Scholar). Does the cell growth retardation of the phosphorylated HSP27-overexpressed cells correlate with the ERK activity? The basal levels of phosphorylated ERK were similar among all HSP27 cDNA-transfected cells (Fig. 3A). Although the ERK phosphorylation levels in all HSP27 cDNA-transfected cells were similarly increased after 2 h of stimulation with 1 nm TNFα, phospho-ERK levels in both WT and the 3D HSP27-overexpressed HuH7 cells significantly decreased in comparison with those in 3A HSP27 cDNA or empty vector-transfected HuH7 cells after 72 h of stimulation with TNFα (Fig. 3A). Total ERK proteins were expressed at almost the same levels among all HSP27 cDNA-transfected cells regardless of whether or not they were stimulated with TNFα. The ERK activity is regulated by upstream kinases MEK and c-Raf. As shown in Fig. 3B, a significant decline of MEK activity in WT and the 3D HSP27-overexpressed HCC cells was observed in comparison with that in 3A HSP27 cDNA or empty vector-transfected cells after 72 h of stimulation of TNFα. Furthermore, significant attenuation of c-Raf activity was also shown in WT and the 3D HSP27-overexpressed HCC cells following 48 h of TNFα stimulation (Fig. 3C). Therefore, phosphorylated HSP27 might act as a repressor for prolonged activation of ERK signaling pathway at a point upstream of c-Raf in the HCC cells. Transactivation Activities of AP-1 and Cyclin D1 Expression Were Suppressed in the 3D HSP27-overexpressed HCC Cells—ERK contributes to the induction of AP-1 transcriptional activity, and AP-1 activates the cyclin D1 promoter to induce cell proliferation (13Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 24Su B. Karin M. Curr. Opin. Immunol. 1996; 8: 402-411Crossref PubMed Scopus (714) Google Scholar). Therefore, the effect of the phosphorylated HSP27 on AP-1 transactivation activity was assessed (Fig. 4A). After 72 h of stimulation with TNFα, WT and the 3D HSP27-overexpressed HuH7 cells expressed significantly less transactivation activity of AP-1 than 3A HSP27-introduced cells (Fig. 4A, columns 6 and 8, in comparison with column 7). A remarkable decrease of AP-1 transactivation activity was observed especially in 3D HSP27 cDNA-transfected cells (Fig. 4A, column 8). To confirm that the attenuation of this AP-1 transactivation activity occurred because of the ERK signaling pathway, constitutive active MEK1 cDNA was transfected into the 3D HSP27-overexpressed HuH7 cells. The active MEK1 restored AP-1 transactivation activity of the 3D HSP27-overexpressed HuH7 cells to t