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

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

doi:10.1074/jbc.m801301200

Abstract

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-alpha, 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.

Highlights

10 members of the human low molecular weight heat shock protein (HSP) family
The results showed that phosphorylated HSP27 repressed the Hepatocellular carcinoma (HCC) cell proliferation in the presence of proinflammatory cytokine, TNF␣, via inhibition of the Raf-extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling pathway and the activation of p38 mitogen-activated protein kinase (MAPK)-MAPK phosphatase-1 (MKP-1) pathway
HuH7 cells were transfected with wild-type (WT) HSP27 cDNA or an empty pcDNA3.1 vector

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Wild-type (WT) and mutant human HSP27s subcloned into pcDNA3.1 mammalian expression vector were kindly provided by Dr C. 4 ϫ 105 HuH7 cells were cultured in 6-well dishes and transfected with 2 ␮g of the WT or mutant HSP27 plasmids that expresses geneticin (G418) resistance using 12 ␮l of UniFECTOR transfection reagent (B-Bridge International, Mountain View, CA) in 1 ml of RPMI 1640 medium without fetal calf serum per well. Cell Growth Assay—Empty vector-transfected, WT, or mutant HSP27s stably expressing HuH7 cells were plated on 96-well dishes (1 ϫ 103 cells/well). Western Blotting—The cultured cells, which overexpressed WT or mutant HSP27s, were stimulated with or without TNF␣ for the indicated time. 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 ␤-actin or totalERK, respectively. A Pearson correlation coefficient ofr Ͼ 0.400 was accepted as a positive correlation

RESULTS

B Cyclin D1

B Phospho-MKP-1