Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor alpha expression via activation of nuclear factor-kappaB.
BACKGROUND
Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-kappaB (NF-kappaB). The downstream target molecule(s) of NF-kappaB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) alpha, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade.


OBJECTIVE
To explore the possibility that TGFalpha might be a target molecule for NF-kappaB activated by the HCV core, and that TGFalpha participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway.


METHODS
A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-kappaB activity was examined by an electrophoretic mobility shift assay. TGFalpha transcription was assessed by a luciferase reporter assay. TGFalpha protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay.


RESULTS
In the HCV core transfectants, NF-kappaB bound to the kappaB site in the TGFalpha proximal promoter region, resulting in an increase in TGFalpha transcription. Immunoblot as well as ELISA showed increased TGFalpha expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-kappaB, cancelled HCV core-induced TGFalpha expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFalpha antibodies.


CONCLUSIONS
These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFalpha transcription via activation of NF-kappaB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.
Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan.
Y
Y
Sato
J
J
Kato
R
R
Takimoto
K
K
Takada
Y
Y
Kawano
K
K
Miyanishi
M
M
Kobune
Y
Y
Sato
T
T
Takayama
T
T
Matunaga
Y
Y
Niitsu
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D002455:Cell Division / Q000502:physiology; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D016328:NF-kappa B; D009363:Neoplasm Proteins; D010455:Peptides; C118258:SN50 peptide; D016211:Transforming Growth Factor alpha; D014758:Viral Core Proteins; D020928:Mitogen-Activated Protein Kinases