| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
To identify the molecules whose mRNA expression were up-regulated/down-regulated by hepatitis C virus (HCV) core protein, we examined the expression profile of down-stream molecules of Raf/MEK/MAP kinase (ERK1/2) by using RT-PCR analysis and RNase protection assay. In HepΔNCTH cells, the HepG2 cells stably expressing HCV core protein, the expression of c-fos mRNA was markedly down-regulated : c-myc and heparin-binding EGF-like growth factor (HB-EGF) were up-regulated. Western blot analysis showed that expression of c-Fos protein was decreased in cellular extract from HepΔNCTH cells.
In the present study, we concentrated upon the molecular analysis of HB-EGF induced by HCV core protein, since it functions in a number of signal transduction pathway. By using ELISA assay, we identified the soluble HB-EGF fraction was significantly increased in culture medium for HepΔNCTH cells (starved for 24 hours). Introduction of plasmid expressing antisense RNA for HCV core gene inhibited secretion of soluble HB-EGF from HepΔNCTH cells, suggesting that HCV core protein specifically induced autocrine secretion of HB-EGF. Moreover, PD08059, a specific MEK inhibitor, also inhibited secretion of soluble HB-EGF from HepΔNCTH cells, suggesting that the activation of Raf-1 kinase by HCV core protein is essential for increased secretion of HB-EGF. It has been described that phosphorylated Raf-1 kinase induced HB-EGF secretion and subsequently activated c-Jun NH2-terminus kinase (JNK). Therefore we investigated whether JNK was activated via autocrine secretion of soluble HB-EGF by HCV core protein. JNK was hyperphosphorylated in HepΔNCTH cells, as compared to HepG2 cells. Antibody-neutralization of soluble HB-EGF in culture medium essentially decreased the phosphorylation of JNK in HepΔNCTH cells, suggesting that activation of JNK by HCV core protein indirectly activated JNK via autocrine secretion of HB-EGF induced by Raf-1 kinase activation.
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