Phosphorylation of hepatic stimulator substance on mitogen-activated protein kinase in BEL-7402 hepatoma cells.

OBJECTIVE

To obtain more insight into information of signal transduction of EGF-receptor-mediated pathway response to the stimulation of hepatic stimulator substance (HSS).

METHODS

HSS was extracted from weanling rat liver and partially purified. Bioactivity of HSS was confirmed with its ability to proliferate hepatoma cell in vitro. Meanwhile, a rat recombinant HSS vector was constructed and expressed in BL-21 E. Coli. Mitogen-activated protein kinase (MAPK) activation marked by phosphorylation at Thr202/Tyr204 was determined by Western blot.

RESULTS

The molecular weight of the biochemically purified HSS was found identically to that of the recombined HSS as expressed in the prokaryotic cells. After the treatment of HSS, cellular MAPK phosphorylation was initiated obviously at 15 min and maintained to 30 min. In comparison with EGF, MAPK phosphorylation as stimulated by HSS appeared less intensive and later time-kinetics as well. The HSS induction on cellular MAPK phosphorylation was gradually inhibited by PD98059, a specific inhibitor of MAPK kinase (MEK). A complete blockade was seen at 100 micromol/L of PD98059.

CONCLUSIONS

The involvement of HSS on MAPK activation implies that this liver-specific growth factor might take part in, either individually or as combined with other growth factors, the regulation of TPK signaling cascade during hepatocyte proliferation.