PHORBOL ESTER-INDUCED LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE-EXPRESSION IN HEPG2 CELLS INVOLVES PROTEIN-KINASE C-MEDIATED P42 44 MAP KINASE ACTIVATION/

The signaling pathway involved in low density lipoprotein (LDL) recept or gene expression induced by tile phorbol ester 12-O-tetradecanoylpho rbol-13-acetate (TPA) was investigated in the human hepatoma HepG2 cel l line. Treatment of HepG2 cells with 100 nM TPX resulted in an approx imately 20-fold increase in LDL receptor MRNA level, as determined by RT-PCR, which peaked at 2-4 of treatment and subsequently declined. Ti le protein kinase C (PRC) inhibitors calphostin C and staurosporine pr evented TPX-mediated LDL receptor mRNA induction. In contrast, TPA did not affect squalene synthase mRNA expression, Immunoblotting of cell extracts with isozyme-specific PKC antibodies revealed that HepG2 cell s expressed PKC alpha, which was mainly cytosolic, and PKC beta, PK ep silon, and PKC zeta, all of which were present in both the cytosolic a nd particulate fractions. Treatment of HepG2 cells with 100 nM TPA res ulted in translocation of cytosolic PKC alpha to the particulate fract ion, with a maximum at SO min-2 h of treatment, but was without effect on tile subcellular distribution of the other isozymes. TPX treatment also led to activation of the mitogen-activated protein kinase (MAPK) ERK cascade. The specific MAPK pathway inhibitor PD98059 blocked TPA- induced ERK activation. Furthermore, pretreatment of cells with PD9805 9 inhibited TPA-induced LDL receptor mRNA induction. Mat-cover, pretre atment of cells with calphostin C inhibited TPA-mediated ERK activatio n and LDL receptor-mRNA induction in a dose-dependent fashion LDL Base d on a close kinetic correlation between PKC alpha translocation and E RR activation, and the erects of specific inhibitors, these findings s uggest that translocation/activation of PRC(x. and subsequent activati on of the Raf-1/MEK/ERK MAPK cascade, represent kev events in the tran scriptional induction of LDL receptor-gene by TPA in HepG2 cells.