Signaling pathways involved in the induction of PAI-1 by VLDL in HEPG2 cells

We have previously shown that very low density lipoproteins (VLDL) enhance the biosynthesis of plasminogen activator inhibitor type 1 (PAl-1) in HepG2 cells. In this study we have investigated the mechanism(s) responsible for the induction of PAI-1 biosynthesis by this lipoprotein fraction. To this end subconfluent HepG2 cells were incubated for 16 h with 100 µg/ml VLDL in the presence of different inhibitors of signalling pathways. Mepacrine (15µM), a phospholipase inhibitor, completely prevented the enhancing effect of VLDL on PAI-I secretion. Protein kinase C involvement was investigated using a specific inhibitor (H7, 50µM) or by enzyme downregulation by cell pretreatment with PMA (100nM). In these conditions VLDL induced PAI-1 biosynthesis was reduced by 80% and 40%, respectively. The role of calcium was investigated by the use of specific inhibitors added to cell cultures before VLDL. TMB8 (20 µM), which prevents Ca2+ release from intracellular stores, reduced PAI-1 secretion by 30%, whereas EGTA (1mM) plus thapsigargin (1-2µM), which induce Ca2+ depletion from internal membrane stores, inhibited it by 50%. In contrast, removal of calcium from the cell culture medium with EGTA (1mM), or blocking ions influx with Nifedipine (50µM) did not not prevent P AI-1 induction by VLDL. Overall the data indicate that several secondary messenger-generating pathways, as the phospholipases, protein kinase C and the release of calcium from internal membrane stores form networks of coregulation which result in the induction of PAI-1 biosynthesis by VLDL.