Tyrosine kinase mediated regulation of PAI-1 production in HEL299 fibroblasts and HepG2 hepatoma cells

Abstract

Summary Objective: To determine why interleukin-6 (IL-6) regulates plasminogen activator inhibitor-1 (PAI-1) production in HEL299 but not in HepG2 hepatoma cells. Design: Since HEL299 and HepG2 cells are IL-6-responsive in various ways, both cells must have IL-6 receptors. Therefore the difference in the PAI-1 response could be due to differences in numbers or affinities of IL-6 receptors on the two cell types, or to differences manifested later in the IL-6 signaling pathway. IL-6 receptors were characterized in labeled ligand binding assays. Potential differences in tyrosine kinase signaling pathways in the two cell types were probed using different tyrosine kinase inhibitors (e.g. Genistein, Tyrphostin-25, etc). Cells were treated with or without IL-6 in the presence or absence of the inhibitors and PAI-1 was measured by ELISA. Results: IL-6 increases PAI-1 levels 2-fold over basal levels in HEL299 cells, but has no effect on HepG2 cells. In contrast, IL-1β does induce PAI-1 levels in HepG2 cells, indicating that PAI-1 is capable of cytokine response in the cells. HEL299 cells have 1250 low affinity (1.2 nM) receptors per cell. Since HepG2 cells have four times as many low affinity receptors, the inability of IL-6 to stimulate PAI-1 production in HepG2 cells is not due to the absence of insufficient number of IL-6 receptors in the HepG2 cells. All tyrosine kinase inhibitors tested reduce both basal and IL-6-stimulated PAI-1 levels in HEL299 cells. Only Genistein blocks basal levels in HepG2 cells. Conclusions: IL-6 induces PAI-1 gene expression in HEL299 cells via a signaling pathway mediated by tyrosine kinases sensitive to multiple inhibitors. Likewise basal PAI-1 production also involves tyrosine phosphorylation sensitive to all tyrosine kinase inhibitors tested. HepG2 cell basal PAI-1 production involves a Genistein sensitive tyrosine kinase(s) that is not sensitive to other tyrosine kinase inhibitors. Therefore HEL299 cells may have one or more unique kinases sensitive to all the inhibitors different from those in HepG2 cells.