John Richard White

Effect of protein kinase inhibitors on IL-8/NAP-1 release from human umbilical vein endothelial cells

Several protein kinase inhibitors (PKIs) were investigated for their effects on IL-1β, TNFα and PMA-induced IL-8 production from human umbilical vein endothelial cells (HUVEC). IL-1β (ED50 0.07 ng/ml), TNFα (ED50 100 ng/ml) and PMA (ED50 20 ng/ml) induced IL-8 production that could be detected as early as 2 h following stimulation. Staurosporine, a potent but non-specific inhibitor of protein kinases, inhibited PMA-induced (IC50 2 nM) but not IL-1β or TNFα (IC50>200 nM) induced IL-8 production. Neither the cAMP-dependent PKI, KT5720, nor the tyrosine PKIs, genistein, tyrphostin (1–100 μM) or lavendustin A (0.0001–1 μM), inhibited IL-8 production elicited by IL-1β. However, the macrolide protein kinase inhibitor geldanamycin (IC50=30 nM), but not the closely related analog herbimycin A (5–500 nM), inhibited IL-8 production by 60%. Northern blot analysis of IL-8 mRNA revealed that staurosporin suppressed mRNA increase following stimulation by PMA but not by IL-1. It is proposed that a novel protein kinase susceptible to geldanamycin inhibition may be involved in IL-1-mediated signal transduction.