LPS-Induced NF-κB Activation and TNF-α Release in Human Monocytes Are Protein Tyrosine Kinase Dependent and Protein Kinase C Independent

Abstract

Background.Tumor necrosis factor alpha (TNF-α) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-κB) and TNF-α release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-κB activation and TNF-α production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-κB DNA binding and TNF-α release in human monocytes.Materials and methods.Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-α release in culture supernatants was measured by an ELISA. NF-κB DNA binding was evaluated by electrophoretic mobility shift assay.Results.LPS increased NF-κB DNA binding and TNF-α release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-κB activation and TNF-α release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-κB DNA binding or TNF-α release. PTK inhibition with Gen attenuated both LPS-induced NF-κB DNA binding and TNF-α production in human monocytes. Direct activation of PKC with PMA induced both NF-κB activation and TNF-α production by human monocytes.Conclusions.These results suggest that LPS-induced NF-κB activation and TNF-α release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-κB activation and TNF-α release in human monocytes and thus could be a potential therapeutic target in inflammatory states.