Fibronectin cell-binding domain triggered transmembrane signal transduction in human monocytes

Fibronectin (Fn) fragments have recently been shown to stimulate tumor necrosis factor (TNF) secretion by human monocytes. In this study, we investigated the signal transduction mechanisms involved in Fn-induced TNF secretion. Treatment of human monocytes with Fn120, a chymotryptic cell-binding fragment of plasma Fn, failed to cause a detectable rise in Ca2+ mobilization. Fn120-induced TNF secretion could be inhibited with Ca2+ channel blockers. The protein kinase C (PKC) inhibitors H-7 and sphingosine inhibited the TNF-inducing activity of Fn120. HA1004 was used as a control for the isoquinoline sulfonamide derivatives and did not change Fn120-induced TNF secretion by monocytes. H-8 inhibited TNF secretion at higher concentrations. A calmodulin-dependent kinase inhibitor, W-7, was found to be effective, with 50% inhibition of Fn120-induced TNF secretion at 5 microM. The activation and translocation of PKC were measured directly. In unstimulated monocytes, approximately 70% of PKC activity was found in the cytosol and 30% in the membrane. Following the stimulation of monocytes with phorbol myristate acetate (100 nM), rapid and sustained translocation of PKC from the cytosol to the membrane was observed. The stimulation of monocytes with Fn120 triggered a rapid translocation of PKC within 2 to 5 min, followed by a return to normal levels within 8 min. These findings support the conclusion that Fn120-induced TNF secretion requires the activation of PKC.