| Ligation of CD40 expressed by monocytes/macrophages and cells of the vasculature by its ligand CD 154 expressed predominantly on activated T cells leads to the induction of inflammatory gene expression, enhancement of co-stimulatory molecules and adhesion molecule expression. All these events play a critical role in the regulation of the atherogenic process. This study was undertaken to evaluate the signaling events initiated through CD40 ligation on monocytes, macrophages and vascular smooth muscle cells.; CD40 signaling of monocytes and macrophages is dependent on Src family protein tyrosine kinase activity and the subsequent activation of the mitogen-activated protein kinase family members, ERK1/2. We next explored the role of TRAF family members in facilitating this CD40 signaling pathway in macrophages. We stably expressed either wild type human CD40 or CD40 with disrupted TRAF binding sites into a CD40-deficient macrophage cell line. Ligation of wild type CD40 and a mutant unable to bind TRAF2/3/5, but not CD40 lacking a functional TRAF6 binding site, resulted in the stimulation of inflammatory cytokine production. Likewise, introduction of a dominant negative TRAF6 into a wild type (CD40+) murine macrophage cell line resulted in abrogation of CD40-mediated induction of cytokine synthesis. The TRAF6 binding mutant was also found to be defective in CD40-mediated ERK1/2, IKK and NFkappaB activation. Lastly, treatment of monocytes with a cell permeable peptide corresponding to the TRAF6 binding motif of CD40 inhibits CD40-mediated ERK1/2 activation and inflammatory cytokine production. These data indicate that CD40 induction of macrophage inflammatory cytokine production, mediated by a Src/ERK1/2 pathway, requires the adaptor protein TRAF6.; In identifying the Src kinase(s) that may mediate CD40 signaling, we evaluated the role of Lyn as a mediator of CD40 signaling in monocytes/macrophages. Our data indicates that although Lyn is not critical for CD40-induced macrophage proinflammatory function it is required for CD40-induced TGFbeta2 synthesis.; CD40 ligation of VSMC resulted in the phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IkappaB kinase. An evaluation of Src kinases that may be important in CD40 signaling identified Lyn as a potential candidate. |
