Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cδ activation

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor kappa B (NF-kappa B)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-kappa B activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10(-7) M) induced an early (1 min) phosphorylation of the PKC isoforms PKC delta, PKC theta, and PKC epsilon, followed by I-kappa B kinase, I kappa B alpha, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 mu M) blocked SP-induced I kappa B alpha and p65 phosphorylation and IL-8 production. The PKC delta inhibitor rottlerin at a low concentration (1 mu M), but not pseudosubstrate PKC theta and PKC epsilon inhibitors (10 mu M), significantly reduced IL-8 secretion. PKC delta silencing by RNA interference reduced PKC delta protein expression and SP-induced PKC delta phosphorylation that was associated with diminished IL-8 promoter and NF-kappa B luciferase activities in response to SP. Moreover, overexpression of wildtype PKC delta increased SP-induced IL-8 promoter- and NF-kappa B driven luciferase activities that were rottlerin-sensitive. We conclude that PKC delta plays an important role in SP-induced proinflammatory signaling in human colonocytes.