Substance P-Stimulated Interleukin-8 Expression in Human Colonic Epithelial Cells Involves Protein Kinase Cδ Activation

Abstract

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 B (NF-B)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-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 Ϫ7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKC␦, PKC, and PKC⑀, followed by I-B kinase, IB␣, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 M) blocked SP-induced IB␣ and p65 phosphorylation and IL-8 production. The PKC␦ inhibitor rottlerin at a low concentration (1 M), but not pseudosubstrate PKC and PKC⑀ inhibitors (10 M), significantly reduced IL-8 secretion. PKC␦ silencing by RNA interference reduced PKC␦ protein expression and SP-induced PKC␦ phosphorylation that was associated with diminished IL-8 promoter and NF-B luciferase activities in response to SP. Moreover, overexpression of wildtype PKC␦ increased SP-induced IL-8 promoter-and NF-Bdriven luciferase activities that were rottlerin-sensitive. We conclude that PKC␦ plays an important role in SP-induced proinflammatory signaling in human colonocytes.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

“…In nontransformed human colonic epithelial NCM 460 cells overexpressing NK-1R (full-length NK1R), SP rapidly induces threonine phosphorylation of PKC␦, and this activation is critical for SP-induced NF-B activation and IL-8 gene expression (14). Our work demonstrates that the activation of truncated NK1R affects threonine phosphorylation of PKC␦ differently than activation of the full-length NK1R.…”

“…NF-B is a ubiquitous transcription factor that governs the expression of genes involved in cell-to-cell communication, cell-to-cell interaction, cell cycle, and cell growth regulation (12). SP-NK1R interaction stimulates PKC␦ and activates NF-B, resulting in up-regulation of IL-8 expression (10,11,(13)(14)(15)(16)(17). SP induces IL-8 expression via NK1R in different cell systems, including mast cells (18), NCM 460 cells, a nontransformed human colonic epithelial cell line (13,14,19), human mesenteric preadipocytes (15), human corneal epithelial cells (20), human astrocytoma cell lines (10, 21, 22), but not in human blood mononuclear cells (22), which express only the truncated NK1R (6).…”

Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals

“…IL-8 Assays-NCM-NK 1 R cells were incubated with SP (10 nM) or vehicle (control) for 4 -8 h. IL-8 in medium was measured by ELISA (12).…”

“…Cell (12). NCM-NK 1 R cells were transiently transfected with ECE-1a-d-GFP, ␤ARR1-or 2-GFP, or ␤ARR 319 -418 -GFP as described previously (9,17).…”

“…Because there is increased mucosal expression of the NK 1 R in the inflamed intestine (11), we studied human colonocytes overexpressing the NK 1 R that respond to SP by release of NF-Bdriven proinflammatory cytokines, including IL-8 (12,13). We defined the importance of ␤ARRs and ECE-1 in regulating plasma membrane, endosomal, and inflammatory signaling.…”