Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cdelta 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-kappaB)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-kappaB 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 PKCdelta, PKC, and PKCepsilon, followed by I-kappaB kinase, IkappaBalpha, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 microM) blocked SP-induced IkappaBalpha and p65 phosphorylation and IL-8 production. The PKCdelta inhibitor rottlerin at a low concentration (1 microM), but not pseudosubstrate PKC and PKCepsilon inhibitors (10 microM), significantly reduced IL-8 secretion. PKCdelta silencing by RNA interference reduced PKCdelta protein expression and SP-induced PKCdelta phosphorylation that was associated with diminished IL-8 promoter and NF-kappaB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCdelta increased SP-induced IL-8 promoter- and NF-kappaB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCdelta plays an important role in SP-induced proinflammatory signaling in human colonocytes.
Koon
Hon-Wai
HW
Gastrointestinal Neuropeptide Center, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Zhao
Dezheng
D
Zhan
Yanai
Y
Simeonidis
Simos
S
Moyer
Mary P
MP
Pothoulakis
Charalabos
C
D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics*; D007413:Intestinal Mucosa / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013373:Substance P / Q000494:pharmacology*; D000099221:Protein Kinase C zeta
D020672:I-kappa B Proteins; D016209:Interleukin-8; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013373:Substance P; D000099221:Protein Kinase C zeta; C000606204:NFKBIA protein, human; C497258:PRKCD protein, human; C497255:PRKCE protein, human