Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases

Abstract

Interaction of the neuropeptide substance P (SP) and its neurokinin-1 receptor (NK-1R) plays an important role in the pathophysiology of intestinal inflammation. SP is known to stimulate production of interleukin (IL)-6 and IL-8 in the U-373-MG human astrocytoma cell line via activation of p38 MAPK (mitogen-activated protein kinase) and nuclear factor (NF)-kappaB, respectively. However, the signalling mechanisms by which SP-NK-1R interaction induces NF-kappaB activation and IL-8 expression are still not clear. In this study we demonstrate that SP stimulates IL-8 secretion and IL-8 promoter activity in the NCM460 non-transformed human colonic epithelial cell line transfected with NK-1R cDNA. Our results indicate that inhibition of endogenous Rho family proteins (RhoA, Rac1 and Cdc42) by their respective dominant negative mutants significantly decreases SP-induced IL-8 secretion and IL-8 promoter activity. We also demonstrate that SP rapidly activates RhoA, Rac1 and Cdc42 and that co-expression of the dominant negative mutants of RhoA, Rac1 and Cdc42 in NK-1R cDNA-transfected NCM460 cells significantly inhibits SP-induced NF-kappaB-dependent gene expression. These results demonstrate that Rho family small GTPases RhoA, Rac1 and Cdc42 are novel signal transducers for SP-stimulated IL-8 expression.

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

“…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).…”

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

“…The NK1R has been shown to mediate SP activation of inflammatory pathways, including IL-8 production (15). Therefore, an IL-8 secretion assay to functionally characterize the lentivirally introduced wild-type and mutant NK1R was undertaken.…”

“…SP was identified as a sialogogic peptide, but has also been shown to function as a neurotransmitter, a neuromodulator, and a key mediator of inflammatory processes (14). In nontransformed mucosal epithelial cells, SP can induce secretion of proinflammatory cytokines, such as IL-6, IL-8, and TNF-␣, and this induction requires activation of the transcription factor NF-B (15,16). SP binding to the NK1R elicits inositol production (17), mobilizes Ca 2ϩ (18), and activates the ERK1 and ERK2 members of the MAPK family (19).…”

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

“…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.…”