No abstract

Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Cθ (PKCθ), but not PKCε and PKCδ, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.

Mesenteric fat is known to undergo inflammatory changes after 2,4,6,-trinitrobenzensulphonic acid (TNBS)-induced colitis. Neurotensin (NT) and neurotensin receptor 1 (NTR1) have been shown to play a major role in the pathogenesis of intestinal inflammation. This led us to explore whether NT and NTR1 are expressed in the mesenteric fat depots during TNBS-induced colitis and whether NT participates in the increased interleukin (IL)-6 secretion in this inflammatory response. TNBS-induced inflammation in the colon increases NT and NTR1 expression in mesenteric adipose tissues, including mesenteric preadipocytes. Compared with wild-type mice, NT knockout (KO) mice have reduced TNBS-induced colitis accompanied by diminished inflammatory responses in mesenteric adipose tissue. Specifically, IL-6 and p65 phosphorylation levels in mesenteric fat of NT KO mice are also reduced compared with wild-type mice. Mouse 3T3-L1 preadipocytes express NTR1 and its expression is increased after stimulation of preadipocytes with proinflammatory cytokines. NT stimulation of 3T3-L1 preadipocytes overexpressing NTR1 causes PKC␦ phosphorylation and IL-6 secretion in a time-and dose-dependent fashion. Moreover, NT-mediated IL-6 expression is nuclear factor-B and PKC␦ dependent. We also found that supernatants from NT-exposed 3T3-L1-NTR1 preadipocytes and mesenteric fat obtained from wild-type mice 2 days after TNBS administration stimulate an IL-6 -dependent macrophage migration measured by a macrophage migration assay, whereas this response is reduced when mesenteric fat from NT KO mice is used. These results demonstrate an important role for NT in acute colitis and adipose tissue inflammation associated with experimental colitis that involves direct NT proinflammatory responses in preadipocytes.cytokine ͉ intestinal inflammation ͉ macrophages ͉ neuropeptide