Corticotropin-releasing factor (CRF) and the closely related family of neuropeptides urocortins (Ucns) are ancient paracrine-signaling peptides secreted in both the central and peripheral neural circuits. CRF and Ucns released from the CNS (central) regulate a plethora of physiological processes that include food intake, inflammation, and bowel motility and permeability. In the gastrointestinal tract, CRF actions are largely proinflammatory, whereas the effects of the Ucn subtypes can be either pro-or antiinflammatory. Central (intracerebroventricular) or peripheral (i.p.) administration of CRF or Ucns inhibits gastric emptying and promotes colonic motility. To ascertain the role of peripherally expressed CRF and UcnII in gastrointestinal inflammation and motility, we generated ileumspecific phenotypic knockouts of these peptides by using RNA interference. Long dsRNA effectively silenced basal expression of CRF and UcnII in ileum. Control dsRNA or saline treatment did not affect CRF or UcnII expression. In an experimental model of toxininduced intestinal inflammation, inhibition of CRF ablated the inflammatory response (measured by epithelial damage, mucosal edema, and neutrophil infiltration). UcnII dsRNA treatment did not alter the inflammatory response to toxin. Furthermore, ileal motility was increased after site-specific inhibition of both CRF and UcnII. Thus, we demonstrate that ileal-specific CRF promotes inflammation and both CRF and UcnII modulate bowel motility.corticotropin-releasing factor ͉ long dsRNA ͉ RNA interference

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-κB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21WAF1/CIP1, and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1β in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

It is becoming increasingly evident that the urocortins (Ucns) and their receptors are involved in the initiation and development of inflammation in the gastrointestinal (GI) tract. There has not been a systematic study of the basal expression of Ucns or their receptors in the GI tract. Here, we examined basal expression of Ucn 2 and its high-affinity receptor, CRF-R2 in the rat GI tract. Ucn 2 mRNA was expressed throughout the small and large intestine. Surprisingly, CRF-R2 mRNA expression was detected in only a subset of GI regions that expressed Ucn 2. Immunohistochemical study showed that both Ucn 2 immuno-reactivity (Ucn 2-IR) and CRF-R2-IR were consistently seen in the neurons of the myenteric plexus and the nerve fibers innervating the circular muscle. By and large, Ucn 2-IR was detected in all layers, including the mucosal and the submucosal layers throughout the GI regions. In contrast, CRF-R2-IR was very low or undetectable in the mucosal layers of all regions examined. The role of Ucn 2 and CRF-R2 was then examined in a rat model of chemically-induced colitis. In the early phase of colitis, Ucn 2 mRNA levels peaked, whereas, in striking contrast, CRF-R2 mRNA expression decreased ∼2.5-fold below control levels. At the peptide level, Ucn 2-IR was specifically induced in a large population of immune cells that infiltrated the lamina propria and submucosa of the distal colon, whereas CRFR2-IR was detected in only a small fraction of infiltrated immune cells. CRF-R2-IR was dramatically reduced in the neurons of the myenteric plexus. Thus, we show, for the first time, that in the acute phase of inflammation, Ucn 2 levels are increased whereas expression levels of its only identified receptor, CRF-R2, are decreased. This suggests that Ucn 2 exerts its effects only in part via CRF-R2.

A. Role of calcitonin receptor-like receptor in colonic motility and inflammation. Am J Physiol Gastrointest Liver Physiol 293: G36-G44, 2007. First published March 15, 2007 doi:10.1152/ajpgi.00464.2006.-Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to ϳ2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLRinjected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acidinduced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline-or control dsRNAinjected rats. Levels of the proinflammatory cytokines TNF-␣ and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-␣ mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP.calcitonin gene-related peptide; RNA interference; trinitrobenzene sulfonic acid; cytokines; colon-specific; tumor necrosis factor-␣ CALCITONIN GENE-RELATED PEPTIDE (CGRP) is widely distributed in both the central and peripheral nervous systems. In the mammalian gastrointestinal (GI) tract, CGRP immunoreactivity is present in the myenteric and submucosal plexuses as well as in the sensory neurons, whose cell bodies are located in the nodose and dorsal root ganglia (13, 30). The functional CGRP receptor has been proposed to be a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein (RAMP) 1. Association of CLR with RAMP1 or RAMP2 confers specificity for binding either CGRP or adrenomedullin, respectively (23). Previous colocalization studies have demonstrated the presence of CLR and RAMP1 in enteric neurons, including the neurons in the myenteric plexus (5). Additionally, nerve fibers containing CGRP are found in close association with the CLR-positive neurons, suggesting that the effects of CGRP may be mediated via the CLR-RAMP1 heterodimer complex (5).CLR knockout mice are embryonic lethal with severe cardiovascular defects (6). Pharmacological antagonists for CLR have been reported, but systemic administration of antagonists does not result in tissue-specific inhibition of r...