MiR-21 in Substance P-induced exosomes promotes cell proliferation and migration in human colonic epithelial cells.

Exosomes are cellular vesicles involved in intercellular communication via their specialized molecular cargo, such as miRNAs. Substance P (SP), a neuropeptide/hormone, and its high-affinity receptor, NK-1R, are highly expressed during colonic inflammation. Our previous studies show that SP/NK-1R signaling stimulates differential miRNA expression and promotes colonic epithelial cell proliferation. In this study, we examined whether SP/NK-1R signaling regulates exosome biogenesis and exosome-miRNA cargo sorting. Moreover, we examined the role of SP/NK-1R signaling in exosome-regulated cell proliferation and migration. Exosomes produced by human colonic NCM460 epithelial cells overexpressing NK-1R (NCM460-NK1R) were isolated from culture media. Exosome abundance and uptake were assessed by Western blot analysis (abundance) and Exo-Green fluorescence microscopy (abundance and uptake). Cargo-miRNA levels were assessed by RT-PCR. Cell proliferation and migration were assessed using xCELLigence technology. Colonic epithelial exosomes were isolated from mice pretreated with SP for 3 days. Cell proliferation in vivo was assessed by Ki-67 staining. SP/NK-1R signaling in human colonic epithelial cells (in vitro) and mouse colons (in vivo) increased 1) exosome production, 2) the level of fluorescence in NCM460s treated with Exo-Green-labeled exosomes, and 3) the level of miR-21 in exosome cargo. Moreover, our results showed that SP/NK-1R-induced cell proliferation and migration are at least in part dependent on intercellular communication via exosomal miR-21 in vitro and in vivo. Our results demonstrate that SP/NK-1R signaling regulates exosome biogenesis and induces its miR-21 cargo sorting. Moreover, exosomal miR-21 promotes proliferation and migration of target cells.NEW & NOTEWORTHY Substance P signaling regulates exosome production in human colonic epithelial cells and colonic crypts in wild-type mice. MiR-21 is selectively sorted into exosomes induced by Substance P stimulation and promotes cell proliferation and migration in human colonocytes and mouse colonic crypts.

Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis.

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), that damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has been relatively understudied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A), which was induced in vivo by infection with TcdA- and/or TcdB-producing C. difficile strains but not with a TcdA-TcdB- isogenic mutant. TcdA or TcdB also induced the expression of VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor signalling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Both neutralization of VEGF-A and inhibition of its signalling pathway attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A that subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis and may also point to the pathophysiological significance of the gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approaches for severe, refractory CDI.

Neurotensin Promotes the Development of Colitis and Intestinal Angiogenesis via Hif-1α-miR-210 Signaling.

Neurotensin (NT) via its receptor 1 (NTR1) modulates the development of colitis, decreases HIF-1α/PHD2 interaction, stabilizes and increases HIF-1α transcriptional activity, and promotes intestinal angiogenesis. HIF-1α induces miR-210 expression, whereas miR-210 is strongly upregulated in response to NT in NCM460 human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1). In this study, we examined whether NT activates a NTR1-HIF-1α-miR-210 cascade using in vitro (NCM460-NTR1 cells) and in vivo (transgenic mice overexpressing [HIF-1α-OE] or lacking HIF-1α [HIF-1α-knockout (KO)] in intestinal epithelial cells and mice lacking NTR1 [NTR1-KO]) models. Pretreatment of NCM460-NTR1 cells with the HIF-1α inhibitor PX-478 or silencing of HIF-1α (small interfering HIF-1α) attenuated miR-210 expression in response to NT. Intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS) administration (2-d model) increased colonic miR-210 expression that was significantly reduced in NTR1-KO, HIF-1α-KO mice, and wild-type mice pretreated intracolonically with locked nucleic acid anti-miR-210. In contrast, HIF-1α-OE mice showed increased miR-210 expression at baseline that was further increased following TNBS administration. HIF-1α-OE mice had also exacerbated TNBS-induced neovascularization compared with TNBS-exposed wild-type mice. TNBS-induced neovascularization was attenuated in HIF-1α-KO mice, or mice pretreated intracolonically with anti-miR-210. Intracolonic anti-miR-210 also reduced colitis in response to TNBS (2 d). Importantly, miR-210 expression was increased in tissue samples from ulcerative colitis patients. We conclude that NT exerts its proinflammatory and proangiogenic effects during acute colitis via a NTR1-prolyl hydroxylase 2/HIF-1α-miR-210 signaling pathway. Our results also demonstrate that miR-210 plays a proinflammatory role in the development of colitis.

Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) microRNA-221-5p inflammatory network in human colonic epithelial cells.

BACKGROUND & AIMS: Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. However, whether SP modulates expression of microRNAs in human colonic epithelial cells remains unknown. METHODS: We performed microRNA profiling analysis of SP-stimulated human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NCM460-NK-1R). Targets of SP-regulated microRNAs were validated by real time polymerase chain reaction (RT-PCR). Functions of miRNAs were tested in NCM460-NK-1R cells and the TNBS and DSS models of colitis. RESULTS: SP stimulated differential expression of 29 microRNAs, including miR-221-5p, the highest up regulated miR (by 12.6-fold) upon SP stimulation. Bioinformatic and luciferase reporter analyses identified interleukin 6 receptor (IL-6R) mRNA as a direct target of miR-221-5p in NCM460 cells. Accordingly, SP exposure of NCM460-NK-1R cells increased IL-6R mRNA expression, while overexpression of miR-221-5p reduced IL-6R expression. NF-κB and JNK inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and colonic biopsies from Ulcerative Colitis, but not Crohn's Disease subjects, compared to controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor, exacerbated TNBS-and DSS-induced colitis, and increased colonic TNF-α, Cxcl10, and Col2 α 1 mRNA expression. In situ hybridization in TNBS-and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes. CONCLUSIONS: Our results reveal a novel NK-1R-miR-221-5p-IL-6R network that protects from colitis. The use of miR-221-5p mimics may be a promising approach for colitis treatment.

Substance P mediates pro-inflammatory cytokine release form mesenteric adipocytes in Inflammatory Bowel Disease patients.

BACKGROUND & AIMS: Substance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions. METHODS: Mesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients. RESULTS: Tacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients. CONCLUSIONS: Preadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.

INTRODUCTION: Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model. METHODS: Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression. RESULTS: Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo. CONCLUSION: Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

Anti-fibrogenic effects of the anti-microbial peptide cathelicidin in murine colitis-associated fibrosis.

BACKGROUND AND AIMS: Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. METHODS: C57BL/6J mice (n=6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6-7 weeks) colitis with fibrosis. mCRAMP peptide (5 mg/kg every 3 day, week 5-7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) were administered intracolonically or intravenously, respectively. 129Sv/J mice were infected with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp expressing lentivirus (107 infectious units day 11) was administered intravenously. RESULTS: TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice, compared to vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-beta1 (TGF-beta1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, that was reduced by LL-37 (3-5 µM) through a MAP kinase-dependent mechanism. CONCLUSION: Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts.

BACKGROUND: Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. METHODS: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial-mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. RESULTS: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. CONCLUSION: Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast-supported colon cancer cell proliferation.

Neurotensin--regulated miR-133α is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.

OBJECTIVE: Neurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo. DESIGN: miR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro. RESULTS: NT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-κB activation, as well as IL-6, IL-8 and IL-1ß messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls. CONCLUSIONS: NT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.

The neurotensin-HIF-1α-VEGFα axis orchestrates hypoxia, colonic inflammation, and intestinal angiogenesis.

The expression of neurotensin (NT) and its receptor (NTR1) is up-regulated in experimental colitis and inflammatory bowel disease; NT/NTR1 interactions regulate gut inflammation. During active inflammation, metabolic shifts toward hypoxia lead to the activation of hypoxia-inducible factor (HIF)-1, which enhances vascular endothelial growth factor (VEGF) expression, promoting angiogenesis. We hypothesized that NT/NTR1 signaling regulates intestinal manifestations of hypoxia and angiogenesis by promoting HIF-1 transcriptional activity and VEGFα expression in experimental colitis. We studied NTR1 signaling in colitis-associated angiogenesis using 2,4,6-trinitrobenzenesulfonic acid-treated wild-type and NTR1-knockout mice. The effects of NT on HIF-1α and VEGFα were assessed on human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1) and human intestinal microvascular-endothelial cells. NTR1-knockout mice had reduced microvascular density and mucosal integrity score compared with wild-type mice after 2,4,6-trinitrobenzenesulfonic acid treatment. VEGFα mRNA levels were increased in NCM460-NTR1 cells treated with 10(-7) mol/L NT, at 1 and 6 hours post-treatment. NT exposure in NCM460-NTR1 cells caused stabilization, nuclear translocation, and transcriptional activity of HIF-1α in a diacylglycerol kinase-dependent manner. NT did not stimulate tube formation in isolated human intestinal macrovascular endothelial cells but did so in human intestinal macrovascular endothelial cells cocultured with NCM460-NTR1 cells. Our results demonstrate the importance of an NTR1-HIF-1α-VEGFα axis in intestinal angiogenic responses and in the pathophysiology of colitis and inflammatory bowel disease.

Chronic unpredictable stress regulates visceral adipocyte-mediated glucose metabolism and inflammatory circuits in male rats.

Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte-associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high-throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High-throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress-related disease states.

Adipose tissue and inflammatory bowel disease pathogenesis.

Creeping fat has long been recognized as an indicator of Crohn's disease (CD) activity. Although most patients with CD have normal or low body mass index (BMI), the ratio of intraabdominal fat to total abdominal fat is far greater than that of controls. The obesity epidemic has instructed us on the inflammatory nature of hypertrophic adipose tissue and similarities between mesenteric depots in obese and CD patients can be drawn. However, several important physiological differences exist between these two depots as well. While the molecular basis of the crosstalk between mesenteric adipose and the inflamed intestine in CD is largely unknown, novel evidence implicates neuropeptides along with adipocyte-derived paracrine mediators (adipokines) as potential targets for future investigations and highlight adipose tissue physiology as a potential important determinant in the course of IBD.

Neurotensin-induced proinflammatory signaling in human colonocytes is regulated by ß-arrestins and endothelin-converting enzyme-1-dependent endocytosis and resensitization of neurotensin receptor 1.

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, ß-arrestin-1 (ßARR1), and ß-arrestin-2 (ßARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of ßARR1 or ßARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves ßARRs and ECE-1, respectively. Our results also indicate that ßARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

Role of substance P in the regulation of glucose metabolism via insulin signaling-associated pathways.

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1(-/-)) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1(-/-) mice and wild-type littermates, Tac1(-/-) mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase C, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase C pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes.

Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

BACKGROUND & AIMS: Neurotensin promotes inflammation and colon cancer via the neurotensin-1 receptor (NTR1). MicroRNAs (miR) regulate protein synthesis by degrading or preventing translation of mRNAs. We analyzed expression of 365 different microRNAs by human colonic epithelial cells (NCM460) after activation of NTR1. METHODS: We performed microarray analysis of mRNA expression by neurotensin-stimulated NCM460 cells that overexpressed NTR1. Nuclear factor-κB (NF-κB) binding sites were identified and tumorigenesis was assessed using soft agar assays and xenograft analysis of severe combined immunodeficiency mice. Targets of neurotensin-regulated microRNAs were identified via bioinformatic, real-time polymerase chain reaction, and immunoblot analyses. We analyzed RNA samples from human normal colon and tumor samples. RESULTS: Neurotensin stimulated differential expression of 38 microRNAs, including miR-21 and miR-155, which have been associated with tumor growth and contain NF-κB binding sites. Neurotensin expression increased colony formation by HCT-116 cells. Blocking miR-21 and/or miR-155 prevented colony formation (P < .001). In mice, intraperitoneal administration of neurotensin increased the growth rate of HCT-116 xenograft tumors; blocking miR-21 and/or miR-155 slowed this tumor growth. Neurotensin activated Akt in HCT-116 cells; this effect was inhibited by blocking miR-21 and/or miR-155 (P < .001). Neurotensin activated AKT through miR-155-mediated suppression of the phosphatase protein phosphatase 2A catalytic subunit alpha (PPP2CA). Levels of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 1 (SOCS1) mRNA, potential targets of miR-21 and miR-155, respectively, were down-regulated by these miRs. Levels of NTR1, miR-21, and miR-155 increased significantly in human colon tumor samples, compared with normal tissues, whereas PPP2CA, SOCS1, and PTEN mRNAs were reduced significantly. CONCLUSIONS: NTR1 activation stimulates expression of miR-21 and miR-155 in colonocytes, via Akt and NF-κB, to down-regulate PTEN and SOCS1 and promote growth of tumors in mice. Levels of NTR1, miR-21, and miR-155 increase in human colon tumor samples and correlate with tumor stage.

Cathelicidin signaling via the Toll-like receptor protects against colitis in mice.

BACKGROUND & AIMS: Cathelicidin (encoded by Camp) is an antimicrobial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease, and we investigated its signaling mechanisms. METHODS: Quantitative, real-time, reverse-transcription polymerase chain reaction (PCR), bacterial 16S PCR, immunofluorescence, and small interfering RNA (siRNA) analyses were performed. Colitis was induced in mice using dextran sulfate sodium (DSS); levels of cathelicidin were measured in human primary monocytes. RESULTS: Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to Escherichia coli DNA induced expression of Camp messenger RNA, which required signaling by extracellular signal-regulated kinase (ERK); expression was reduced by siRNAs against Toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9(-/-) mice with DSS-induced colitis. Compared with wild-type mice, Camp(-/-) mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. CONCLUSIONS: Cathelicidin protects against induction of colitis in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA. This pathway might be involved in the pathogenesis of ulcerative colitis.

Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats.

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.

Neuropeptide - adipose tissue communication and intestinal pathophysiology.

Until recently, fat was considered a relatively inactive tissue serving only as a depot for the storage of excess lipid around the body. Over the last decade, however, several studies have established fat as a metabolically active endocrine organ able to affect human pathophysiology at multiple levels. During this time adipose tissue has been shown to produce a number of hormones and inflammatory mediators collectively termed as adipokines. These molecules have been shown to be involved in the etiology of a number of inflammation-associated pathological conditions ranging from atherosclerosis and hypertension to diabetes and cancer. Despite the close physical association of abdominal fat and the intestine in the visceral cavity and the significant paracrine functions now attributed to adipose tissue, very little is known on the potential interactions between these tissues as they may relate to intestinal homeostasis. Considering the dramatic alterations in mesenteric fat depot size and placement during at least one intestinal disease, Crohn's disease, the potential involvement of fat tissue in the development as well as the progression of this and other pathological conditions should be considered. In this review we discuss the latest knowledge on neuropeptide-adipose tissue communication and the potential changes such interaction may induce in intra-abdominal fat tissue physiology. Finally we will discuss evidence on the potential pathways by which such changes in fat physiology may affect the development and progress of intestinal pathological conditions such as inflammatory bowel disease.

Substance P (SP)-neurokinin-1 receptor (NK-1R) alters adipose tissue responses to high-fat diet and insulin action.

Peripheral administration of a specific neurokinin-1 receptor (NK-1R) antagonist to mice leads to reduced weight gain and circulating levels of insulin and leptin after high-fat diet (HFD). Here, we assessed the contribution of substance P (SP) and NK-1R in diet-induced obesity using NK-1R deficient [knockout (KO)] mice and extended our previous findings to show the effects of SP-NK-1R interactions on adipose tissue-associated insulin signaling and glucose metabolic responses. NK-1R KO and wild-type (WT) littermates were fed a HFD for 3 wk, and obesity-associated responses were determined. Compared with WT, NK-1 KO mice show reduced weight gain and circulating levels of leptin and insulin in response to HFD. Adiponectin receptor mRNA levels are higher in mesenteric fat and liver in NK-1 KO animals compared with WT, after HFD. Mesenteric fat from NK-1R KO mice fed with HFD has reduced stress-activated protein kinase/c-Jun N-terminal kinase and protein kinase C activation compared with WT mice. After glucose challenge, NK-1R KO mice remove glucose from the circulation more efficiently than WT and pair-fed controls, suggesting an additional peripheral effect of NK-1R-mediated signaling on glucose metabolism. Glucose uptake experiments in isolated rat adipocytes showed that SP directly inhibits insulin-mediated glucose uptake. Our results further establish a role for SP-NK-1R interactions in adipose tissue responses, specifically as they relate to obesity-associated pathologies such as glucose intolerance and insulin resistance. Our results highlight this pathway as an important therapeutic approach for type 2 diabetes.