Transient Receptor Potential Vanilloid 4 Activated Inflammatory Signals by Intestinal Epithelial Cells and Colitis in Mice

doi:10.1053/j.gastro.2010.09.045

Gastroenterology

Volume 140, Issue 1, January 2011, Pages 275-285.e3

https://doi.org/10.1053/j.gastro.2010.09.045 Get rights and content

Background & Aims

Ligand-gated calcium channels have been reported to be involved in the pathogenesis of inflammatory bowel disease. One family member, transient receptor potential vanilloid 4 (TRPV4), is activated by arachidonic acid derivatives that might be released on inflammation, yet its role in gastrointestinal inflammation has not been characterized. We investigated whether TRPV4 activation participates in intestinal inflammation and its expression and functions in the gastrointestinal tract.

Methods

TRPV4 expression was studied in human colon samples, human intestinal epithelial cell lines (Caco-2 and T84), and inflamed colons of mice. Calcium mobilization and cytokine release were analyzed in intestinal epithelial cells exposed to the selective TRPV4 agonist 4α-phorbol-12,13-didecanoate (4αPDD). Mice were killed 3, 6, or 24 hours after intracolonic administration of 4αPDD; inflammatory parameters were measured in their colon tissues, and paracellular colonic permeability was measured by the passage of ⁵¹Cr-EDTA from the colon lumen to the blood.

Results

High levels of TRPV4 were detected in Caco-2 cells and in epithelial cells of human colon tissue samples; its expression was up-regulated in colons from inflamed mice compared with noninflamed control mice. Administration of 4αPDD to Caco-2 and T84 cells caused a dose-dependent increase in intracellular calcium concentration and chemokine release. In mice, intracolonic administration of 4αPDD caused colitis to develop 3 to 6 hours later; inflammation resolved by 24 hours. Increased colonic permeability was observed in vivo 3 hours after intracolonic administration of 4αPDD.

Conclusions

TRPV4 is expressed and functional in intestinal epithelial cells; its activation in the gastrointestinal tract causes increases in intracellular calcium concentrations, chemokine release, and colitis.

Section snippets

Cell Culture

T84 human intestinal epithelial cells (CCL-248; American Type Culture Collection, Manassas, VA) were cultured in a mixture of Dulbecco's modified Eagle medium and Ham's F-12 (D6421; Sigma, Saint Quentin Fallavier, France) supplemented with 5% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mmol/L l-glutamine (Invitrogen, Cergy Pontoise, France). Caco-2 human intestinal epithelial cells (HTB-37; American Type Culture Collection) were grown in Dulbecco's

TRPV4 Expression in the Gut

As seen in Figure 1, cytokeratin 18 (in red), a type I intermediate filament protein widely expressed in epithelial cells, colocalized with TRPV4 (in green) in human colonic resections from non-IBD patients (controls) or in tissues from patients with IBD (both Crohn's disease [CD] and ulcerative colitis [UC]). A strong signal for TRPV4 immunostaining was detected in cytokeratin 18–positive epithelial cells (Figure 1A). Using a marker of calcium binding proteins that are localized in the

Discussion

Our study provides evidence for a proinflammatory role of the calcium channel TRPV4 in the intestine and more particularly on intestinal epithelial cells. We identified TRPV4 as a previously unknown mediator of epithelial cell biology, raising evidence that its activation alerts innate immunity.

TRPV4 is not only detected in mouse colonic tissues and human intestinal cell lines but also is strongly expressed in human colonic tissues both from controls (non-IBD patients) and patients with IBD. We

Acknowledgments

The authors thank Dr Alexandre De Nadai Souza, Dr André Buret, Dr Daniela Balz-Hara, Dr Gilles Dietrich, and Tamia Lapointe for technical assistance; the IFR-150 core facilities for microscopy and histopathology; and Dr Nigel Bunnett and Dr Graham Cottrell for providing HEK cells expressing TRPV4.

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Liver diseases constitute a major healthcare burden globally, including acute hepatic injury resulted from acetaminophen overdose, ischemia–reperfusion or hepatotropic viral infection and chronic hepatitis, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). Attainable treatment strategies for most liver diseases remain inadequate, highlighting the importance of substantial pathogenesis. The transient receptor potential (TRP) channels represent a versatile signalling mechanism regulating fundamental physiological processes in the liver. It is not surprising that liver diseases become a newly explored field to enrich our knowledge of TRP channels. Here, we discuss recent findings revealing TRP functions across the fundamental pathological course from early hepatocellular injury caused by various insults, to inflammation, subsequent fibrosis and hepatoma. We also explore expression levels of TRPs in liver tissues of ALD, NAFLD and HCC patients from Gene Expression Omnibus (GEO) or The Cancer Genome Atlas (TCGA) database and survival analysis estimated by Kaplan–Meier Plotter. At last, we address the therapeutical potential and challenges by pharmacologically targeting TRPs to treat liver diseases. The aim is to provide a better understanding of the implications of TRP channels in liver diseases, contributing to the discovery of novel therapeutic targets and efficient drugs.
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DSS-induced acute colitis model was successfully established in mice; photodynamic therapy (PDT) treatment partially improved DSS-induced colonic damages and cell inflammation. Microarray assays and integrative bioinformatics analysis identified PPARG coactivator 1 alpha (PPARGC1A) as a significantly differentially-expressed gene in PDT-treated IBD compared with non-treated IBD. PPARGC1A expression was downregulated in IBD clinical samples, DSS-induced colitis mice colons, and DSS-stimulated colonic epithelial cells, whereas partially upregulated by PDT treatment in DSS-stimulated cells. Single DSS stimulation significantly promoted cellular inflammation; PDT partially attenuated, whereas sh-PPARGC1A transduction further enhanced DSS effects on cancer cell inflammation. In colitis mice, DSS decreased PPRA-α and PPRA-γ proteins in mice colons; the in vivo effects of DSS were partially attenuated by PDT treatment, whereas amplified by sh-PPARGC1A transduction. Upstream miR-301a-3p targeted and inhibited PPARGC1A expression.
Collectively, PPARGC1A, which is downregulated in DSS-induced acute colitis and DSS-stimulated colonic epithelial cells, could be upregulated by PDT treatment. PPARGC1A knockdown could attenuate PDT therapeutic effects on DSS-induced acute colitis and DSS-stimulated colonic epithelial cells.
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Citation Excerpt :
Finally, it is known that the expression and activity of TRPV4 are enhanced with the reduced Na+ absorption but increased Cl- secretion in UC, leading to colitis diarrhea. Therefore, it has been established that TRPV4 channels activate inflammatory signals by IECs and colitis in humans and mice (17–19). Our findings are in line with the previous notion that small numbers of TRPV4 channels lead to local Ca2+ increase that may play physiological roles, but excessive activation of TRPV4 channels lead to rapid global Ca2+ overload that may cause pathological disorders (27).
Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (I_sc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca²⁺ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal I_sc but significantly inhibited carbachol- and caffeine-induced intestinal I_sc in WT mice. Capsaicin similarly inhibited the intestinal I_sc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca²⁺ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal I_sc via Na⁺/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic I_sc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive I_sc. We therefore conclude that capsaicin inhibits intestinal Cl^- secretion and promotes Na⁺ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.

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: Conflicts of interest The authors disclose no conflicts.

: Funding N.V. is supported by grants from the Fondation Bettencourt-Schueller, INSERM-AVENIR, Agence Nationale pour la Recherche, Fondation pour la Recherche Medicale, and Fondation Schlumberger.

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Basic—Alimentary TractTransient Receptor Potential Vanilloid 4 Activated Inflammatory Signals by Intestinal Epithelial Cells and Colitis in Mice

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell Culture

TRPV4 Expression in the Gut

Discussion

Acknowledgments

Autoimmun Rev

Gastroenterology

J Biol Chem

Am J Pathol

World J Gastroenterol

J Biol Chem

Exp Cell Res

Gastroenterology

Permeation and selectivity of TRP channels

Annu Rev Physiol

TRPV4 calcium entry channel: a paradigm for gating diversity

Am J Physiol Cell Physiol

Vanilloid transient receptor potential cation channels: an overview

Curr Pharm Des

Transient receptor potential vanilloid-4 has a major role in visceral hypersensitivity symptoms

Gastroenterology

Modulation of the transient receptor potential vanilloid channel TRPV4 by 4alpha-phorbol esters: a structure-activity study

J Med Chem

Basic—Alimentary Tract
Transient Receptor Potential Vanilloid 4 Activated Inflammatory Signals by Intestinal Epithelial Cells and Colitis in Mice