Olfactory receptor-78 (Olfr-78) is a recently identified G protein-coupled receptor activated by short-chain fatty acids acetate and propionate. A suggested role for this receptor exists in the prostate where it may influence chronic inflammatory response leading to intraepithelial neoplasia. Olfr-78 has also been shown to be expressed in mouse colon. Short-chain fatty acids and their receptors are well known to modulate inflammation in the gut. Considering this possibility, we first explored if colitis regulated Olfr-78 expression in the gut, where we observed a significant reduction in the expression of Olfr-78 transcript in mouse models of dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. To more directly test this, mice deficient in Olfr-78 were administered with DSS in water for 7 days and were found to have increased expression of IL-1β and inflammatory signs in colon compared with control mice. Next, we explored the expression of its human counterpart olfactory receptor family 51, subfamily E, member 2 (OR51E2) in human intestinal samples and observed that it was in fact also expressed in human colon samples. RNA sequence analysis revealed significant changes in the genes involved in infection, immunity, inflammation, and colorectal cancer between wild-type and Olfr-78 knockout mice. Collectively, our findings show that Olfr-78 is highly expressed in colon and downregulated in DSS- and TNBS-induced colitis, and DSS-treated Olfr-78 null mice had increased colonic expression of cytokine RNA levels, suggesting a potential role for this receptor in intestinal inflammation. Future investigations are needed to understand how Olfr-78/OR51E2 in both mouse and human intestine modulates gastrointestinal pathophysiology.
DRA or SLC26A3 is the key transporter for chloride absorption in the mammalian intestine. Our recent studies demonstrated that DRA deficiency increased paracellular permeability and altered tight and adherens junction protein expression in mouse colon. However, the mechanisms underlying increased paracellular permeability and the role of intercellular junctional proteins (desmosomes) that play a critical role in the maintenance of intestinal barrier integrity in DRA KO mice are not well understood. Aim The current studies were undertaken to examine the role of desmosomes and Rho-ROCK-MLC phosphorylation pathway in colonic barrier integrity in DRA KO mice.DRA KO and WT mice (M/F, 8-10 weeks old) were used as an in-vivo model. Protein expression was measured by immunoblotting and immunofluorescence, whereas mRNA expression was measured by QRT-PCR and Rho A activity was examined utilizing the G-LISA kit.Our results showed that the mRNA expression of desmosome proteins, desmoglein-2 (DSG2) and desmocollin-2 (DSC2) was significantly reduced (50-70%, p<0.05) in DRA KO colons compared to wild-type mice. Similarly, DSG2 protein expression was also significantly downregulated in DRA KO mice colon (~ 50%, p<0.05) as assessed by immunoblotting and immunostaining. We also examined the role of Rho-ROCK-MLC2 phosphorylation pathway in DRA KO mice. Interestingly, DRA KO mice colon showed a significant increase in Rho A protein expression (~5-fold, p<0.01) and Rho A activity (~ 3.5-fold, p<0.05) compared to WT. Furthermore, DRA KO mice also exhibited a significant decrease in ezrin phosphorylation (a downstream target of Rho A signaling) (~ 2-fold, p<0.05) compared to WT mice. In addition, another downstream target of the Rho A pathway, MYPT-1 (Myosin Phosphatase Target Subunit 1), also showed a significant decrease (~ 60%, p<0.05) in its protein expression in DRA KO mice. MYPT-1 downregulation in DRA KO mice was associated with a significant increase in MLC-2 phosphorylation (~ 2-fold, p<0.05), which may have contributed to barrier dysfunction in colon of DRA KO mice. Since, our recent studies showed microbial dysbiosis in DRA KO mice, it was of interest to examine the role of gut microbiome by co-housing WT and KO mice for 4 weeks for microbial exchange. Interestingly, co-housed DRA KO mice also showed a significant but lower increase in Rho A expression (~ 3-fold, p<0.05) and downregulation of MYPT-1 expression (~ 60%, p<0.05) compared to co-housed WT mice indicating that the regulation of Rho A and MYPT-1 expression was only partly dependent on microbial dysbiosis observed in DRA KO mice.Our studies highlight the novel role of desmosome dysregulation and increased MLC phosphorylation in compromised colonic barrier integrity in DRA KO mice.
Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in the nervous system and peripheral organs including the intestine. This peptide exerts its action via binding to VPAC1, VPAC2 and PAC1, which are G‐protein coupled receptors (GPCR). VPAC1 is the major receptor constitutively expressed in many cell types and is responsible for majority of VIP's biological activity. However, the distribution of VPAC1 along the length of the gastrointestinal tract and its sub cellular localization in intestinal epithelial cells has not been fully defined. Therefore, the current studies were undertaken to determine VPAC1 distribution and localization so that VIP based therapies can be targeted to specific regions of the intestine. Methods Real time PCR, immunoblotting and immuno‐fluorescence staining were used to determine the mRNA, protein and cellular localization of VPAC1 respectively. Intestinal tissues from male C57Bl/6 mice (6–8 weeks) and human tissues as well as colonic adenocarcinoma cell lines were used. The results indicated that the mRNA levels of VPAC1 in m ouse intestine were highest in colon followed by jejunum and ileum. In parallel, the VPAC1 protein levels were higher in the mouse colon (proximal > distal) followed by the jejunum and ileum. Immuno‐fluorescence studies in mouse colonic tissue sections demonstrated that the receptor was specifically localized to the luminal surface as evident by co‐localization of the VPAC1 protein with the apical marker villin but not with the basolateral marker sodium potassium ATPase. In the human intestine, VPAC1 mRNA expression exhibited an expression pattern similar to mouse intestine (colon > jejunum> ileum). Furthermore, in human colon, VPAC1 predominantly showed apical localization as observed by immuno fluorescence staining. Western analysis demonstrated almost equal protein levels of VPAC1 in human adenocarcinoma cell lines e.g. Caco2, T84, HT‐29 and SKC015 cells. The luminal localization was further confirmed by relatively high apical staining for VPAC1 in differentiated Caco2 monolayers. Our current data showed differential expression of VPAC1 along the length of intestine and demonstrated its localization on the luminal membrane of colonocytes. The physiological relevance of this expression and apical localization of VPAC1 remains elusive. We speculate that apical localization of VPAC1 in intestinal epithelial cells has physiological relevance in recognizing secreted peptides in the intestinal lumen and supports the potential therapeutic use of VIP nanomedicine via oral route to target gut inflammation. Characters with spaces: 2901 Support or Funding Information Supported by NIDDK and VA Merit
Microvillus inclusion disease (MVID) is a rare congenital severe malabsorptive and secretory diarrheal disease characterized by blunted or absent microvilli with accumulation of secretory granules and inclusion bodies in enterocytes. The typical clinical presentation of the disease is severe chronic diarrhea that rapidly leads to dehydration and metabolic acidosis. Despite significant advances in our understanding of the causative factors, to date, no curative therapy for MVID and associated diarrhea exists. Prognosis mainly relies on life-long total parenteral nutrition (TPN) and eventual small bowel and/or liver transplantation. Both TPN and intestinal transplantation are challenging and present with many side effects. A breakthrough in the understanding of MVID emanated from seminal findings revealing mutations inMYO5Bas a cause for MVID. During the last decade, many studies have thus utilized cell lines and animal models with knockdown ofMYO5Bto closely recapitulate the human disease and investigate potential therapeutic options in disease management. We will review the most recent advances made in the research pertaining to MVID. We will also highlight the tools and models developed that can be utilized for basic and applied research to increase our understanding of MVID and develop novel and effective targeted therapies.
Abstract Background Diagnosis and monitoring of inflammatory bowel diseases (IBDs) utilize invasive methods including endoscopy and tissue biopsy, with blood tests being less specific for IBDs. Substantial evidence has implicated involvement of the neurohormone serotonin (5-hydroxytryptamine, 5-HT) in the pathophysiology of IBDs. The current study investigated whether serum 5-HT is elevated in patients with active ulcerative colitis (UC) or Crohn’s disease (CD). Methods Serum samples were obtained from a German cohort of 96 CD and UC patients with active disease, refractory disease, or remission of disease based upon their disease activity index (DAI) and disease history. High pressure liquid chromatography with tandemmass spectrometry was used to measure 5-HT, tryptophan (TRP), and kynurenine (KYN) levels in the serum samples, and Luminex Multiplex ELISA was used to measure cytokine levels. Intestinal mucosal biopsies were obtained from a separate cohort of healthy and CD patients, and the immunoreactivity of the serotonin transporter (SERT) was determined. Results There was no statistically significant difference in TRP or KYN levels between disease categories in either UC or CD. Interestingly, 5-HT levels were significantly elevated in patients with active CD but not active UC when compared with the levels in remission or refractory disease. Serum 5-HT was superior to C-reactive protein and circulating cytokines in differentiating between disease categories in CD. Additionally, SERT immunoreactivity was decreased in the ileum and colon of patients with CD compared to healthy controls. Conclusion We have shown that the serum 5-HT can differentiate between active disease and refractory disease or remission among CD patients, emphasizing the potential suitability of serum 5-HT as an auxiliary measure in diagnosing active CD.
Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106 oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and β-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.
Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in many organs including the intestine. Its actions are mediated via G protein-coupled receptors, and vasoactive intestinal peptide receptor 1 (VPAC1) is the key receptor responsible for majority of VIP's biological activity. The distribution of VPAC1 along the length of the gastrointestinal tract and its subcellular localization in intestinal epithelial cells have not been fully characterized. The current studies were undertaken to determine VPAC1 distribution and localization so that VIP-based therapies can be targeted to specific regions of the intestine. The results indicated that the mRNA levels of VPAC1 showed an abundance pattern of colon > ileum > jejunum in the mouse intestine. In parallel, the VPAC1 protein levels were higher in the mouse colon, followed by the ileum and jejunum. Immunofluorescence studies in mouse colon demonstrated that the receptor was specifically localized to the luminal surface, as was evident by colocalization with the apical marker villin but not with the basolateral marker Na
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