Emily M. Bradford

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Occludin S408 phosphorylation regulates interactions between occludin, ZO-1, and select claudins to define tight junction molecular structure and barrier function.

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}{/}\mathrm{HCO}_{3}^{-}\) \end{document} cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pHi) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pHi regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} revealed a sharp decrease in both cAMP-stimulated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} uptake during cAMP-stimulated anion secretion in the proximal colon.

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells

We questioned how tight junctions contribute to intestinal barrier function during the cell shedding that is part of physiological cell renewal. Intravital confocal microscopy studied the jejunal villus epithelium of mice expressing a fluorescent zonula occludens 1 (ZO-1) fusion protein. Vital staining also visualized the cell nucleus (Hoechst staining) or local permeability to luminal constituents (Lucifer Yellow; LY). In a cell fated to be shed, ZO-1 redistributes from the tight junction toward the apical and then basolateral cell region. ZO-1 rearrangement occurs 15 ± 6 min (n = 28) before movement of the cell nucleus from the epithelial layer. During cell extrusion, permeation of luminal LY extends along the lateral intercellular spaces of the shedding cell only as far as the location of ZO-1. Within 3 min after detachment from the epithelial layer, nuclear chromatin condenses. After cell loss, a residual patch of ZO-1 remains in the space previously occupied by the departed cell, and the size of the patch shrinks to 14 ± 2% (n = 15) of the original cell space over 20 min. The duration of cell shedding measured by nucleus movement (14 ± 1 min) is much less than the total duration of ZO-1 redistribution at the same sites (45 ± 2 min). In about 15% of cell shedding cases, neighboring epithelial cells also undergo extrusion with a delay of 5-10 min. With the use of normal mice, ZO-1 immunofluorescent staining of fixed tissue confirmed ZO-1 redistribution and the presence of ZO-1 patches beneath shedding cells. Immunostaining also showed that redistribution of ZO-1 occurred without corresponding mixing of apical and basolateral membrane domains as marked by ezrin or E-cadherin. ZO-1 redistribution is the earliest cellular event yet identified as a herald of physiological cell shedding, and redistribution of tight junction function along the lateral plasma membrane sustains epithelial barrier during cell shedding.

AE2 Cl−/HCO3− exchanger is required for normal cAMP-stimulated anion secretion in murine proximal colon

Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.

Reduced NHE3-mediated Na+ absorption increases survival and decreases the incidence of intestinal obstructions in cystic fibrosis mice

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 Na+/H+ exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target.

CLIC5 mutant mice are resistant to diet-induced obesity and exhibit gastric hemorrhaging and increased susceptibility to torpor

Chloride intracellular channel 5 (CLIC5) and other CLIC isoforms have been implicated in a number of biological processes, but their specific functions are poorly understood. The association of CLIC5 with ezrin and the actin cytoskeleton led us to test its possible involvement in gastric acid secretion. Clic5 mutant mice exhibited only a minor reduction in acid secretion, Clic5 mRNA was expressed at only low levels in stomach, and Clic5 mutant parietal cells were ultrastructurally normal, negating the hypothesis that CLIC5 plays a major role in acid secretion. However, the mutants exhibited gastric hemorrhaging in response to fasting, reduced monocytes and granulocytes suggestive of immune dysfunction, behavioral and social disorders suggestive of neurological dysfunction, and evidence of a previously unidentified metabolic defect. Wild-type and mutant mice were maintained on normal and high-fat diets; plasma levels of various hormones, glucose, and lipids were determined; and body composition was studied by quantitative magnetic resonance imaging. Clic5 mutants were lean, hyperphagic, and highly resistant to diet-induced obesity. Plasma insulin and glucose levels were reduced, and leptin levels were very low; however, plasma triglycerides, cholesterol, phospholipids, and fatty acids were normal. Indirect calorimetry revealed increased peripheral metabolism and greater reliance on carbohydrate metabolism. Because Clic5 mutants were unable to maintain energy reserves, they also exhibited increased susceptibility to fasting-induced torpor, as indicated by telemetric measurements showing episodes of reduced body temperature and heart rate. These data reveal a requirement for CLIC5 in the maintenance of normal systemic energy metabolism.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn’s disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin–mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf−/−) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM–derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2−/− BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Establishing clinically meaningful severity levels for the Tampa Scale for Kinesiophobia (TSK-13).

Kinesiophobia is an excessive, irrational and debilitating fear of physical movement and activity resulting from a feeling of vulnerability to painful injury or re‐injury. The Tampa Scale for kinesiophobia (TSK) is a patient‐reported outcome (PRO) measure designed to help identify kinesiophobia. The original version of the TSK had 17 items. A 13‐item version was later found to have better psychometric properties and was used in the present study. Although the TSK‐13 has been widely studied, one shortcoming is the lack of clinically meaningful score categories. The objective of the present study was to develop severity levels to help aid clinical interpretation of TSK‐13 scores.

The combination of ezetimibe and ursodiol promotes fecal sterol excretion and reveals a G5G8-independent pathway for cholesterol elimination

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

Volume Density, Distribution, and Ultrastructure of Secretory and Basolateral Membranes and Mitochondria Predict Parietal Cell Secretory (Dys)function

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2−/− mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2−/−, GHKAα−/−, and NHE4−/− mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.