Biomedical subjects

Michael Fromm

Publications and source records attributed to Michael Fromm.

At least 19 recordsLinked to original sources

PubMed2006

AffyMiner: mining differentially expressed genes and biological knowledge in GeneChip microarray data.

BACKGROUND: DNA microarrays are a powerful tool for monitoring the expression of tens of thousands of genes simultaneously. With the advance of microarray technology, the challenge issue becomes how to analyze a large amount of microarray data and make biological sense of them. Affymetrix GeneChips are widely used microarrays, where a variety of statistical algorithms have been explored and used for detecting significant genes in the experiment. These methods rely solely on the quantitative data, i.e., signal intensity; however, qualitative data are also important parameters in detecting differentially expressed genes. RESULTS: AffyMiner is a tool developed for detecting differentially expressed genes in Affymetrix GeneChip microarray data and for associating gene annotation and gene ontology information with the genes detected. AffyMiner consists of the functional modules, GeneFinder for detecting significant genes in a treatment versus control experiment and GOTree for mapping genes of interest onto the Gene Ontology (GO) space; and interfaces to run Cluster, a program for clustering analysis, and GenMAPP, a program for pathway analysis. AffyMiner has been used for analyzing the GeneChip data and the results were presented in several publications. CONCLUSION: AffyMiner fills an important gap in finding differentially expressed genes in Affymetrix GeneChip microarray data. AffyMiner effectively deals with multiple replicates in the experiment and takes into account both quantitative and qualitative data in identifying significant genes. AffyMiner reduces the time and effort needed to compare data from multiple arrays and to interpret the possible biological implications associated with significant changes in a gene's expression.

Algorithms↗

PubMed2006

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum.

BACKGROUND: Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. AIM: To characterise the inherent pathomechanisms of G lamblia infection. METHODS: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (I(SC)) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. RESULTS: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Omega cm(2); p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm(2)). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal I(SC) increased from 191 (20) in control to 261 (12) microA/h/cm(2) in giardiasis. The bumetanide-sensitive portion of I(SC) in giardiasis was also increased (51 (5) vs 20 (9) microA/h/cm(2) in control; p<0.05). Finally, phlorizin-sensitive Na(+)-glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) microA/h/cm(2)). CONCLUSIONS: G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na(+)-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.

Adult↗

PubMed2006

Restoration of ENaC expression by glucocorticoid receptor transfection in human HT-29/B6 colon cells.

The epithelial sodium channel (ENaC) controls colonic sodium absorption. So far, investigation of ENaC was limited by an unexplained lack of steroid-dependent ENaC expression in cultured intestinal cells, which we aimed to resolve. HT-29/B6 cells constitutively expressed the alpha-ENaC subunit, while beta- and gamma-ENaC subunits could not be detected due to deficient basal as well as corticosteroid-induced transcription. This was due to a lack of expression of both activating and inhibiting isoforms of glucocorticoid receptor (GR-alpha, -beta) and mineralocorticoid receptor. Stable transfection of GR-alpha restored intestine-specific glucocorticoid upregulation of beta- and gamma-ENaC in HT-29/B6 cells, which was followed by intact targeting of ENaC channels to the apical cell membrane and dose-dependent induction of electrogenic sodium absorption. In conclusion, ENaC deficiency is due to a lack of steroid receptors and not the consequence of a crypt-like phenotype of cultured intestinal cells. By stable GR transfection we obtained a model, in which ENaC regulation can be studied.

Colon↗

PubMed2006

Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of Claudin-16.

Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle's loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.

Amino Acid Sequence↗

PubMed2006

TRPV4-mediated regulation of epithelial permeability.

TRPV4 is a calcium-permeable channel activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. We show that TRPV4 is localized in the basolateral membrane of the mouse mammary cell line HC11. Activation of TRPV4 caused an increase in the intracellular Ca(2+) concentration through influx of extracellular Ca(2+), triggering two independent chains of events: 1) a rapid increase in transcellular conductance through the activation of apical large conductance calcium-activated (BK) potassium channels that were blockable by paxilline; 2) a slow increase in paracellular permeability for small solutes. The latter effect was accompanied by a down-regulation of the tight junctional proteins claudin -1, -3, -4, -5, -7, and -8 and by dramatic changes in tight junction morphology, including frequent large breaks in the tight junction strands. This dual modulation of epithelial permeability after TRPV4 activation may be involved in regulating the tonicity across mammary gland epithelia. TRPV4 activation may also be responsible for exudation and edema formation during inflammation processes.-Reiter, B., Kraft, R., Günzel, D., Zeissig, S., Schulzke, J-D., Fromm, M., Harteneck, C. TRPV4-mediated regulation of epithelial permeability.

Animals↗

PubMed2006

Increased bacterial permeation in long-lasting ileoanal pouches.

BACKGROUND AND AIMS: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. MATERIALS AND METHODS: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch-anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na-glucose cotransport and active Cl secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. RESULTS: Bacterial permeation occurred in 2 of 13 pre-IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium-glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long-lasting pouches, but there was no correlation with bacterial permeation. CONCLUSIONS: The long-lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.

3-O-Methylglucose↗

PubMed2006

Endothelin antagonism: effects of FP receptor agonists prostaglandin F2alpha and fluprostenol on trabecular meshwork contractility.

PURPOSE: This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2alpha and fluprostenol (fluprostenol-isopropyl ester [travoprost]), which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1, a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied. METHODS: The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2alpha on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis. RESULTS: The ET-1-induced (10(-8) M) contraction in isolated BTM was inhibited by PGF2alpha (10(-6) M) and fluprostenol (10(-6) M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2alpha or fluprostenol. In cultured TM cells, ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2alpha. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM. CONCLUSIONS: FP receptor agonists operate by direct interaction with ET-1-induced contractility of TM. This effect is mediated by the FP receptor. Thus, FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1-dependent mechanisms.

Animals↗

PubMed2006

Disrupted barrier function through epithelial cell apoptosis.

Epithelial barrier function is determined by trans- and paracellular permeabilities, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. The present article aims to present experimental evidence for a functional role of epithelial apoptoses by means of cell culture models as well as in tissues from patients with inflammatory bowel disease. It is shown that epithelial apoptoses are sites of elevated conductance within the intestinal epithelium and that proinflammatory cytokines like TNF-alpha upregulate both the apoptotic rate and single apoptotic conductivity, making cytokine-induced apoptosis functionally far more relevant than is spontaneous apoptosis. In ulcerative colitis and Crohn's disease (CD), but not in collagenous colitis, apoptotic rates are increased to about 5%, in mild-to-moderately inflamed colon specimens, where as the control apoptotic rate is about 2%. Thus, epithelial apoptoses lead to a loss of ions and water into the intestinal lumen, causing leak flux diarrhea and enabling small antigens of <4,000 Da in the intestinal lumen to enter the intestinal mucosa, thereby perpetuating inflammatory responses. In addition to TNF-alpha, interleukin (IL)-13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF-alpha-antibodies (infliximab) can restore barrier function in Crohn's disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.

Apoptosis↗

PubMed2005

Pharmacological and functional characterization of endothelin receptors in bovine trabecular meshwork and ciliary muscle.

To clarify the potential role of endothelin-1 (ET-1) in the pathogenesis of glaucoma, the endothelin receptors expressed in bovine trabecular meshwork (TM) and ciliary muscle (CM) were identified. TM and CM strips were subjected to ET-1 as well as to specific endothelin receptor antagonists. In both tissues BQ123, a specific ET-A receptor antagonist, substantially inhibited ET-1-induced contraction. BQ788, a specific ET-B receptor antagonist, showed only moderate effects. Both ET receptor types were detected in bovine TM and CM using Western blot analysis. ET-1 produced an increase in intracellular calcium in cultured TM cells. This effect was inhibited by BQ123, but not by BQ788. Thus, although both receptors are present, the ET-A receptor appears to play the predominant role in mediating contraction in both the TM and CM, while the ET-B receptor seems to contribute little to the overall ET-1 effect.

Animals↗

PubMed2005

E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions.

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.

Animals↗

PubMed2005

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.

Blotting, Western↗

PubMed2005

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Adult↗

PubMed2005

The HIV protease inhibitors saquinavir, ritonavir, and nelfinavir induce apoptosis and decrease barrier function in human intestinal epithelial cells.

BACKGROUND AND AIMS: Diarrhoea is a frequent adverse effect of HIV protease inhibitors (PIs) which may be due to intestinal barrier disruption. We investigated whether tight junction dysregulation, apoptosis or necrosis are responsible for this epithelial damage. METHODS: Saquinavir, nelfinavir, and ritonavir were added to the mucosal or serosal side of HT-29/B6 colon cell monolayers. Transepithelial resistance was monitored for 72 h to assess epithelial barrier function. Apoptosis and necrosis were investigated by light and electron microscopy and quantified by nucleosome ELISA and LDH measurement, respectively. Tight junction components were analysed by Western blots of occludin and zonula occludens. Apoptosis induction in normal human intestinal epithelium was examined by measurement of poly(ADP-ribose) polymerase (PARP) cleavage in Western blots of mucosal tissue explants cultured with PIs for 24 h. RESULTS: HIV PIs decreased transepithelial resistance by more than 44% in HT-29/B6 monolayers. Histology revealed massive apoptotic body formation but no evidence for necrosis after PI treatment. Correspondingly, LDH release was lower than 0.2%/h of total LDH, independent of PI treatment, and nucleosomes were increased up to 22-fold after drug treatment versus control. Occludin and zonula occludens-1 expression in the membrane were not diminished. PARP cleavage increased in normal human intestinal tissue treated with PIs. CONCLUSIONS: PI-induced barrier disruption in intestinal epithelial cells is not due to necrosis or tight junction alterations, but to induction of massive apoptosis which may lead to leak-flux diarrhoea in vivo. Our findings suggest that induction of apoptosis by PIs could have potential for antitumour therapy.

Adult↗

PubMed2004

IL-1beta and TNFalpha regulate sodium absorption in rat distal colon.

The epithelial Na+ channel (ENaC) provides the main absorptive pathway of the distal large intestine. This study aimed to characterize regulatory influences of cytokines in rat late distal colon. After 6 h incubation with either IL1beta, TNFalpha, IFNgamma, or combinations of TNFalpha and IFNgamma, ENaC was measured as electrogenic Na+ transport after 8 h induction by 3 nM aldosterone (JNa) in totally stripped specimens in the Ussing chamber. Subsequently, alpha-, beta-, and gamma-ENaC subunit mRNAs were analyzed by Northern blotting. The gamma-ENaC promoter was cloned and characterized by reporter gene assays. IL-1beta and TNFalpha, but not interferon-gamma, decreased JNa. In parallel, beta- and gamma-ENaC transcription was inhibited, whereas alpha-ENaC was unaffected. gamma-ENaC promoter activity was inhibited by IL-1beta and TNFalpha but not by IFNgamma. We conclude that the pro-inflammatory cytokines IL-1beta and TNFalpha inhibit electrogenic sodium absorption in rat distal colon by mRNA expression regulation of the beta- and gamma-ENaC subunits.

Aldosterone↗

PubMed2004

The specific fates of tight junction proteins in apoptotic epithelial cells.

The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp(320) within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

Animals↗

PubMed2004

Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity.

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and beta-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/beta-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.

Base Sequence↗

PubMed2004

Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis.

BACKGROUND & AIMS: The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). METHODS: Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays. RESULTS: In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha. CONCLUSIONS: We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.

Antineoplastic Agents↗

PubMed2002

Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.

Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells (MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kOhms cm(2)), compared with C11 cells (<100 Ohms cm(2)). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However, confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2, was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na(+) identical with 1, K(+)=1.02, NMDG(+)=0.79, choline(+)=0.71, Cl(-)=0.12, Br(-)=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na(+) conductivity was 0.2 mS cm(-2) in vector controls and 1.7 mS cm(-2) in claudin-2-transfected cells, while Cl(-) conductivity was 0.2 mS cm(-2) in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a 'tight' tight junction into a leaky one.

Animals↗