Dorothee Günzel

Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease

Background: Epithelial barrier function is impaired in Crohn’s disease. Aim: To define the underlying cellular mechanisms with special attention to tight junctions. Methods: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn’s disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4′,6-diamidino-2-phenylindole staining. Results: Patients with active Crohn’s disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn’s disease compared with active ulcerative colitis and was inducible by tumour necrosis factor α. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn’s disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn’s disease in remission. Conclusion: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn’s disease.

Claudins and the Modulation of Tight Junction Permeability

Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions.

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.

Claudin-2, a component of the tight junction, forms a paracellular water channel

Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na+ gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na+-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.

Molecular Basis for Cation Selectivity in Claudin-2―based Paracellular Pores: Identification of an Electrostatic Interaction Site

Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin-expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.

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 Henles 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.

Claudins and Other Tight Junction Proteins

Epithelial transport relies on the proper function and regulation of the tight junction (TJ), other-wise uncontrolled paracellular leakage of solutes and water would occur. They also act as a fence against mixing of membrane proteins of the apical and basolateral side. The proteins determining paracellular transport consist of four transmembrane regions, intracellular N and C terminals, one intracellular and two extracellular loops (ECLs). The ECLs interact laterally and with counterparts of the neighboring cell and by this achieve a general sealing function. Two TJ protein families can be distinguished, claudins, comprising 27 members in mammals, and TJ-associated MARVEL proteins (TAMP), comprising occludin, tricellulin, and MarvelD3. They are linked to a multitude of TJ-associated regulatory and scaffolding proteins. The major TJ proteins are classified according to the physiological role they play in enabling or preventing paracellular transport. Many TJ proteins have sealing functions (claudins 1, 3, 5, 11, 14, 19, and tricellulin). In contrast, a significant number of claudins form channels across TJs which feature selectivity for cations (claudins 2, 10b, and 15), anions (claudin-10a and -17), or are permeable to water (claudin-2). For several TJ proteins, function is yet unclear as their effects on epithelial barriers are inconsistent (claudins 4, 7, 8, 16, and occludin). TJs undergo physiological and pathophysiological regulation by altering protein composition or abundance. Major pathophysiological conditions which involve changes in TJ protein composition are (1) effects of pathogens binding to TJ proteins, (2) altered TJ protein composition during inflammation and infection, and (3) altered TJ protein expression in cancers.

Microbial butyrate and its role for barrier function in the gastrointestinal tract

Butyrate production in the large intestine and ruminant forestomach depends on bacterial butyryl‐CoA/acetate‐CoA transferase activity and is highest when fermentable fiber and nonstructural carbohydrates are balanced. Gastrointestinal epithelia seem to use butyrate and butyrate‐induced endocrine signals to adapt proliferation, apoptosis, and differentiation to the growth of the bacterial community. Butyrate has a potential clinical application in the treatment of inflammatory bowel disease (IBD; ulcerative colitis). Via inhibited release of tumor necrosis factor α and interleukin 13 and inhibition of histone deacetylase, butyrate may contribute to the restoration of the tight junction barrier in IBD by affecting the expression of claudin‐2, occludin, cingulin, and zonula occludens poteins (ZO‐1, ZO‐2). Further evaluation of the molecular events that link butyrate to an improved tight junction structure will allow for the elucidation of the cofactors affecting the reliability of butyrate as a clinical treatment tool.

Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function

The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.