Tight Junctions as Targets and Effectors of Mucosal Immune Homeostasis

Defective epithelial barrier function is present in maladies including epidermal burn injury, environmental lung damage, renal tubular disease, and a range of immune-mediated and infectious intestinal disorders. When the epithelial surface is intact, the paracellular pathway between cells is sealed by the tight junction. However, permeability of tight junctions varies widely across tissues and can be markedly impacted by disease. For example, tight junctions within the skin and urinary bladder are largely impermeant and their permeability is not regulated. In contrast, tight junctions of the proximal renal tubule and intestine are selectively permeable to water and solutes on the basis of their biophysical characteristics and, in the gut, can be regulated by the immune system with remarkable specificity. Conversely, modulation of tight junction barrier conductance, especially within the gastrointestinal tract, can impact immune homeostasis and diverse pathologies. Thus, tight junctions are both effectors and targets of immune regulation. Using the gastrointestinal tract as an example, this review explores current understanding of this complex interplay between tight junctions and immunity. Defective epithelial barrier function is present in maladies including epidermal burn injury, environmental lung damage, renal tubular disease, and a range of immune-mediated and infectious intestinal disorders. When the epithelial surface is intact, the paracellular pathway between cells is sealed by the tight junction. However, permeability of tight junctions varies widely across tissues and can be markedly impacted by disease. For example, tight junctions within the skin and urinary bladder are largely impermeant and their permeability is not regulated. In contrast, tight junctions of the proximal renal tubule and intestine are selectively permeable to water and solutes on the basis of their biophysical characteristics and, in the gut, can be regulated by the immune system with remarkable specificity. Conversely, modulation of tight junction barrier conductance, especially within the gastrointestinal tract, can impact immune homeostasis and diverse pathologies. Thus, tight junctions are both effectors and targets of immune regulation. Using the gastrointestinal tract as an example, this review explores current understanding of this complex interplay between tight junctions and immunity. SummaryParacellular transport across the selectively permeable mucosal barrier is essential for health. Two distinct pathways, pore and leak, mediate transport across the tight junction, which is the rate-limiting step in paracellular flux. The permeabilities of these routes can be differentially regulated by immune and other stimuli and, conversely, have distinct effects on intestinal and systemic immune function. Paracellular transport across the selectively permeable mucosal barrier is essential for health. Two distinct pathways, pore and leak, mediate transport across the tight junction, which is the rate-limiting step in paracellular flux. The permeabilities of these routes can be differentially regulated by immune and other stimuli and, conversely, have distinct effects on intestinal and systemic immune function. Mucosal surfaces are lined by epithelial cells that, depending on the site, mediate and regulate nutrition absorption,1Chen L. Tuo B. Dong H. Regulation of intestinal glucose absorption by ion channels and transporters.Nutrients. 2016; 8Crossref Scopus (40) Google Scholar, 2Kato A. Romero M.F. Regulation of electroneutral NaCl absorption by the small intestine.Annu Rev Physiol. 2011; 73: 261-281Crossref PubMed Scopus (83) Google Scholar, 3Kellett G.L. Brot-Laroche E. Mace O.J. Leturque A. Sugar absorption in the intestine: the role of GLUT2.Annu Rev Nutr. 2008; 28: 35-54Crossref PubMed Scopus (269) Google Scholar, 4Pappenheimer J.R. On the coupling of membrane digestion with intestinal absorption of sugars and amino acids.Am J Physiol. 1993; 265: G409-G417PubMed Google Scholar, 5Turnberg L.A. Absorption and secretion of salt and water by the small intestine.Digestion. 1973; 9: 357-381Crossref PubMed Google Scholar secretion,5Turnberg L.A. Absorption and secretion of salt and water by the small intestine.Digestion. 1973; 9: 357-381Crossref PubMed Google Scholar, 6Binder H.J. Rajendran V. Sadasivan V. Geibel J.P. Bicarbonate secretion: a neglected aspect of colonic ion transport.J Clin Gastroenterol. 2005; 39: S53-S58Crossref PubMed Google Scholar, 7Barrett K.E. Keely S.J. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects.Annu Rev Physiol. 2000; 62: 535-572Crossref PubMed Scopus (0) Google Scholar physical barrier protection,8Liang G.H. Weber C.R. Molecular aspects of tight junction barrier function.Curr Opin Pharmacol. 2014; 19C: 84-89Crossref Scopus (36) Google Scholar,9Koch S. Nusrat A. Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions.Ann N Y Acad Sci. 2009; 1165: 220-227Crossref PubMed Scopus (0) Google Scholar transcellular transport,4Pappenheimer J.R. On the coupling of membrane digestion with intestinal absorption of sugars and amino acids.Am J Physiol. 1993; 265: G409-G417PubMed Google Scholar,10Khanal R.C. Nemere I. Regulation of intestinal calcium transport.Annu Rev Nutr. 2008; 28: 179-196Crossref PubMed Scopus (0) Google Scholar and environmental sensing.11Smith K. Karimian Azari E. LaMoia T.E. Hussain T. Vargova V. Karolyi K. Veldhuis P.P. Arnoletti J.P. de la Fuente S.G. Pratley R.E. Osborne T.F. Kyriazis G.A. T1R2 receptor-mediated glucose sensing in the upper intestine potentiates glucose absorption through activation of local regulatory pathways.Mol Metab. 2018; 17: 98-111Crossref PubMed Scopus (5) Google Scholar, 12Wang S. Charbonnier L.M. Noval Rivas M. Georgiev P. Li N. Gerber G. Bry L. Chatila T.A. MyD88 adaptor-dependent microbial sensing by regulatory T cells promotes mucosal tolerance and enforces commensalism.Immunity. 2015; 43: 289-303Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 13Knoop K.A. McDonald K.G. McCrate S. McDole J.R. Newberry R.D. Microbial sensing by goblet cells controls immune surveillance of luminal antigens in the colon.Mucosal Immunol. 2015; 8: 198-210Crossref PubMed Scopus (90) Google Scholar, 14Oh J.Z. Ravindran R. Chassaing B. Carvalho F.A. Maddur M.S. Bower M. Hakimpour P. Gill K.P. Nakaya H.I. Yarovinsky F. Sartor R.B. Gewirtz A.T. Pulendran B. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination.Immunity. 2014; 41: 478-492Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 15Pacheco A.R. Curtis M.M. Ritchie J.M. Munera D. Waldor M.K. Moreira C.G. Sperandio V. Fucose sensing regulates bacterial intestinal colonization.Nature. 2012; 492: 113-117Crossref PubMed Scopus (225) Google Scholar At these sites, the plasma membranes of epithelial cells, along with extracellular components (eg, mucin), establish a barrier that prevents free exchange of materials between the lumen and subepithelial tissues (ie, the lamina propria). Nevertheless, a potential shunt pathway between adjacent epithelial cells must also be sealed. This requires structural support by desmosomes and adherens junctions, which link epithelial cells to one another, and tight junctions, which limit paracellular flux. Importantly, tight junctions are not absolute seals, but are selectively permeable barriers that discriminate between water and solutes on the basis of size and charge. Two distinct pathways across the tight junction have been described and can be separately regulated by immune signals. Conversely, changes in the permeability of each pathway can differentially modulate mucosal immune activation. Thus, the interaction between tight junctions and mucosal immune system is a dynamic conversation with signals being transmitted in both directions. Some forms of immune activation and other stimuli reduce intestinal barrier function by directly damaging the epithelium, thereby creating a flux route termed the unrestricted pathway. Finally, any analysis of signaling between the immune system and epithelium must consider the means by which luminal materials, including microbiota and their metabolites, interact with the epithelium. The complete molecular composition and structure of tight junctions remain to be defined. However, a great deal of progress has been made over the half-century since tight junctions were initially described.16Farquhar M. Palade G. Junctional complexes in various epithelia.J Cell Biol. 1963; 17: 375-412Crossref PubMed Google Scholar, 17Staehelin L.A. Mukherjee T.M. Williams A.W. Freeze-etch appearance of the tight junctions in the epithelium of small and large intestine of mice.Protoplasma. 1969; 67: 165-184Crossref PubMed Scopus (0) Google Scholar, 18Machen T.E. Erlij D. Wooding F.B. Permeable junctional complexes. The movement of lanthanum across rabbit gallbladder and intestine.J Cell Biol. 1972; 54: 302-312Crossref PubMed Google Scholar This includes discovery of zonula occludens (ZO)-119Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.J Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1142) Google Scholar and the related cytoplasmic scaffolding proteins ZO-220Jesaitis L.A. Goodenough D.A. Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.J Cell Biol. 1994; 124: 949-961Crossref PubMed Google Scholar and ZO-321Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin.J Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (460) Google Scholar; cingulin22Citi S. Sabanay H. Jakes R. Geiger B. Kendrick-Jones J. Cingulin, a new peripheral component of tight junctions.Nature. 1988; 333: 272-276Crossref PubMed Google Scholar; the tight junction associated Marvel proteins occludin,23Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions.J Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (1921) Google Scholar tricellulin,24Ikenouchi J. Furuse M. Furuse K. Sasaki H. Tsukita S. Tsukita S. Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells.J Cell Biol. 2005; 171: 939-945Crossref PubMed Scopus (511) Google Scholar and marvelD325Steed E. Rodrigues N.T. Balda M.S. Matter K. Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family.BMC Cell Biol. 2009; 10: 95Crossref PubMed Scopus (0) Google Scholar,26Raleigh D.R. Marchiando A.M. Zhang Y. Shen L. Sasaki H. Wang Y. Long M. Turner J.R. Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions.Mol Biol Cell. 2010; 21: 1200-1213Crossref PubMed Scopus (182) Google Scholar; claudins27Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.J Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1514) Google Scholar, 28Furuse M. Furuse K. Sasaki H. Tsukita S. Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.J Cell Biol. 2001; 153: 263-272Crossref PubMed Scopus (575) Google Scholar, 29Van Itallie C. Rahner C. Anderson J.M. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability.J Clin Invest. 2001; 107: 1319-1327Crossref PubMed Google Scholar, 30Amasheh S. Meiri N. Gitter A.H. Schoneberg T. Mankertz J. Schulzke J.D. Fromm M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.J Cell Sci. 2002; 115: 4969-4976Crossref PubMed Scopus (542) Google Scholar, 31Mineta K. Yamamoto Y. Yamazaki Y. Tanaka H. Tada Y. Saito K. Tamura A. Igarashi M. Endo T. Takeuchi K. Tsukita S. Predicted expansion of the claudin multigene family.FEBS Lett. 2011; 585: 606-612Crossref PubMed Scopus (308) Google Scholar; and others.32Higashi T. Tokuda S. Kitajiri S. Masuda S. Nakamura H. Oda Y. Furuse M. Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis.J Cell Sci. 2013; 126: 966-977Crossref PubMed Scopus (0) Google Scholar,33Kolosov D. Kelly S.P. Tricellular tight junction-associated angulins in the gill epithelium of rainbow trout.Am J Physiol Regul Integr Comp Physiol. 2018; 315: R312-R322Crossref PubMed Scopus (5) Google Scholar Beyond these compositional proteins, the tight junction is functionally and structurally linked to the subcortical terminal web of actin microfilaments and the perijunctional actomyosin ring.34Bentzel C.J. Hainau B. Edelman A. Anagnostopoulos T. Benedetti E.L. Effect of plant cytokinins on microfilaments and tight junction permeability.Nature. 1976; 264: 666-668Crossref PubMed Scopus (0) Google Scholar, 35Hull B.E. Staehelin L.A. The terminal web. A reevaluation of its structure and function.J Cell Biol. 1979; 81: 67-82Crossref PubMed Google Scholar, 36Madara J.L. Intestinal absorptive cell tight junctions are linked to cytoskeleton.Am J Physiol. 1987; 253: C171-C175Crossref PubMed Google Scholar, 37Yu D. Marchiando A.M. Weber C.R. Raleigh D.R. Wang Y. Shen L. Turner J.R. MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function.Proc Natl Acad Sci U S A. 2010; 107: 8237-8241Crossref PubMed Scopus (138) Google Scholar, 38Graham W.V. He W. Marchiando A.M. Zha J. Singh G. Li H.S. Biswas A. Ong M. Jiang Z.H. Choi W. Zuccola H. Wang Y. Griffith J. Wu J. Rosenberg H.J. Wang Y. Snapper S.B. Ostrov D. Meredith S.C. Miller L.W. Turner J.R. Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.Nat Med. 2019; 25: 690-700Crossref PubMed Scopus (9) Google Scholar, 39Turner J.R. Rill B.K. Carlson S.L. Carnes D. Kerner R. Mrsny R.J. Madara J.L. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.Am J Physiol. 1997; 273: C1378-C1385Crossref PubMed Google Scholar Solutes and water cross the tight junction by two distinct pathways that can be distinguished on the basis of their size-selectivity, charge-selectivity, and capacity (Figure 1A).40Turner J.R. Intestinal mucosal barrier function in health and disease.Nat Rev Immunol. 2009; 9: 799-809Crossref PubMed Scopus (1528) Google Scholar,41Anderson J.M. Van Itallie C.M. Physiology and function of the tight junction.Cold Spring Harb Perspect Biol. 2009; 1: a002584Crossref PubMed Scopus (496) Google Scholar The pore pathway is a high-conductance route that is charge-selective and extremely size-selective, with an upper limit of 6- to 8-Å diameter. In contrast, the less well-defined upper size limit of the lower conductance, charge nonselective leak pathway has been estimated to be ∼100-Å diameter.42Buschmann M.M. Shen L. Rajapakse H. Raleigh D.R. Wang Y. Wang Y. Lingaraju A. Zha J. Abbott E. McAuley E.M. Breskin L.A. Wu L. Anderson K. Turner J.R. Weber C.R. Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux.Mol Biol Cell. 2013; 24: 3056-3068Crossref PubMed Scopus (74) Google Scholar This model is consistent with in vivo studies of mucosal permeability along the villus-crypt axis, which identified distinct paracellular flux routes that could be distinguished on the basis of size-selectivity; that work concluded that 12-Å diameter pores were present in the villus but that larger, 100- to 120-Å diameter pores populated the crypts.43Fihn B.M. Sjöqvist A. Jodal M. Permeability of the rat small intestinal epithelium along the villus-crypt axis: effects of glucose transport.Gastroenterology. 2000; 119: 1029-1036Abstract Full Text Full Text PDF PubMed Google Scholar The pore pathway was identified in parallel by 2 sets of experiments. Van Itallie et al44Van Itallie C.M. Holmes J. Bridges A. Gookin J.L. Coccaro M.R. Proctor W. Colegio O.R. Anderson J.M. The density of small tight junction pores varies among cell types and is increased by expression of claudin-2.J Cell Sci. 2008; 121: 298-305Crossref PubMed Scopus (242) Google Scholar analyzed flux of polyethylene glycols across pig ileum and monolayers of Caco-2 intestinal epithelial cells and 2 distinct clones of Madin-Darby canine kidney (MDCK) cells; all demonstrated a size-restrictive pore with a sharp size cutoff at ∼8-Å diameter. When the 2 MDCK lines, which had markedly different transepithelial electrical resistances (TERs), were compared, increased flux of 7-Å diameter polyethylene glycol correlated with increased ion conductance (ie, reduced TER). Analysis of tight junction protein expression showed that MDCK II, the MDCK line with greater polyethylene glycol flux, expressed claudin-2 but that the less permeable MDCK C7 line did not. Expression of claudin-2 in MDCK C7 cells reduced TER and enhanced paracellular flux of 7-Å diameter polyethylene glycol, but not larger polyethylene glycols, consistent with previous work showing that forced claudin-2 expression in MDCK C7 monolayers increased Na+, but not 4-kDa dextran, flux.30Amasheh S. Meiri N. Gitter A.H. Schoneberg T. Mankertz J. Schulzke J.D. Fromm M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.J Cell Sci. 2002; 115: 4969-4976Crossref PubMed Scopus (542) Google Scholar Van Itallie et al44Van Itallie C.M. Holmes J. Bridges A. Gookin J.L. Coccaro M.R. Proctor W. Colegio O.R. Anderson J.M. The density of small tight junction pores varies among cell types and is increased by expression of claudin-2.J Cell Sci. 2008; 121: 298-305Crossref PubMed Scopus (242) Google Scholar therefore concluded that claudin-2 expression increased the number of small tight junction pores. Concurrently, Weber et al45Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity.J Biol Chem. 2010; 285: 12037-12046Crossref PubMed Scopus (157) Google Scholar treated T84 intestinal epithelial cell monolayers with interleukin (IL)-13 and found that this increased paracellular cation permeability but did not affect flux of 4-kDa dextran. Detailed study showed that IL-13 selectively induced claudin-2 expression and that siRNA-mediated blockade of claudin-2 up-regulation prevented IL-13-induced conductance increases.45Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity.J Biol Chem. 2010; 285: 12037-12046Crossref PubMed Scopus (157) Google Scholar This confirmed observations in MDCK C7 cells, as described previously, and further demonstrated that IL-13 selectively enhances paracellular permeability by the high conductance, charge, and size-selective pore pathway.45Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity.J Biol Chem. 2010; 285: 12037-12046Crossref PubMed Scopus (157) Google Scholar Further understanding of claudin-2-mediated pore pathway conductance was provided by a series of mutagenesis studies that identified specific residues that define the claudin-2 pore.46Angelow S. Yu A.S. Cysteine mutagenesis to study the structure of claudin-2 paracellular pores.Ann N Y Acad Sci. 2009; 1165: 143-147Crossref PubMed Scopus (15) Google Scholar, 47Li J. Angelow S. Linge A. Zhuo M. Yu A.S. Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines.Am J Physiol Cell Physiol. 2013; 305: C190-C196Crossref PubMed Scopus (24) Google Scholar, 48Li J. Zhuo M. Pei L. Yu A.S. Conserved aromatic residue confers cation selectivity in claudin-2 and claudin-10b.J Biol Chem. 2013; 288: 22790-22797Crossref PubMed Scopus (17) Google Scholar, 49Yu A.S. Cheng M.H. Angelow S. Gunzel D. Kanzawa S.A. Schneeberger E.E. Fromm M. Coalson R.D. Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.J Gen Physiol. 2009; 133: 111-127Crossref PubMed Scopus (199) Google Scholar These were all within the first extracellular loop of claudin-2 (Figure 1B) and could be mapped to narrower and wider portions of the channel. Subsequent patch clamp analyses demonstrated that claudin-2 channels are actively gated and have single channel conductances of ∼9 pA.50Weber C.R. Liang G.H. Wang Y. Das S. Shen L. Yu A.S. Nelson D.J. Turner J.R. Claudin-2-dependent paracellular channels are dynamically gated.Elife. 2015; 4e09906Crossref PubMed Scopus (50) Google Scholar Together, these data indicate that, although claudin-2 channels are located between cells and are oriented parallel to plasma membranes, they have significant similarities to traditional transmembrane ion channels. The data described above focus on claudin-2, a member of the claudin protein family. Alternative splicing of the 27 claudin genes allows expression of an even greater number of proteins. Individual claudin proteins are differentially expressed within specific tissues and cell types; the patterns of expression are also modified during development and in response to extracellular stimuli, including immune cells and their products. In general, claudin proteins have been subdivided into pore-forming and barrier-forming classes. Claudin-2 is a pore-forming claudin, as are claudins 10a, 10b, 15, 16, and 17; these form channels that are either cation- or anion-selective. Conversely, claudin- expression in MDCK II monolayers reduces paracellular flux of Na+ and 7-Å diameter polyethylene glycol. More detailed discussion of claudin proteins, their functions, and interactions are available.51Gunzel D. Fromm M. Claudins and other tight junction proteins.Compr Physiol. 2012; 2: 1819-1852Crossref PubMed Scopus (0) Google Scholar, 52Gunzel D. Yu A.S. Claudins and the modulation of tight junction permeability.Physiol Rev. 2013; 93: 525-569Crossref PubMed Scopus (527) Google Scholar, 53Tsukita S. Tanaka H. Tamura A. The claudins: from tight junctions to biological systems.Trends Biochem Sci. 2019; 44: 141-152Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 54Suzuki H. Tani K. Fujiyoshi Y. Crystal structures of claudins: insights into their intermolecular interactions.Ann N Y Acad Sci. 2017; 1397: 25-34Crossref PubMed Scopus (13) Google Scholar, 55Rosenthal R. Gunzel D. Theune D. Czichos C. Schulzke J.D. Fromm M. Water channels and barriers formed by claudins.Ann N Y Acad Sci. 2017; 1397: 100-109Crossref PubMed Scopus (27) Google Scholar, 56Garcia-Hernandez V. Quiros M. Nusrat A. Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation.Ann N Y Acad Sci. 2017; 1397: 66-79Crossref PubMed Scopus (66) Google Scholar, 57France M.M. Turner J.R. The mucosal barrier at a glance.J Cell Sci. 2017; 130: 307-314Crossref PubMed Scopus (60) Google Scholar, 58Liu F. Koval M. Ranganathan S. Fanayan S. Hancock W.S. Lundberg E.K. Beavis R.C. Lane L. Duek P. McQuade L. Kelleher N.L. Baker M.S. Systems proteomics view of the endogenous human claudin protein family.J Proteome Res. 2016; 15: 339-359Crossref PubMed Scopus (10) Google Scholar, 59Gunzel D. Claudins: vital partners in transcellular and paracellular transport coupling.Pflugers Arch. 2017; 469: 35-44Crossref PubMed Scopus (0) Google Scholar The tremendous efficacy of transmembrane ion channel inhibitors in many disorders suggests that development of specific means to modulate pore pathway tight junction channels may also be therapeutic. One approach to claudin-2 channel inhibition involves inhibition of casein kinase 2. This results in dephosphorylation of serine 408 within the C-terminal occludin tail and assembly of a tripartite complex composed of occludin, ZO-1, and claudin-2.60Raleigh D.R. Boe D.M. Yu D. Weber C.R. Marchiando A.M. Bradford E.M. Wang Y. Wu L. Schneeberger E.E. Shen L. Turner J.R. Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function.J Cell Biol. 2011; 193: 565-582Crossref PubMed Scopus (151) Google Scholar Incorporation into this complex de-anchors claudin-2 at the tight junction and disrupts channel function. For example, casein kinase-2 inhibition acutely reversed IL-13-induced increases in paracellular permeability of T84 monolayers.60Raleigh D.R. Boe D.M. Yu D. Weber C.R. Marchiando A.M. Bradford E.M. Wang Y. Wu L. Schneeberger E.E. Shen L. Turner J.R. Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function.J Cell Biol. 2011; 193: 565-582Crossref PubMed Scopus (151) Google Scholar Although translation to in vivo applications has not been reported and will likely require more specificity than casein kinase-2 inhibition provides,61Koch S. Capaldo C.T. Hilgarth R.S. Fournier B. Parkos C.A. Nusrat A. Protein kinase CK2 is a critical regulator of epithelial homeostasis in chronic intestinal inflammation.Mucosal Immunol. 2013; 6: 136-145Crossref PubMed Scopus (30) Google Scholar these data indicate that molecular targeting of protein interactions has the potential to modulate claudin channels and pore pathway permeability. In contrast to the pore pathway, the specific sites of leak pathway flux have not been defined. One possibility is that transient breaks within tight junction strands allow macromolecules (>8-Å diameter) to pass.62Sasaki H. Matsui C. Furuse K. Mimori-Kiyosue Y. Furuse M. Tsukita S. Dynamic behavior of paired claudin strands within apposing plasma membranes.Proc Natl Acad Sci U S A. 2003; 100: 3971-3976Crossref PubMed Scopus (170) Google Scholar, 63Van Itallie C.M. Fanning A.S. Bridges A. Anderson J.M. ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton.Mol Biol Cell. 2009; 20: 3930-3940Crossref PubMed Scopus (243) Google Scholar, 64Van Itallie C.M. Tietgens A.J. Anderson J.M. Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1.Mol Biol Cell. 2017; 28: 524-534Crossref PubMed Scopus (34) Google Scholar This hypothesis proposes that, as strands reform, macromolecules are trapped in interstrand spaces until a break in the next strand allows them to continue to move across the tight junction. As discussed later, tricellular tight junctions, where 3 cells meet, have also been proposed as specialized sites of paracellular, macromolecular flux.65Krug S.M. Amasheh S. Richter J.F. Milatz S. Gunzel D. Westphal J.K. Huber O. Schulzke J.D. Fromm M. Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability.Mol Biol Cell. 2009; 20: 3713-3724Crossref PubMed Scopus (202) Google Scholar Despite lack of structural understanding, components of the signal transduction machinery that regulates leak pathway permeability have been studied extensively.66Buckley A. Turner J.R. Cell biology of tight junction barrier regulation and mucosal disease.Cold Spring Harb Perspect Biol. 2018; 10Crossref PubMed Scopus (52) Google Scholar The most well-characterized of these is myosin light chain kinase (MLCK), which regulates paracellular permeability during physiological, Na+-nutrient cotransport.39Turner J.R. Rill B.K. Carlson S.L. Carnes D. Kerner R. Mrsny R.J. Madara J.L. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.Am J Physiol. 1997; 273: C1378-C1385Crossref PubMed Google Scholar,67Atisook K. Carlson S. Madara J.L. Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia.Am J Physiol. 1990; 258: C77-C85Crossref PubMed Google Scholar Expression of constitutively-active MLCK is sufficient to increase leak pathway permeability in vitro68Shen L. Black E.D. Witkowski E.D. Lencer W.I. Guerriero V. Schneeberger E.E. Turner J.R. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure.J Cell Sci. 2006; 119: 2095-2106Crossref PubMed Scopus (287) Google Scholar and in vivo.69Su L. Shen L. Clayburgh D.R. Nalle S.C. Sullivan E.A. Meddings J.B. Abraham C. Turner J.R. Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis.Gastroenterology. 2009; 136: 551-563Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar Based on the hypothesis that tight junction signaling mechanisms triggered by physiological stimuli mediate transduction by pathophysiological stimuli, Zolotarevsky et al70Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar asked if MLCK was involved in tight junction barrier loss induced by tumor necrosis factor (TNF). They showed that a highly specific MLCK inhibitor, PIK, was able to reverse both increased myosin II regulatory light chain (MLC) phosphorylation and reduced TER induced by TNF in vitro.70Zolotarevsky Y. Hecht G