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At the blood–brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5wild type in MDCK-II cells—but not that of mutants R145A, Y148A, Y158A or E159Q—increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5Y₁₄₈A, Y₁₅₈A or E₁₅₉Q enhanced permeability of FITC-dextran₁₀ kDa, which was unchanged in cells expressing claudin-5wild type or claudin-5R₁₄₅A. In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

Sealing of the paracellular cleft by tight junctions is of central importance for epithelia and endothelia to function as efficient barriers between the extracellular space and the inner milieu. Occludin and claudins represent the major tight junction components involved in establishing this barrier function. A special situation emerges at sites where three cells join together. Tricellulin, a recently identified tetraspan protein concentrated at tricellular contacts, was reported to organize tricellular as well as bicellular tight junctions. Here we show that in MDCK cells, the tricellulin C-terminus is important for the basolateral translocation of tricellulin, whereas the N-terminal domain appears to be involved in directing tricellulin to tricellular contacts. In this respect, identification of homomeric tricellulin-tricellulin and of heteromeric tricellulin-occludin complexes extends a previously published model and suggests that tricellulin and occludin are transported together to the edges of elongating bicellular junctions and get separated when tricellular contacts are formed.

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis -Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans -interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans -interactions were analyzed in cocultures of differently monotransfected cells. trans -Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.

Background Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. Results Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca 2+ -switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. Conclusions These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400–408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.

Vedolizumab has been shown to induce clinical remission in patients with active ulcerative colitis. Treatment with anti-integrin vedolizumab leads to clinical remission in 16.9% and clinical response in 47.1% of cases after 6 weeks. However, in clinical practice, no decision to discontinue or continue vedolizumab therapy is made until 14 weeks at the earliest. The aim of this study is to develop an algorithm for optimizing vedolizumab administration in patients with moderate-to-severe ulcerative colitis by calculating the probability of clinical response at week 14, on the basis of the data from week 6. This is a prospective, single-arm, multicentric, noninterventional, observational study with no interim analyses and a sample size of 35 evaluable patients. The enrollment started in August 2018 and was still open at the date of submission. The study is expected to complete in September 2020. The early identification of patients who are responding to an integrin antibody is therapeutically beneficial. At the same time, patients who are not responding can be identified earlier. The development of a therapeutic algorithm for identifying patients as responders or nonresponders can thus help prescribing physicians avoid ineffective treatments and stop these very early.

Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca.sup.2+-switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400-408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.