Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

Introduction: The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Methods: Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Results: Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. Conclusion: These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn's disease.

OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Tight junction channel regulation by interclaudin interference.

Tight junctions form selectively permeable seals across the paracellular space. Both barrier function and selective permeability have been attributed to members of the claudin protein family, which can be categorized as pore-forming or barrier-forming. Here, we show that claudin-4, a prototypic barrier-forming claudin, reduces paracellular permeability by a previously unrecognized mechanism. Claudin-4 knockout or overexpression has minimal effects on tight junction permeability in the absence of pore-forming claudins. However, claudin-4 selectively inhibits flux across cation channels formed by claudins 2 or 15. Claudin-4-induced loss of claudin channel function is accompanied by reduced anchoring and subsequent endocytosis of pore-forming claudins. Analyses in nonepithelial cells show that claudin-4, which is incapable of independent polymerization, disrupts polymeric strands and higher order meshworks formed by claudins 2, 7, 15, and 19. This process of interclaudin interference, in which one claudin disrupts higher order structures and channels formed by a different claudin, represents a previously unrecognized mechanism of barrier regulation.

Electrophysiologic Analysis of Tight Junction Size and Charge Selectivity.

Tight junctions form selectively permeable barriers that limit paracellular flux across epithelial-lined surfaces. Rather than being absolute barriers, tight junctions in many tissues allow ions, water, and other small molecules to cross on the basis of size and charge selectivity via the high-capacity pore pathway. Most probes currently used to assess tight junction permeability exceed the maximum size capacity of the pore pathway. As a result, available analytical tools have generally been limited to measurement of transepithelial electrical resistances. These provide no information regarding size selectivity and, therefore, cannot be used to distinguish between the pore pathway and the leak pathway, a low-capacity route that accommodates larger macromolecules. This article describes use of dilution potential and bi-ionic potential measurements for analysis of tight junction size and charge selectivity within monolayers of cultured epithelial cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Culture of MDCK monolayers on semipermeable supports and induction of claudin-2 expression Basic Protocol 2: Configuring voltage/current clamp and other equipment Basic Protocol 3: Measuring dilution and bi-ionic potentials Basic Protocol 4: Calculating ion permeabilities and pore diameter Support Protocol: Preparation of agar bridges and electrophysiology rig setup.

Inactivation of paracellular cation-selective claudin-2 channels attenuates immune-mediated experimental colitis in mice.

The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.

Inflammation-induced Occludin Downregulation Limits Epithelial Apoptosis by Suppressing Caspase-3 Expression.

BACKGROUND & AIMS: Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. METHODS: Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. RESULTS: Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. CONCLUSIONS: The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.

Epithelial Organization: The Gut and Beyond.

Epithelial cells are essential to the survival and homeostasis of complex organisms. These cells cover the surfaces of all mucosae, the skin, and other compartmentalized structures essential to physiological function. In addition to maintenance of barriers that separate internal and external compartments, epithelia display a variety of organ-specific differentiated functions. Function is reflected in overall epithelial structure and organization, shape of individual cells, and proteins expressed by these cells. More than one epithelial cell type is often present within a single organ and, in many cases, individual cells differentiate to change their functional behaviors as part of normal development or in response to extracellular stimuli. This article discusses the diversity of epithelial structure and function in general terms and explores representative tissues in greater depth to highlight organ specific functions and their contributions to physiology and disease. © 2017 American Physiological Society. Compr Physiol 7:1497-1518, 2017.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity.

Ca2+ ions are critical to cadherin ectodomain rigidity, which is required for the activation of adhesive functions. Therefore, changes in Ca2+ concentration, both in vivo and in vitro, can affect cadherin conformation and function. We employed single-molecule tracking to measure the diffusion of cadherin ectodomains tethered to supported lipid bilayers at varying Ca2+ concentrations. At a relatively high Ca2+ concentration of 2 mM, cadherin molecules exhibited a fast diffusion coefficient that was identical to that of individual lipid molecules in the bilayer (Dfast ≈ 3 µm2/s). At lower Ca2+ concentrations, where cadherin molecules were less rigid, the ensemble-average cadherin diffusion coefficient was systematically smaller. Individual cadherin trajectories were temporally heterogeneous, exhibiting alternating periods of fast and slow diffusion; the periods of slow diffusion (Dslow ≈ 0.1 µm2/s) were more prevalent at lower Ca2+ concentration. These observations suggested that more flexible cadherin ectodomains at lower Ca2+ concentration alternated between upright and lying-down conformations, where the latter interacted with more lipid molecules and experienced greater viscous drag.

Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions.

The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions.

Allosteric Regulation of E-Cadherin Adhesion.

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

α-catenin cytomechanics--role in cadherin-dependent adhesion and mechanotransduction.

The findings presented here demonstrate the role of α-catenin in cadherin-based adhesion and mechanotransduction in different mechanical contexts. Bead-twisting measurements in conjunction with imaging, and the use of different cell lines and α-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin- and α-catenin-dependent manner. The modest effect of α-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that force-activated adhesion strengthening is due to enhanced cadherin-cytoskeletal interactions rather than to α-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although α-catenin alters traction force generation, it is not the sole regulator of cell contractility on compliant cadherin-coated substrata.

N-glycosylation alters cadherin-mediated intercellular binding kinetics.

We present direct evidence that the N-glycosylation state of neural cadherin impacts the intrinsic kinetics of cadherin-mediated intercellular binding. Micropipette manipulation measurements quantified the effect of N-glycosylation mutations on intercellular binding dynamics. The wild-type protein exhibits a two-stage binding process in which a fast, initial binding step is followed by a short lag and second, slower transition to the final binding stage. Mutations that ablate N-glycosylation at three sites on the extracellular domains 2 and 3 of neural cadherin alter this kinetic fingerprint. Glycosylation does not affect the affinities between the adhesive N-terminal domains, but instead modulates additional cadherin interactions, which govern the dynamics of intercellular binding. These results, together with previous findings that these hypo-glycosylation mutations increase the prevalence of cis dimers on cell membranes, suggest a binding mechanism in which initial adhesion is followed by additional cadherin interactions, which enhance binding but are modulated by N-glycosylation. Given that oncogene expression drives specific changes in N-glycosylation, these results provide insight into possible mechanisms altering cadherin function during tumor progression.