Regulation Of Epithelial Tight Junctions Research Articles

Cochlear Explantation Prevents Hair Cell Degeneration in Transmembrane Serine Protease 3 (Tmprss3) Deficient Mice

Background/Objective: Transmembrane serine protease 3 (TMPRSS3) is the most common causative hearing loss gene in adults undergoing cochlear implantation and a significant cause of non-syndromic hearing loss. However, the function of TMPRSS3 within the inner ear is unknown. In murine models, Tmprss3Y260X/Y260X mutants (Tmprss3-/-) have normal hair cell development followed by rapid hair cell degeneration from postnatal day 12 (P12) to P14. The endocochlear potential in vivo rises from P7 to a peak at P12, when mice begin to hear, which temporally corresponds to the HC degeneration in Tmprss3-/- mice. We tested if hair cell death occurs after removal of the endocochlear potential through cochlear explantation.Methods: P7 organ of Corti explants from control (Tmprss3+/-) and Tmprss3-/- mice were cultured in 5 mM potassium solution for 7 days in vitro (equivalent to P14). Whole mounts of cultured (P7+7DIV) and in vivo P14 cochlea were immunostained for MYO7A (hair cell marker) and DAPI followed by quantification of inner and outer hair cell (IHC/OHC) counts per 20 μm. Statistical analysis included two tailed t-test with p-value of (<0.01, n=6).Results: As expected, there is complete loss of hair cells at P14 in vivo in Tmprss3-/- mice. Compared to P14 in vivo, Tmprss3-/- OC explants displayed significantly improved IHC and OHC survival (P<0.001). IHC and OCH survival was similar between control and Tmprss3-/- OHC explants in vitro (P=0.99, n=6)Conclusion: These results suggest that degeneration of Tmprss3-/- hair cells is due to factors related to the endocochlear potential and implicate TMPRSS3 function in regulation of epithelial tight junctions. Future directions include confirming that hair cell death is potassium-mediated by crossing Tmprss3-/- mice with Pou3f4del-J mice, which have a decreased endocochlear potential without hair cell degeneration.

Emerging neuropeptide targets in inflammation: NPY and VIP

The enteric nervous system (ENS), referred to as the "second brain," comprises a vast number of neurons that form an elegant network throughout the gastrointestinal tract. Neuropeptides produced by the ENS play a crucial role in the regulation of inflammatory processes via cross talk with the enteric immune system. In addition, neuropeptides have paracrine effects on epithelial secretion, thus regulating epithelial barrier functions and thereby susceptibility to inflammation. Ultimately the inflammatory response damages the enteric neurons themselves, resulting in deregulations in circuitry and gut motility. In this review, we have emphasized the concept of neurogenic inflammation and the interaction between the enteric immune system and enteric nervous system, focusing on neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). The alterations in the expression of NPY and VIP in inflammation and their significant roles in immunomodulation are discussed. We highlight the mechanism of action of these neuropeptides on immune cells, focusing on the key receptors as well as the intracellular signaling pathways that are activated to regulate the release of cytokines. In addition, we also examine the direct and indirect mechanisms of neuropeptide regulation of epithelial tight junctions and permeability, which are a crucial determinant of susceptibility to inflammation. Finally, we also discuss the potential of emerging neuropeptide-based therapies that utilize peptide agonists, antagonists, siRNA, oligonucleotides, and lentiviral vectors.

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

Occludin phosphorylation in regulation of epithelial tight junctions.

Occludin is the first transmembrane protein of the tight junction to be discovered. While numerous studies emphasized the important role of occludin in assembly and maintenance of tight junctions, occludin knockout studies indicated that it was not required for tight junction assembly in different epithelia. However, a detailed characterization of the occludin knockout mouse concluded that the occludin gene is indispensable, and plays a complex role in regulation of epithelial tight junctions in different organs. This article describes the role of occludin phosphorylation in the regulation of its assembly into the tight junctions. Occludin is highly phosphorylated on Ser and Thr residues, while Tyr-phosphorylation is kept at minimum in the intact epithelium. During the disruption of tight junctions by various factors, occludin undergoes dephosphorylation on Ser/Thr residues and elevated phosphorylation on Tyr residues. The phosphorylation of occludin on Tyr, Ser, and Thr residues appears to be regulated by the balance between protein kinases such as c-Src, PKCzeta, and PKClambda/iota, and protein phosphatases such as PP2A, PP1, and PTP1B. The precise mechanism of regulation of the tight junction by occludin phosphorylation is unclear at this time. However, an in vitro study indicated that Tyr-phosphorylation of occludin C-terminal domain attenuates its interaction with ZO-1. Therefore, phosphorylation of specific Ser/Thr/Tyr residues in occludin may regulate its interactions with ZO-1 and possibly other tight junction proteins. It is likely that occludin plays a regulatory role in tight junctions rather than a role in the de novo assembly of tight junctions.

PKCη regulates occludin phosphorylation and epithelial tight junction integrity

PKC eta is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC eta phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC eta by specific pseudo substrate inhibitor or knockdown of PKC eta by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC eta(K394R) disrupts the TJ and barrier function, whereas wild-type PKC eta and constitutively active PKC eta(A161E) enhance the TJ integrity. Inhibition and knockdown of PKC eta or expression of PKC eta(K394R) induce dephosphorylation of occludin on threonine residues, whereas active PKC eta elevates occludin phosphorylation. PKC eta directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC eta inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC eta.

826 T403/404d Mutation of Occludin Attenuates PKCη-Mediated Regulation of Epithelial Tight Junctions (TJS)

826 T403/404d Mutation of Occludin Attenuates PKCη-Mediated Regulation of Epithelial Tight Junctions (TJS)

Mammary gland homeostasis employs serotonergic regulation of epithelial tight junctions

Homeostatic control of volume within the alveolar spaces of the mammary gland has been proposed to involve a feedback system mediated by serotonin signaling. In this article, we describe some of the mechanisms underlying this feedback based on studies of a human normal mammary epithelial cell line (MCF10A) and mouse mammary epithelium. Mammary serotonin was elevated during lactation and after injection of 5-hydroxytryptophan (5-HTP). The genes encoding the serotonin reuptake transporter (SERT) and the type 7 serotonin receptor (5-HT(7)) were expressed in human and mouse mammary epithelial cells, and serotonin caused a concentration-dependent increase of cAMP in MCF10A cells. Mouse and human mammary epithelial cells formed polarized membranes, in which tight junction activity was monitored. Treatment of mammary epithelial membranes with serotonin receptor antagonists increased their transepithelial electrical resistance (TEER). Antagonist and agonist effects on TEER were mediated by receptors on the basolateral face of the membranes. Our results suggest a process in which serotonin accumulates in the interstitial fluid surrounding the mammary secretory epithelium and is detected by 5-HT(7) receptors, whereupon milk secretion is inhibited. One mechanism responsible for this process is serotonin-mediated opening of tight junctions, which dissipates the transepithelial gradients necessary for milk secretion.

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. The effects of LIGHT and interferon-gamma on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers. LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-gamma, which induced lymphotoxin beta receptor (LT beta R) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LT beta R knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis. T cell-derived LIGHT activates intestinal epithelial LT beta R to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.

A unique role for the nonmuscle myosin IIA in regulation of epithelial apical junctions

The integrity of epithelial monolayers depends on the formation of tight junctions (TJ) and adherens junctions (AJ). TJ and AJ associate with underlying actomyosin bundles that are critical for their biogenesis and functions. We investigated roles of three nonmuscle myosin II (NMM II) isoforms, A, B, and C in the regulation of epithelial TJ and AJ. NMM IIA, NMM IIB and NMM IIC were all expressed in SK-CO15 and Caco-2 colonic epithelial cell lines, and all isoforms colocalized with perijunctional F-actin. Using isoform-specific siRNAs, we selectively down-regulated expression of the NMM IIA, NMM IIB, or NMM IIC in SK-CO15 cells. Down-regulation of the NMM IIA caused the following morphological and functional changes: transformation from an orthogonal epithelial to a protrusive fibroblast-like cell shape; a three fold decrease in transepithelial electrical resistance; attenuation of TJ and AJ disassembly and cell contraction induced by extracellular calcium depletion; inhibition of TJ and AJ reassembly after calcium repletion. None of the above changes were observed after down-regulation of either the NMM IIB or NMM IIC. These findings suggest that the NMM IIA plays a unique role in regulation of the dynamics and barrier properties of epithelial apical junctions. Supported by the Crohn’s and Colitis Foundation of America (AII) and NIH (AN and CAP).

Review article: In vitro models in inflammatory bowel disease research--a critical review.

Research efforts in inflammatory bowel disease (IBD) have been directed towards the epithelium as it has become clear that epithelial cells play a critical role in inflammatory response. Most research involving IBD employs in vitro techniques. In vitro epithelial cell studies have played and are continuing to play a major role in providing specific information relevant to IBD. Thus, such studies have provided irrefutable evidence that epithelial responses can be induced by microbes/microbial products and by immune activation. Culture experiments have provided insights into the effects of individual cytokines and other inflammatory mediators on epithelial pathophysiology, injury and repair, apoptosis, necrosis, and other processes that may be involved in IBD. Activated epithelial cells can participate in and even orchestrate immune responses, by stimulating T cells (and possibly others) and by producing cytokines that recruit specific inflammatory cells. Physiological regulation of epithelial tight junctions has been demonstrated by in vitro studies; the implication of this information for treating IBD is just beginning to be explored. It is becoming increasingly clear that epithelial processing and presentation of antigens is critical to the outcome of the immune response.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.