Occludin Phosphorylation Research Articles

A phosphorylation hotspot within the occludin C‐terminal domain

Tight junctions (TJs) form paracellular barriers defining the permeability characteristics of epithelial and endothelial cell layers in our body. Tetraspanin integral membrane proteins, including occludin, tricellulin, MarvelD3, and a set of claudins, form a network of anastomosing strands bringing the membranes of neighboring cells into close contact. Occludin is assumed to play an important role in the regulation of TJ formation, structure, and function, and is tightly regulated by phosphorylation. We here summarize the role of occludin phosphorylation on assembly/disassembly and function of TJs and specifically focus on a cluster of 11 amino acids in the C-terminal cytoplasmic domain of occludin (Tyr398-Ser408), including highly conserved phosphorylation sites for c-Src, PKCs, and CK2. Phosphorylation by these kinases affects occludin localization, dynamics, and interaction with other TJ proteins. Interestingly, this phosphorylation hotspot is localized in an unstructured region close to the ZO-1 binding site, and a cysteine residue which is involved in intermolecular disulfide-bond formation thus contributing to occludin dimerization. We discuss potential consequences and open questions in respect to the functional role of this phosphorylation hotspot.

Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier

ObjectiveRecently, the authors demonstrated altered gene expression in the jejunal mucosa of diarrhoea-predominant irritable bowel syndrome patients (IBS-D); specifically, the authors showed that genes related to mast cells and the...

Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.

Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.

Abstract 383: Characterization of Inflammatory Cytokine Effects on the Blood-Brain Barrier Using an in Vitro Human Brain Microvascular Endothelial Cell Model

Introduction: The blood-brain barrier (BBB) represents a restrictive interface between the circulation and CNS. Within the multicellular neurovascular unit (NVU), the microvascular endothelium resides at the forefront of this dynamic barrier in direct contact with blood. Following cerebrovascular injury (e.g. ischemic stroke, trauma, infection), inflammatory cytokines are released by NVU cells (neurons, astrocytes, pericytes, endothelial cells) and circulating immune cells, often leading to elevated microvessel permeability and pathology. Much has yet to be uncovered however about how cytokines specifically impact endothelial BBB phenotype at the molecular level. In the present study, we investigated how TNF Alpha and IL-6 affect adherens/tight junction assembly using a human brain microvascular endothelial cell (HBMVEC) model in vitro . Methods: Cultured HBMVECs were exposed to either TNF Alpha or IL-6 over a range of doses (1-100 ng/ml) and times (6 and 18 hours). Cytokine levels were monitored by both standard and multiplex ELISA. Endothelial barrier phenotype was routinely monitored with respect to paracellular permeability (transwell monolayer assay) and junctional protein expression (qRT-PCR & Western blotting). Results: (i) Both TNF Alpha and IL6 treatments increased HBMVEC permeability in a dose- and time-dependent manner. Treatments also increased cell proliferation whilst having minimal impact on cell viability (as monitored by FACS, MTS, LDH and xCelligence assays); (ii) In parallel with permeability increases, cytokine treatments dose-/time-dependently decreased mRNA and protein levels for tight junction-associated occludin, zonnula occludens-1 and claudin-5, as well as for adherens junction-associated VE-cadherin; (iii) TNF Alpha treatment was also seen to dose-/time-dependently induce IL-6 expression and release from HBMVECs. Conclusions: Inflammatory cytokines (TNF Alpha and IL-6) reduce microvascular endothelial BBB integrity in-part through a mechanism involving reduced synthesis of junctional proteins. Cytokine-mediated post-translational modification of junctional proteins (e.g. occludin phosphorylation and ubiquitination) is also suspected in these events. Moreover, the TNF Alpha-stimulated production/release of IL-6 may reflect a putative mechanism through which the former cytokine elicits BBB compromise.

Modulation of Tight Junction Structure and Function by Kinases and Phosphatases Targeting Occludin

Tight junctions (TJs) typically represent the most apical contacts in epithelial and endothelial cell layers where they play an essential role in the separation of extracellular or luminal spaces from underlying tissues in the body. Depending on the protein composition, TJs define the barrier characteristics and in addition maintain cell polarity. Two major families of integral membrane proteins form the typical TJ strand network, the tight junction-associated MARVEL protein (TAMP) family members occludin, tricellulin, and MarvelD3 as well as a specific set of claudins. Occludin was the first identified member of these tetraspanins and is now widely accepted as a regulator of TJ assembly and function. Therefore, occludin itself has to be tightly regulated. Phosphorylation of occludin appears to be of central importance in this context. Here we want to summarize current knowledge on the kinases and phosphatases directly modifying occludin, and their role in the regulation of TJ structure, function, and dynamics.

Liver X Receptor Activation Enhances Blood–Brain Barrier Integrity in the Ischemic Brain and Increases the Abundance of ATP‐Binding Cassette Transporters ABCB1 and ABCC1 on Brain Capillary Cells

The blood-brain barrier (BBB) consists of dense contacts between endothelial cells, the tight junctions, which are complemented by membrane-bound transporters belonging to the ATP-binding cassette (ABC) transporter family. Liver X receptors (LXR) have previously been shown to stabilize the integrity of atherosclerotic noncerebral arteries. Their effects on ischemic cerebral vessels are still unknown. By delivering LXR agonists, T0901317 and GW3965, to mice submitted to 30 minutes intraluminal middle cerebral artery occlusion, we show that LXR activation reduces brain swelling and decreases BBB permeability by upregulating LXR's target calpastatin that deactivates calpain-1/2, stabilizing p120 catenin. p120 catenin specifically interacts with RhoA and Cdc42, inactivating the former and overactivating the latter, thus restoring the postischemic expression, phosphorylation and interaction of the tight junction proteins occludin and zona occludens-1. Moreover, LXR activation deactivates matrix metalloproteases-2/9 and inhibits microvascular apoptosis by deactivating JNK1/2 and caspase-3. In addition to the cholesterol transporters ABCA1 and ABCG1, which have previously been shown to be upregulated by LXR in noncerebral vessels, LXR activation increases the abundance of the drug transporters ABCB1 and ABCC1 on ischemic brain capillaries, as we further show. That LXR activation promotes endothelial integrity in different ways makes this receptor attractive as target for stroke therapies.

Mechanisms involved in vein occlusion related macular edema

AbstractPurpose To describe the pathophysiological processes involved in vein occlusion related macular edema.Methods Review of the experimental and clinical published data explaining the pathophysiological mechanisms involved in vein occlusion related macular edema.Results Venous stasis lead to internal blood‐retinal barrier breakdown, extravasation of blood vessel content and finally to macular edema. Tissular hypoxia of the internal retinal layers develops secondary to arterial blood flow decrease, followed by Na+/K+ ATPase pump dysfunction, intracellular edema, and neuronal cell death by necrosis and apoptosis. Many vasopermeability factors will be subsequently released, as inflammatory mediators and VEGF. The rationale for clinical treatment of macular edema is based on the understanding and the inhibition of these pathophysiological mechanisms. On the medical side, nonsteroidal anti‐inflammatory drugs inhibit the production of prostaglandins and leukotrienes, and modulate fluid movement coupled to chloride movement. Corticosteroids block cyclooxygenase and interleukin, downregulate VEGF and decrease the phosphorylation of occludin, thereby increasing the tightness of the blood‐retinal barrier. Anti‐VEGF agents restore occludin proteins in the blood‐retinal barrier and reduce protein kinase C activation.Conclusion New advances in understanding the mechanisms involved vein occlusion related macular edema lead to development of new therapeutical strategies which have to be confirmed by clinical randomized trials.

CaV1.3 Channels and Intracellular Calcium Mediate Osmotic Stress-induced N-terminal c-Jun Kinase Activation and Disruption of Tight Junctions in Caco-2 Cell Monolayers

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.

Correlation between the destruction of tight junction by patulin treatment and increase of phosphorylation of ZO-1 in Caco-2 human colon cancer cells

Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24h of treatment with 50μM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24h and decreased from the cell–cell contact regions of TJs after 48h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72h. Phosphorylation of ZO-1 was detected by 24h and increased markedly after 72h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48h and was completely absent after 72h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.

Quantitative MRI Reveals the Elderly Ischemic Brain is Susceptible to Increased Early Blood—Brain Barrier Permeability Following Tissue Plasminogen Activator Related to Claudin 5 and Occludin Disassembly

Great uncertainty exists as to whether aging enhances the detrimental effects of tissue plasminogen activator (tPA) on vascular integrity of the ischemic brain. We hypothesized that tPA treatment would augment ischemic injury by causing increased blood-brain barrier (BBB) breakdown as determined by quantitative serial T(1) and T(2) magnetic resonance imaging (MRI), and the transfer constant for gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) from blood to brain in aged (18 to 20 months) compared with young (3 to 4 months) Wistar rats after middle cerebral artery occlusion, mediated through the acute disassembly of claudin 5 and occludin. Increased T(2) values over the first hour of postreperfusion were independently augmented following treatment with tPA (P<0.001) and aging (P<0.01), supporting a synergistic effect of tPA on the aged ischemic brain. Blood-brain barrier permeability for Gd-DTPA (K(Gd)) was substantial following reperfusion in all animal groups and was exacerbated by tPA treatment in the elderly rat (P<0.001). The frequency of hematoma formation was proportionately increased in the elderly ischemic brain (P<0.05). Both tPA and age independently increased claudin 5 and occludin phosphorylation during ischemia. Early BBB permeability detected by quantitative MRI following ischemic stroke is enhanced by increased age and tPA and is related to claudin 5 and occludin phosphorylation.

Occludin Protein Family: Oxidative Stress and Reducing Conditions

The occludin-like proteins belong to a family of tetraspan transmembrane proteins carrying a marvel domain. The intrinsic function of the occludin family is not yet clear. Occludin is a unique marker of any tight junction and is found in polarized endothelial and epithelial tissue barriers, at least in the adult vertebrate organism. Occludin is able to oligomerize and to form tight junction strands by homologous and heterologous interactions, but has no direct tightening function. Its oligomerization is affected by pro- and antioxidative agents or processes. Phosphorylation of occludin has been described at multiple sites and is proposed to play a regulatory role in tight junction assembly and maintenance and, hence, to influence tissue barrier characteristics. Redox-dependent signal transduction mechanisms are among the pathways modulating occludin phosphorylation and function. This review discusses the novel concept that occludin plays a key role in the redox regulation of tight junctions, which has a major impact in pathologies related to oxidative stress and corresponding pharmacologic interventions.

Apolipoprotein E Regulates the Integrity of Tight Junctions in an Isoform-dependent Manner in an in Vitro Blood-Brain Barrier Model

Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ε4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3- and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC)η in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKCη, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner.

Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

BackgroundThe endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term “blood-labyrinth-barrier”. This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na+/K+-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.Methodology/Principal FindingsUsing a mass-spectrometry, shotgun-proteomics approach combined with a novel “sandwich-dissociation” method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.Conclusions/SignificanceThe results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier.

Expression of the Lactobacillus Plantarum Surface Layer MIMP Protein Protected NCM460 Epithelial Cells from Enteroinvasive Escherichia Coli Infection

Our previous studies demonstrated that the micro integral membrane protein (MIMP) located within the integral membrane protein of Lactobacillus plantarum CGMCC 1258 protected the intestinal barrier from injury. To further analyze the protective effects conferred to intestinal epithelial cells by MIMP, we established transient MIMP-expressing NCM460 cells (NCM460/MIMP) as a means of assessing their susceptibility to infection. We constructed a recombinant eukaryotic expression vector using pcDNA3.1(-) and the MIMP gene. The recombinant vector was then transduced into NCM460 cells and the anti-infective properties of the transient MIMP-expressing NCM460/MIMP cells assessed. Flow cytometric analysis revealed that 82.16% ±12.22% of NCM460/MIMP cells expressed MIMP and Western blot analysis confirmed high levels of MIMP expression. Attachment assays showed that the ability of enteropathogenic Escherichia coli (EPEC) to attach to NCM460/MIMP cells decreased significantly compared to adhesion observed to NCM460 cells. Western blot and quantitative RT-PCR showed that the expression levels of tight junction (TJ) proteins, including claudin-1, occludin, JAM-1 and ZO-1 in NCM460/MIMP cells infected with EPEC were similar to levels observed in uninfected NCM460 cells. Fluorescence further showed that NCM460/MIMP cells had significantly higher TJ protein staining intensity ocompared to NCM460 cells and transmission electron microscopy indicated that TJ structure was unchanged in NCM460/MIMP cells infected with EPEC compared to NCM460 cells after EPEC infection. Expression levels of PKC-η and phosphorylated occludin were also higher after EPEC infection in NCM460/MIMP cells compared to expression levels in EPEC-infected NCM460 cells. These data demonstrated that NCM460/MIMP cells possessed EPEC anti-infective properties related to the activation of protein kinase C-η and occludin phosphorylation.

Prednisolone restores blood brain barrier damages in dystrophic MDX mouse

Although the glucocorticoids delay the progression of Duchenne muscular dystrophy (DMD) their mechanism of action is unknown. In our previous studies we demonstrated that in the mdx mice, an animal model of DMD, besides the muscle degeneration, serious damages of the blood-brain barrier (BBB) occur taking to enhanced vessels permeability and brain edema (1). Moreover, we observed that the mdx mice after α–methyl-prednisolone (PDN) treatment ameloriated the histopathological profiles and the excitation-contraction of the myofibers (2). In this study, we evaluated the effects of the PDN on the BBB of the mdx mice, by estimating the immunocytochemical and biochemical expression of endothelial ZO-1 and occludin, pericyte desmin, and glial GFAP and short dystrophin isoform Dp 71 proteins, used as BBB markers. In addition, we analyzed the expression of dystrophin associate proteins (DAPs) aquaporin-4 (AQP4) and α-β dystroglycan in parallel in both brain and muscles of PDN treated mdx as well as in control mice. Results showed in mdx PDN treated mice a significant increase of the mRNA and protein content of all the glial, pericyte and endothelial proteins as compared to untreated mdx. Moreover, by immunoprecipitation we demonstrated that the BBB alteration in the mdx mice were coupled with enhanced occludin and AQP4 phosphorylation degree which, instead, was reduced after PDN treatment. Finally we observed that AQP4 and α-β dystroglycan complex increases its mRNA and protein content in both PDN mdx brain and muscle fibers, compared with mdx mice where the perivascular glial membranes and the myofibers showed a light staining after immunofluorescence analysis . These data indicate that the PDN restores the BBB damages in the mdx mice by inducing in the glial cell the expression of GFAP, AQP4 and Dp71 proteins and in the pericytes and endothelial cells, of the desmin and ZO-1 proteins, which are deficient in the distrophic mice. Moreover, the reduction in the AQP4 and occludin phosphorylation degree coupled with their ankoring to glial and endothelial membranes in the PDN mdx mice suggests that the glial and endothelial cells may be a cellular target of the drug. Finally, the enhanced expression of DAPs AQP4 and α-β dystroglycan in both brain and myofibers of PDN treated mdx mice compared to untreated mdx ones suggest the PDN might ameliorate the brain vessels and muscles functions of the dystrophic mice by a restoring a correct links between DAPs proteins and the extracellular matrix. 1. Nico B et al. Glia, 42: 235-251. (2003). 2. Cozzoli A. et al., Neuropathol. Appl. Neurobiol. 37, 243-256 (2011).

Potentiation of Polarized Intestinal Caco-2 Cell Responsiveness to Probiotics Complexed with Secretory IgA

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Prostanoid EP 1 receptor antagonist reduces blood–brain barrier leakage after cerebral ischemia

Disruption of the blood–brain barrier (BBB) after cerebral ischemia is considered to be the initial step in the development of brain injuries, and an increase in the tyrosine phosphorylation of the tight junctional protein occludin has been shown to cause an increase in BBB permeability. Prostaglandin E 2 (PGE 2) appears to be associated with both toxic and protective effects on neuronal survival in vitro. However, it remains to be determined whether the prostanoid EP 1 receptor is involved in the disruption of the BBB after cerebral ischemia. So we examined the effect of a prostanoid EP 1 receptor antagonist, SC51089, on BBB leakage and tyrosine phosphorylation of occludin after cerebral ischemia. We demonstrated that SC51089 attenuated the increase in the tyrosine phosphorylation of occludin in isolated brain capillaries, which was coincident with a decrease in BBB leakage. These results suggest that the prostanoid EP 1 receptor is involved in the tyrosine phosphorylation of occludin at tight junction, which may lead to disruption of the BBB and be linked to the development of cerebral infarctions.

Na‐K‐ATPase activity involved in noise induced blood‐labyrinth‐barrier leakage

Na+/K+‐ATPase plays an important role in mediating barrier integrity in the polarized epithelial cells. In the cochlea, we found that Na+/K+‐ATPase subunit alpha 1 was the most abundant protein of the vascular tissue in the blood‐labyrinth‐barrier (BLB) and was expressed predominantly at the luminal side of the endothelium. Na‐K‐ATPase is known to be required for controlling the tight junctions in epithelial cells. In this study, we investigated the involvement of Na+/K+‐ATPase in noise induced BLB leakage. In mice, the expression and activity of Na+/K+‐ATPase were found to be correlated with the changes of expression of tight junction proteins as well as the change in BLB permeability. Increased expression of Na+/K+‐ATPase, at both transcriptional and protein levels, occurred after noise exposure at 120 dB SPL for 3h per day for two days. However, Na+/K+‐ATPase enzymatic activity was significantly decreased. There was a significant decrease in endothelial tight junction membrane contact points. In vitro inhibition of Na+, K+‐ATPase by ouabain corroborated the in vivo studies and showed swelling of capillary endothelial cells and increased ratio between occludin phosphorylation and dephosphorylation. The results support a functional link between Na+, K+‐ATPase and occludin in the BLB as one of the mechanisms for noise induced in the BLB.Support: NIDCD DC 00888, NIDCD DC 00105, NIDCD DC 005983 (p30)

Casein kinase 2 (CK2) phosphorylates occludin at Ser408 to increase intra‐tight junction diffusion and reduce paracellular barrier function

CK2 phosphorylates the carboxy‐terminal cytoplasmic tail of the tight junction protein occludin. We sought to determine the consequences of CK2‐mediated occludin phosphorylation and identify critical target residues(s). The CK2 inhibitors TBB and DMAT each elevated transepithelial resistance (TER) of Caco‐2 monolayers. This regulation was CK2‐ and occludin‐dependent, as it did not occur after siRNA knockdown (KD) of either protein. To assess the functional relevance of previously identified CK2 targets Thr404 and Ser408, wild type or T408D/S408D occludin was expressed in occludin KD cell lines. Both constructs restored TER, which was reduced by occludin KD, but only wild type occludin re‐established the TER response to CK2 inhibition. Fluorescence recovery after photobleaching (FRAP) showed a reduced occludin mobile fraction after either chemical‐ or siRNA‐mediated CK2 inhibition, which was associated with increased occludin density at the tight junction. DMAT similarly reduced occludin FRAP in vivo. In vitro, T404A, T404D, and S408D occludin mutants had FRAP behavior indistinguishable from wild type occludin, but S408D was unaffected by CK2 inhibition. Conversely, S408A occludin FRAP at the tight junction was reduced, relative to wild type, and was unaffected by CK2 inhibition. Thus, CK2‐mediated phosphorylation of occludin S408 accelerates tight junction remodeling and increases permeability.

Metalloproteinase mediated occludin cleavage in the cerebral microcapillary endothelium under pathological conditions

Reactive oxygen species (ROS) as well as matrix metalloproteinases (MMPs) induce modifications in the tight junction (TJ) protein occludin which is crucial for the blood–brain barrier (BBB) function. We investigated the role of ROS and MMPs in endothelial autoregulatory response on oxidative stress with respect to occludin and the BBB integrity. The ROS hydrogen peroxide (H 2O 2) was applied to our well-established BBB cell culture model based on primary porcine brain capillary endothelial cells (PBCEC). At low concentrations (2.5 mM), H 2O 2-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H 2O 2 concentrations (≥ 10 mM) occludin cleavage occurred and elevated levels of active MMP-2 were detected. Under those conditions intercellular gaps were formed within the monolayer visualizing the barrier breakdown also determined by impedance analysis. A primary structure sequence analysis revealed potential type IV collagenase sensitive motives in the first extracellular loop, thus providing evidence that occludin might be an MMP-2 substrate. MMP inhibition by the metalloproteinase inhibitor GM6001 prevented occludin degradation and reduced the intercellular gap formation. However, the barrier function quantified by impedance measurement could not be maintained despite MMP inhibition. When we applied an enzymatic activity level which caused occludin cleavage in injured PBCEC to intact PBCEC, neither occludin cleavage nor barrier impairment was observed. Thus, in our model occludin cleavage is not an autoregulatory mechanism of microcapillary endothelium in barrier modulation under oxidative stress, but only occurs upon endothelial damage.