Zona occludens-1 (ZO-1) is a key component of tight junctions that govern the function of the endothelial barrier against tumor metastasis. Factors secreted by tumor cells contribute to the maintenance of tumor vascular networks. How tumor cell-derived protein signals regulate ZO-1 expression is unclear. Here, we explored the effect of tumor cell-secreted asparaginyl endopeptidase (AEP) on the permeability of endothelial cells in the tumor microenvironment. First, we confirmed the existence of AEP in conditioned medium (CM) from AEP-overexpressing MDA-MB-231 and 4T1 cells. Treatment with CM from AEP-overexpressing tumor cells increased the permeability and tumor cell transversal of an endothelial monolayer. Furthermore, CM from AEP-overexpressing tumor cells suppressed endothelial ZO-1 expression, as well as ZO-1-associated nucleic acid binding protein ZONAB. In addition, the level of phosphorylated STAT3 was increased by treatment with AEP-containing CM. A mutation of RGD or blocking integrin αvβ3 with antibody recovered the ZO-1 downregulation induced by AEP. In vivo, a lung metastatic mouse model showed increased endothelial permeability in the AEP-overexpressing group compared with the control group. An orthotopic tumor transplantation model was established using AEP-overexpression and compared with mice receiving control 4T1 cells. Compared with controls, overexpression of AEP increased lung metastatic foci and area, as well as vascular instability in primary tumors or lung metastatic sites. Moreover, endothelial ZO-1 was decreased in the AEP-overexpressing group. Taken together, our data show that tumor cell-derived AEP increases the permeability of endothelial barriers. Interactions between RGD and endothelial integrin αvβ3 mediate this effect by downregulating ZO-1.

Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability.

Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (∼Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.

Moxifloxacin (MOX) suppresses cell movement in human corneal fibroblasts (HCFs). Zonula occludens-1 (ZO-1) is localized to the leading edge of migrating HCFs. This study explored the role of ZO-1 in MOX-suppressed cell migration in HCFs. A single-cell trajectory analysis revealed that MOX negatively regulated the migratory properties of HCFs including migration distance, migration velocity, and directionality (P < 0.001, P < 0.001, and P = 0.018, respectively). MOX increased endogenous ZO-1 in HCFs in a concentration-dependent manner (P = 0.083, P = 0.005, and P = 0.001 at 10, 50, and 100 μg/ml, respectively), but decreased the phosphorylation of endogenous ZO-1 at serines, threonines, and tyrosines. In contrast, MOX did not alter the expression of protein kinase C epsilon (PKCε), Rac-1, Cdc42, and MRCKβ. However, MOX did also reduce the phosphorylation level of PKCε at serines and threonines (P < 0.001 at 100 μg/ml). In addition, MOX increased the phosphorylation level of Rac-1 in a concentration-dependent manner (P < 0.001 at 100 μg/ml). Compared with the mock cells, the directionality of cell movement increased significantly in ZO-1-expressing HCFs (P = 0.012) and decreased significantly in ZO-1-silenced HCFs (P = 0.002). The directionality did not change significantly in Rac-1-silenced HCFs. ZO-1-expressing HCFs moved faster than mock cells. PKCε, Cdc42, Rac-1, and phosphorylated Rac-1 were decreased in ZO-1-overexpressing HCFs, but increased in ZO-1-silenced HCFs. Finally, silencing ZO-1 blocked MOX hyperactivation of Rac-1. These suggest that MOX might trigger random migration in human corneal stromal cells through PKCε-modulated ZO-1 inactivation and Rac-1 hyperactivation.

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.

Zonula occludens-1 (ZO-1) is a scaffolding protein of tight junctions, which seal adjacent epithelial cells, that is also expressed in adherens junctions. The distribution pattern of ZO-1 differs among stratified squamous epithelia, including that between skin and oral buccal mucosa. However, the causes for this difference, and the mechanisms underlying ZO-1 spatial regulation, have yet to be elucidated. In this study, we showed that epithelial turnover and proliferation are associated with ZO-1 distribution in squamous epithelia. We tried to verify the regulation of ZO-1 by comparing normal skin and psoriasis, known as inflammatory skin disease with rapid turnover. We as well compared buccal mucosa and oral lichen planus, known as an inflammatory oral disease with a longer turnover interval. The imiquimod (IMQ) mouse model, often used as a psoriasis model, can promote cell proliferation. On the contrary, we peritoneally injected mice mitomycin C, which reduces cell proliferation. We examined whether IMQ and mitomycin C cause changes in the distribution and appearance of ZO-1. Human samples and mouse pharmacological models revealed that slower epithelial turnover/proliferation led to the confinement of ZO-1 to the uppermost part of squamous epithelia. In contrast, ZO-1 was widely distributed under conditions of faster cell turnover/proliferation. Cell culture experiments and mathematical modelling corroborated these ZO-1 distribution patterns. These findings demonstrate that ZO-1 distribution is affected by epithelial cell dynamics.

The process of cytoadhesion in Plasmodium falciparum malaria infection causes signaling processes that lead to structural and functional changes at the cellular level. Histopathological changes of acute kidney injury (AKI) in P. falciparum malaria often involve glomerular proliferation, thickening of the glomerular basement membrane, acute tubular necrosis, and interstitial inflammation. Focusing on the glomeruli, this study aimed to investigate glomerular and tight junction-associated protein- zonula occludens-1 (ZO-1) changes in P. falciparum malaria patients.

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/-). ZO-1+/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

Hepatocellular carcinoma (HCC) is a frequent type of primary liver cancer, and its prevalence is increasing worldwide. Indeed, the underlying molecular mechanism is not well understood. Previous studies have shown evidence that tight junction (TJ) components were correlated with carcinogenesis and tumor development. Our aims were to determine the serum levels of tight junction protein Zonula Occludens (ZO)-1 and an inflammatory marker such as high-sensitive C-reactive protein (hs-CRP) in HCC patients compared to healthy volunteers and also to identify the association between ZO-1 and inflammation in HCC.

Allergic rhinitis (AR) is a common disease affecting 400 million of the population worldwide. Nasal epithelial cells form a barrier against the invasion of environmental pathogens. These nasal epithelial cells are connected together by tight junction (TJ) proteins including zonula occludens-1 (ZO-1), ZO-2 and ZO-3. Impairment of ZO proteins are observed in AR patients whereby dysfunction of ZOs allows allergens to pass the nasal passage into the subepithelium causing AR development. In this review, we discuss ZO proteins and their impairment leading to AR, regulation of their expression by Th1 cytokines (i.e., IL-2, TNF-α and IFN-γ), Th2 cytokines (i.e., IL-4 and IL-13) and histone deacetylases (i.e., HDAC1 and HDAC2). These findings are pivotal for future development of targeted therapies by restoring ZO protein expression and improving nasal epithelial barrier integrity in AR patients.

Zonula occludens (ZO)-1, a tight-junction protein (TJP), is expressed in dendritic cells (DCs) but not in monocytes, and 27-hydroxycholesterol (27OHChol) drives the differentiation of monocytes into DCs. Because the effects of 27OHChol on ZO-1 are not yet clearly defined, we investigated whether 27OHChol induces expression of the TJP. The treatment of human THP-1 monocytic cells with 27OHChol resulted in the elevated transcript levels of ZO-1 but not of ZO-2 or -3. 27OHChol increased the total amount of ZO-1 protein in the cells as well as its level on the cells surface. Cholesterol, however, did not influence expression of ZO-1. And, the expression of ZO-1 protein was mediated by endoplasmic reticulum (ER)-to-Golgi body transport system. Pharmacological kinase inhibition with LY294002 (a PI3K inhibitor), U0126 (a MEK/ERK inhibitor), or PP2 (a Src family kinase inhibitor) resulted in impaired ZO-1 expression at both transcript and protein levels. Drugs that are reported to suppress DC differentiation also inhibited 27OHChol-mediated expression and the localization of ZO-1, indicating the coincidence of ZO-1 upregulation and DC differentiation. These results suggest that ZO-1 is differentially expressed while monocytes differentiate into DCs in the presence of 27OHChol via pathways in which distinct signaling molecules are involved.

Ischemic brain injury induces both functional and structural disarray affecting the blood-brain barrier (BBB) which in return aggravates stroke outcomes. Complement system and its bioactive proteins are important molecular responders to ischemia. C5a protein along with its receptor C5a receptor 1 is a key component of this system with potent pro-inflammatory and chemoattractant properties. The purpose of this study is to investigate the role of C5a protein and its receptor which are believed to participate in the inflammatory response that follows ischemic insult.

Hypercholesterolemia and tight junctions play important roles in atherosclerosis. But the relationship between these two factors is unclear. In the present study, we investigated whether hypercholesterolemic serum could change the permeability of endothelial cells through altering expression and/or distribution of tight junction protein zonula occludens-1 (ZO-1). Phosphatidylinositol 3-kinase (PI3K) signaling pathway was also examined.

The aim of this research was to study the alteration of three key tight junction proteins, to explore whether they were involved in the occurrence of prelabor rupture of the membrane (PROM) and to determine the correlation with intrauterine infection.

Blood-brain barrier (BBB) disruption is a key pathophysiological factor of intracerebral hemorrhage (ICH). The level of zonula occludens-1 (ZO-1) has been closely associated with the degree of BBB damage, and is an indicator of BBB destruction. The aim of the present study was to evaluate the effects of rhubarb on BBB function in a rat model of ICH. ICH was induced in rats by treatment with type VII collagenase. Sham-operated rats were administered with an equal volume of saline. Following the administration of rhubarb decoction (20 g/kg), neurobehavioral function evaluation and Evans blue extravasation assays were performed at days 1, 3 and 5 after ICH. ZO-1 expression in the brain of ICH-induced rats were analyzed via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses. The results suggested that rhubarb significantly ameliorated neurological symptoms and attenuated BBB permeability. The results of immunohistochemistry and RT-PCR studies indicated that the expression of ZO-1 expression was robust in the sham-operated group and was weak in the vehicle-treated group at day 3. The present data indicated that rhubarb effectively attenuated ICH-induced BBB damage in rats, raising the possibility that rhubarb or its active components may be considered useful as neuroprotective drugs for ICH. The protective mechanisms appeared to involve the preservation of BBB integrity and elevation of ZO-1 protein expression levels.

Atypical Protein Kinase C zeta (PKCζ) forms Partitioning-defective (PAR) polarity complex for apico-basal distribution of membrane proteins essential to maintain normal cellular junctional complexes and tissue homeostasis. Consistently, tumor suppressive role of PKCζ has been established for multiple human cancers. However, recent studies also indicate pro-oncogenic function of PKCζ without firm understanding of detailed molecular mechanism. Here we report a possible mechanism of oncogenic PKCζ signaling in the context of breast cancer. We observed that depletion of PKCζ promotes epithelial morphology in mesenchymal-like MDA-MB-231 cells. The induction of epithelial morphology is associated with significant upregulation of adherens junction (AJ) protein E-cadherin and tight junction (TJ) protein Zonula Occludens-1 (ZO-1). Functionally, depletion of PKCζ significantly inhibits invasion and metastatic progression. Consistently, we observed higher expression and activation of PKCζ signaling in invasive and metastatic breast cancers compared to non-invasive diseases. Mechanistically, an oncogenic PKCζ- NFκB-p65 signaling node might be involved to suppress E-cadherin and ZO-1 expression and ectopic expression of a constitutively active form of NFκB-p65 (S536E-NFκB-p65) significantly rescues invasive potential of PKCζ-depleted breast cancer cells. Thus, our study discovered a PKCζ - NFκB-p65 signaling pathway might be involved to alter cellular junctional dynamics for breast cancer invasive progression.

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein-protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein-which are pharmacologically active components of Chinese skullcap (Radix scutellariae)-with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein-but not baicalin-induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a "partial" epithelial-mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1-CLD interaction, activation of both transforming growth factor-β (TGF-β) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.