Specific Tight Junction Proteins Research Articles

Research status and hotspots of tight junctions and colorectal cancer: A bibliometric and visualization analysis

BACKGROUND Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death. Over the past two decades, numerous researchers have provided important evidence regarding the role of tight junction (TJ) proteins in the occurrence and progression of CRC. The causal relationship between the presence of specific TJ proteins and the development of CRC has also been confirmed. Despite the large number of publications in this field, a bibliometric study to review the current state of research and highlight the research trends and hotspots in this field has not yet been performed. AIM To analyze research on TJs and CRC, summarize the field’s history and current status, and predict future research directions. METHODS We searched the Science Citation Index Expanded database for all literature on CRC and TJs from 2001-2023. We used bibliometrics to analyze the data of these papers, such as the authors, countries, institutions, and references. Co-authorship, co-citation, and co-occurrence analyses were the main methods of analysis. CiteSpace and VOSviewer were used to visualize the results. RESULTS A total of 205 studies were ultimately identified. The number of publications on this topic has steadily increased since 2007. China and the United States have made the largest contributions to this field. Anticancer Research was the most prolific journal, publishing 8 articles, while the journal Oncogene had the highest average citation rate (68.33). Professor Dhawan P was the most prolific and cited author in this field. Co-occurrence analysis of keywords revealed that “tight junction protein expression”, “colorectal cancer”, “intestinal microbiota”, and “inflammatory bowel disease” had the highest frequency of occurrence, revealing the research hotspots and trends in this field. CONCLUSION This bibliometric analysis evaluated the scope and trends of TJ proteins in CRC, providing valuable research perspectives and future directions for studying the connection between the two. It is recommended to focus on emerging research hotspots, such as the correlations among intestinal microbiota, inflammatory bowel disease, TJ protein expression, and CRC.

Sex‐related differences in the visual function of an animal model of type 1 diabetes

Aims/Purpose: Recent basic and clinical research evidence suggests that male–female differences may influence retinal function and structure. It is also well‐established that diabetes affects retinal function. This work aimed to assess whether type 1 diabetes differentially affects neuroretinal function in male and female Wistar rats.Methods: To induce type 1 diabetes, male and female Wistar rats were injected (i.p.) with streptozotocin (65 mg/kg). Electroretinograms (ERGs) were recorded before and 1, 2, and 4 weeks after diabetes induction (n = 12–13 animals/group). To assess the effect of chronic hyperglycemia on the content of molecular components of the blood‐retinal barrier (BRB), specific tight junction proteins were assessed by Western Blotting. Neuroinflammation was evaluated by measuring the retinal levels of pro‐inflammatory cytokines.Results: Some sex‐related changes in retinal function were detected in the diabetic animals. Overall, in male diabetic rats, compared to control male rats, the differences found under scotopic conditions were mainly in the positive scotopic threshold response (pSTR), with decreased amplitudes (at weeks 2 and 4), and higher latencies (at weeks 1 and 4). In contrast, diabetic female rats, compared to control females, presented a higher increase in the latency value of the negative STR (nSTR; at weeks 1 and 4), a‐wave (at weeks 1, 2, and 4), and oscillatory potentials 2, 3, and 4 (at weeks 1, 2, and 4). Under photopic conditions, male diabetic rats showed higher implicit time values of the fundamental flicker wave (at week 4) and a decrease in the amplitude of the flicker 2nd harmonic (at week 4) compared to control males. Conversely, in diabetic female rats, there were no differences in these parameters compared to control female rats. Concerning the BRB and neuroinflammation, no changes were found in diabetic males and females compared to their respective controls.Conclusions: Our findings suggest that type 1 diabetes differentially affects the retinal function in male and female Wistar rats.Support: FCT‐Portugal: PEst UIDB/04539/2020 and UIDP/04539/2020; 2020.07432.BD.

Wnt7a Decreases Brain Endothelial Barrier Function Via β-Catenin Activation

The blood-brain barrier consists of tightly connected endothelial cells protecting the brain’s microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/β-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated β-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/β-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of β-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by β-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by β-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of β-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease.

Structure-function analysis of tight junction-directed permeation enhancer PIP250

The intestinal paracellular route of absorption is modulated via tight junction (TJ) structures located at the apical neck of polarized intestinal epithelial cells to restrict solute movement through the intercellular space between them. Tight junctions open or close in response to changes in the phosphorylation status of light chain (MLC) at position Ser-19. This phosphorylation event is primarily controlled by MLC kinase (MLCK) and MLC phosphatase (MLCP), the latter being a holoenzyme that involves interaction between protein phosphatase 1 (PP1) and myosin targeting protein 1 (MYPT1). An entirely D-amino acid Permeant Inhibitor of Phosphatase (PIP) peptide sequence designed to disrupt PP1-MYPT1 interactions at the cytoplasmic surface of TJs, PIP250 (rrfkvktkkrk) localized at intracellular TJ structures, altered expression levels of specific TJ proteins, increased cellular phosphorylated MLC (pMLC) levels, binding to PP1, decreased epithelial barrier function, and significantly increased systemic uptake of the poorly absorbed antibiotic gentamicin in vivo. A series of PIP250 peptide analogues showed that positions phe3 and val5 were critical to its functional properties, with some providing opportunities to tune the dynamic actions of its TJ modulation properties. These data confirm the activity of PIP250 as a rationally designed oral permeation enhancer and validated key amino acids involved in its interaction with PP1 that define its overall actions; the magnitude and duration of these enhancing properties were associated with the MYPT1-mimetic properties of the PIP250 peptide analogues described.

Fructooligosaccharide Reduces Weanling Pig Diarrhea in Conjunction with Improving Intestinal Antioxidase Activity and Tight Junction Protein Expression.

This study was to illustrate the effects of fructooligosaccharide (FOS) on the antioxidant capacity, intestinal barrier function, and microbial community of weanling pigs. Results showed that FOS reduced the incidence of diarrhea (6.5 vs. 10.8%) of pigs (p < 0.05) but did not affect growth performance when compared with the control group. A diet supplemented with FOS increased ileal mRNA expression of occludin (1.7 vs. 1.0), claudin-1 (1.9 vs. 1.0), claudin-2 (1.8 vs. 1.0), and claudin-4 (1.7 vs. 1.0), as well as colonic mRNA expression of ZO-1 (1.6 vs. 1.0), claudin-1 (1.7 vs. 1.0), occludin (1.9 vs. 1.0), and pBD-1 (1.5 vs. 1.0) when compared with the control group (p < 0.05). FOS supplementation improved the anti-oxidase activity and expression of nuclear factor erythroid-2 related factor 2 (Nrf2), and decreased concentrations of D-lactate (3.05 U/L vs. 2.83 U/L) and TNF-α (59.1 pg/mL vs. 48.0 pg/mL) in the serum when compared with the control group (p < 0.05). In addition, FOS increased Sharpea, Megasphaera, and Bacillus populations in the gut when compared with the control group (p < 0.05). Association analysis indicated that mRNA expression of occludin and claudin-1 in the ileal mucosa were correlated positively with populations of Sharpea and Bacillus (p < 0.05). Furthermore, mRNA expression of occludin and claudin-1 in the colonic mucosa were correlated positively with abundances of Sharpea, Lactobocillus, and Bifidobacterium (p < 0.05). In conclusion, FOS activated Nrf2 signaling and increased the expression of specific tight junction proteins, which were associated with reduced diarrhea incidence.

Selective blood-nerve barrier leakiness with claudin-1 and vessel-associated macrophage loss in diabetic polyneuropathy

Diabetic polyneuropathy (DPN) is the most common complication in diabetes and can be painful in up to 26% of all diabetic patients. Peripheral nerves are shielded by the blood-nerve barrier (BNB) consisting of the perineurium and endoneurial vessels. So far, there are conflicting results regarding the role and function of the BNB in the pathophysiology of DPN. In this study, we analyzed the spatiotemporal tight junction protein profile, barrier permeability, and vessel-associated macrophages in Wistar rats with streptozotocin-induced DPN. In these rats, mechanical hypersensitivity developed after 2 weeks and loss of motor function after 8 weeks, while the BNB and the blood-DRG barrier were leakier for small, but not for large molecules after 8 weeks only. The blood-spinal cord barrier remained sealed throughout the observation period. No gross changes in tight junction protein or cytokine expression were observed in all barriers to blood. However, expression of Cldn1 mRNA in perineurium was specifically downregulated in conjunction with weaker vessel-associated macrophage shielding of the BNB. Our results underline the role of specific tight junction proteins and BNB breakdown in DPN maintenance and differentiate DPN from traumatic nerve injury. Targeting claudins and sealing the BNB could stabilize pain and prevent further nerve damage.Key messages• In diabetic painful neuropathy in rats:• Blood nerve barrier and blood DRG barrier are leaky for micromolecules.• Perineurial Cldn1 sealing the blood nerve barrier is specifically downregulated.• Endoneurial vessel-associated macrophages are also decreased.• These changes occur after onset of hyperalgesia thereby maintaining rather than inducing pain.Graphical abstract

Blood-brain barrier dysfunction as a potential therapeutic target for neurodegenerative disorders.

The blood-brain barrier (BBB) is composed of specific tight junction proteins and transporters expressed on the lining of endothelial cells of the vasculature in the brain. The structural and functional integrity of the BBB is one of the most critical factors for maintaining brain homeostasis and is mainly regulated by complex interactions between various cell types, such as endothelial cells, pericytes, and astrocytes, which are shaped by their differential responses to changes in microenvironments. Alterations in these cellular components have been implicated in neurodegenerative disorders. Although it has long been considered that BBB dysfunction is a mere ramification of pathological phenomena, emerging evidence supports its critical role in the pathogenesis of various disorders. In epilepsy, heightened BBB permeability has been found to be associated with increased occurrence of spontaneous seizures. Additionally, exaggerated inflammatory responses significantly correlate with increased BBB permeability during healthy aging. Furthermore, it has been previously reported that BBB disruption can be an early marker for predicting cognitive impairment in the progression of Alzheimer's disease. We herein review a potential role of the major cellular components of the BBB, with a focus on the contribution of BBB disruption, in neurodegenerative disease progression.

Organic osmolytes increase expression of specific tight junction proteins in skin and alter barrier function in keratinocytes.

The epidermal barrier is important for water conservation, failure of which is evident in dry-skin conditions. Barrier function is fulfilled by the stratum corneum, tight junctions (TJs, which control extracellular water) and keratinocyte mechanisms, such as organic osmolyte transport, which regulate intracellular water homeostasis. Organic osmolyte transport by keratinocytes is largely unexplored and nothing is known regarding how cellular and extracellular mechanisms of water conservation may interact. We aimed to characterize osmolyte transporters in skin and keratinocytes, and, using transporter inhibitors, to investigate whether osmolytes can modify TJs. Such modification would suggest a possible link between intracellular and extracellular mechanisms of water regulation in skin. Immunostaining and quantitative polymerase chain reaction of organic osmolyte-treated organ-cultured skin were used to identify changes to organic osmolyte transporters, and TJ protein and gene expression. TJ functional assays were performed on organic osmolyte-treated primary human keratinocytes in culture. Immunostaining demonstrated the expression of transporters for betaine, taurine and myo-inositol in transporter-specific patterns. Treatment of human skin with either betaine or taurine increased the expression of claudin-1, claudin-4 and occludin. Osmolyte transporter inhibition abolished this response. Betaine and taurine increased TJ function in primary human keratinocytes invitro. Treatment of skin with organic osmolytes modulates TJ structure and function, which could contribute to the epidermal barrier. This emphasizes a role for organic osmolytes beyond the maintenance of intracellular osmolarity. This could be harnessed to enhance topical therapies for diseases characterized by skin barrier dysfunction.

Effect of ZO Protein Knockdown on Leak Pathway Pore Size in MDCK Renal Epithelial Cell Populations

The tight junction is the primary barrier to the paracellular movement of water and solutes across epithelial cells (paracellular permeability). Paracellular permeability is divided into two pathways: the Pore Pathway (small solute size, high capacity, charge‐selective) and the Leak Pathway (large solute size, low capacity, charge‐nonselective). Much has been learned about the molecular basis, properties, and regulation of the Pore Pathway. The Leak Pathway is much less well understood. Two basic questions about the Leak Pathway that remain controversial are: 1) does the Leak Pathway have a size limit; and 2) if so, how is the Leak Pathway size limit affected by manipulating specific tight junction proteins. We have initiated an investigation of these two questions by measuring the paracellular permeability of a size range of fluorescein dextrans (4 kDa to 70 kDa) and calculating pore size using the mathematical model, the Renkin sieving equation. In this mathematical model, calculation of pore size is independent of total flux rate, which is also affected by the pore volume fraction and pore length. We first used this approach to examine Leak Pathway permeability in the wild type MDCK renal epithelial cell line. The paracellular movement of all fluorescein dextrans across MDCK cell monolayers was linear with time. The apparent permeability (Papp) decreased with increasing solute Stokes radius. Using the Renkin sieving equation, the Leak Pathway pore size in wild type MDCK cell monolayers was calculated to be ~423 Angstroms. We then examined the effect of knockdown of either of the tight junction‐associated cytoplasmic proteins, ZO‐1 or ZO‐2, on pore size. The paracellular flux rates for all fluorescein dextrans were higher across ZO‐1 knockdown MDCK (ZO‐1 KD) cell populations than across either wild type MDCK cell populations or ZO‐2 knockdown MDCK (ZO‐2 KD) cell populations, which were similar. The paracellular flux of each fluorescein dextran was linear with time across populations of either ZO‐1 KD cells or ZO‐2 KD cells. Papp decreased with increasing Stokes radius for both knockdown cell lines. The calculated pore size for ZO‐2 KD cell populations was similar to that calculated for wild type MDCK cell populations. In contrast, the pore size for ZO‐1 KD cell populations was substantially smaller (~171 Angstroms) than was calculated for wild type MDCK cell populations despite the fact that the total flux rate for all fluorescein dextrans was significantly higher across ZO‐1 KD cell populations. These results indicate that knockdown of ZO‐1 in MDCK cells decreases the Leak Pathway pore size, whereas, knockdown of ZO‐2 does not alter Leak Pathway pore size. In addition, since the flux rate across ZO‐1 KD cell monolayers is higher than across monolayers of the other two cell lines, these results suggest ZO‐1 KD cell populations exhibit either a greater pore volume fraction and/or a shorter pore length than do either wild type MDCK cell populations or ZO‐2 KD cell populations.Support or Funding InformationNYITCOM internal funds

Escherichia coli K12: An evolving opportunistic commensal gut microbe distorts barrier integrity in human intestinal cells

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.

Inhibition of mTOR protects the blood-brain barrier in models of Alzheimer's disease and vascular cognitive impairment.

An intact blood-brain barrier (BBB) limits entry of proinflammatory and neurotoxic blood-derived factors into the brain parenchyma. The BBB is damaged in Alzheimer's disease (AD), which contributes significantly to the progression of AD pathologies and cognitive decline. However, the mechanisms underlying BBB breakdown in AD remain elusive, and no interventions are available for treatment or prevention. We and others recently established that inhibition of the mammalian/mechanistic target of rapamycin (mTOR) pathway with rapamycin yields significant neuroprotective effects, improving cerebrovascular and cognitive function in mouse models of AD. To test whether mTOR inhibition protects the BBB in neurological diseases of aging, we treated hAPP(J20) mice modeling AD and low-density lipoprotein receptor-null (LDLR-/-) mice modeling vascular cognitive impairment with rapamycin. We found that inhibition of mTOR abrogates BBB breakdown in hAPP(J20) and LDLR-/- mice. Experiments using an in vitro BBB model indicated that mTOR attenuation preserves BBB integrity through upregulation of specific tight junction proteins and downregulation of matrix metalloproteinase-9 activity. Together, our data establish mTOR activity as a critical mediator of BBB breakdown in AD and, potentially, vascular cognitive impairment and suggest that rapamycin and/or rapalogs could be used for the restoration of BBB integrity. NEW & NOTEWORTHY This report establishes mammalian/mechanistic target of rapamycin as a critical mediator of blood-brain barrier breakdown in models of Alzheimer's disease and vascular cognitive impairment and suggests that drugs targeting the target of rapamycin pathway could be used for the restoration of blood-brain barrier integrity in disease states.

Dietary salt loading and ion-poor water exposure provide insight into the molecular physiology of the rainbow trout gill epithelium tight junction complex.

This study utilized dietary salt loading and ion-poor water (IPW) exposure of rainbow trout (Oncorhynchus mykiss) to further understand the role of fish gill epithelium tight junction (TJ) physiology in salt and water balance. Gill morphology, biochemistry and molecular physiology were examined, with an emphasis on genes encoding TJ proteins. Fish were either fed a control or salt-enriched diet (~10% NaCl) for 4weeks prior to IPW exposure for 24h. Serum [Na(+)], [Cl(-)] and muscle moisture content were unaltered by salt feeding, but changed in response to IPW irrespective of diet. Dietary salt loading altered the morphology (reduced Na(+)-K(+)-ATPase-immunoreactive cell numbers and surface exposure of mitochondrion-rich cells), biochemistry (decreased vacuolar-type H(+)-ATPase activity) and molecular physiology (decreased nkaα1a and cftrII mRNA abundance) of the gill in a manner indicative of reduced active ion uptake activity. But in control fish and not salt-fed fish, gill mRNA abundance of nkaα1c increased and nbc decreased after IPW exposure. Genes encoding TJ proteins were typically either responsive to salt feeding or IPW, but select genes responded to combined experimental treatment (e.g. IPW responsive but only if fish were salt-fed). Therefore, using salt feeding and IPW exposure, new insights into what factors influence gill TJ proteins and the role that specific TJ proteins might play in regulating the barrier properties of the gill epithelium have been acquired. In particular, evidence suggests that TJ proteins in the gill epithelium, or the regulatory networks that control them, respond independently to external or internal stimuli.

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.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation.

Feeding intravenously saves the patient but starves the gut: consequences for the intestinal barrier

During the late 1960s, the medical breakthrough of providing a patient's complete daily nutritional requirement via intravenous catheter was successfully developed (Dudrick, 2009). When coupled with a growing understanding of the role of optimal nutrition in the recovery of patients unable to consume food, total parenteral nutrition (TPN) has saved numerous lives. However, as with any advanced medical therapy, TPN comes with a number of important complications, many of which are life-threatening. One specific consequence of TPN is gut mucosal atrophy, in which the intestinal mucosa is reduced in thickness because of the importance of luminal nutrients on mucosal growth. Furthermore, there is an associated breakdown in intestinal barrier function, which is critical because the mucosal barrier is the most important line of defence against microorganisms within the intestinal lumen. Bacterial translocation can in turn trigger sepsis, an important cause of mortality in gravely ill patients. However, the mechanisms of intestinal barrier breakdown in patients on TPN are poorly understood. The intestinal mucosal barrier is composed of a single layer of columnar epithelial cells attached side-by-side by a series of interepithelial junctions, including the apical-most tight junctions. Over the last 10–15 years, tight junctions have been the subject of intensive study because although they are ‘tight’ they are also the weakest link in the barrier. Tight junctions are composed of interdigitating extracytoplasmic loops of junctional proteins, including occludin, claudins and junction adhesion molecules. Loss of barrier function at the level of the tight junctions is thought to be a pivotal event in the pathophysiology of a number of important disease syndromes, including infectious diarrhoeal disease, ischaemia/reperfusion injury and inflammatory bowel disease (Hering et al. 2012). However, the mechanisms whereby intestinal barrier function is pathologically altered in intestinal disease are not well defined. In this issue of The Journal of Physiology, Feng & Teitelbaum (2013) have built on their previous findings of barrier disruption in experimental animals on TPN by showing that a complex interplay of tumour necrosis factor (TNFα) and its two receptors (TNFR1 and TNFR2) is responsible for the breakdown of intestinal epithelial tight junctions. Experiments using mutant mice in which TNFR1, TNFR2, or both receptors were knocked out revealed that the absence of either TNF receptor partially prevented the effects of TPN on tight junctions. Additionally, genetic deletion of both TNF receptors prevented any notable effect of TPN on intestinal barrier function. This is interesting not only to researchers studying the mechanisms of TPN-induced barrier disruption, but also to others who have focused on mechanisms whereby cytokines such as TNFα disrupt epithelial barrier function. The TPN model is particularly interesting because it involves selective loss of tight junction integrity independent of inflammatory cell infiltration. In this way, TPN models may allow more precise studies assessing the effect of select factors on tight junction integrity in vivo which are complicated in many models by inflammation. From cell studies, it is well known that cytokines, including TFNα, interferon-γ and interleukin-1-β, can disrupt epithelial barrier function. For example, initial studies assessing the role of cytokines in cell culture models revealed that TNFα dose-dependently reduced epithelial barrier function associated with morphological disruption of tight junctions using electron microscopy (Schmitz et al. 1999). With advancing technology, studies of specific tight junction proteins have shown that TNFα induces endocytosis of occludin as a consequence of activation of the cytoskeletal-associated enzyme myosin light chain kinase (MLCK). Interestingly, Feng et al. noted increased phosphorylation of myosin light chain by MLCK in their TPN mouse model. MLCK has a physiological role in ‘opening’ tight junctions in response to sodium-coupled glucose intestinal epithelial transport, but has more recently been closely linked to pathological states involving disruption of tight junctions. TNFα has also been shown to have alternate mechanisms of barrier disruption. For example, recent studies have shown that TNFα disrupts the highly controlled process of cell shedding of the most superficial surface epithelium resulting in excessive cell loss that can be seen in patients with inflammatory bowel disease (Watson et al. 2012). Such cell loss is also associated with perturbation of tight junctions, allowing cell detachment. Which of the actions of TNFα is critical to loss of barrier function in TPN-treated mice is not entirely clear, although the tight junctions and MLCK are certainly involved. As far as the translational relevance of TNFα in disruption of the gut barrier is concerned, Feng & Teitelbaum (2013) have additionally shown that the TNFα inhibitor etanercept prevents loss of barrier function in TPN-infused mice. Etanercept is a TNFR fusion protein that was originally studied for the treatment of shock (Fisher et al. 1996) and has been marketed for the treatment of select autoimmune diseases. Considering the fact that placement of a patient on TPN is elective, there may be the opportunity to intervene prior to onset of intestinal barrier disruption.

HIV-1 Tat Triggers Nuclear Localization of ZO-1 via Rho Signaling and cAMP Response Element-Binding Protein Activation

The human immunodeficiency virus (HIV)-specific protein trans-activator of transcription (Tat) can contribute to the dysfunction of brain endothelial cells and HIV trafficking into the brain by disrupting tight junction (TJ) integrity at the blood-brain barrier (BBB) level. Specific TJ proteins, such as zonula occludens (ZO) proteins, localize not only at the cell-cell borders but are also present in the nuclei. The objective of the present study was to evaluate the mechanisms and significance of Tat-induced nuclear localization of ZO-1. Treatment of a brain endothelial cell line (hCMEC/D3 cells) with Tat resulted in a decrease in total levels of ZO-1 but significantly upregulated ZO-1 protein expression in the nuclei. In addition, exposure to Tat stimulated Rho signaling and induced phosphorylation and activity of transcription factor cAMP response element-binding protein (CREB), binding sites that have been identified in the proximal region of the ZO-1 promoter. Interestingly, inhibition of the Rho cascade protected against Tat-induced upregulation of ZO-1 in the nuclei and activation of CREB. Depletion of CREB by infection of cells with specific shRNA lentiviral particles attenuated both Tat-induced Rho signaling and nuclear targeting of ZO-1. A decrease in CREB levels also attenuated Tat-induced endothelial and BBB hyperpermeability as well as transendothelial migration of monocytic cells. The role of CREB in Tat-mediated alterations of ZO-1 was confirmed in brain microvessels in mice with CREB shRNA lentiviral particles injected into the cerebral circulation. The present results indicate the crucial role of Rho signaling and CREB in modulation of nuclear localization of ZO-1 and maintaining the integrity of endothelial monolayers.

Maternal glucocorticoid exposure alters tight junction protein expression in the brain of fetal sheep

We examined the expression of tight junction (TJ) proteins in the cerebral cortex, cerebellum, and spinal cord of fetuses after maternal treatment with single and multiple courses of dexamethasone. Ewes received either single courses of four 6-mg dexamethasone or placebo injections every 12 h for 48 h between 104 and 107 days or the same treatment once a week between 76-78 and 104-107 days of gestation. TJ protein expression was determined by Western immunoblot analysis on tissue harvested at 105-108 days of gestation. Blood-brain barrier permeability has been previously quantified with the blood-to-brain transfer constant (K(i)) with alpha-aminoisobutyric acid (39). After a single course of dexamethasone, claudin-5 increased (P < 0.05) in the cerebral cortex, occludin and claudin-1 increased in the cerebellum, and occludin increased in the spinal cord. After multiple dexamethasone courses, occludin and zonula occludens (ZO)-1 increased in the cerebral cortex, and occludin and claudin-1 increased in the cerebellum. Junctional adhesion molecule-A and ZO-2 expressions did not change. Linear regression comparing K(i) to TJ proteins showed inverse correlations with claudin-1 and claudin-5 in the cerebral cortex after a single course and ZO-2 in the spinal cord after multiple courses and direct correlations with ZO-1 in the cerebellum and spinal cord after multiple courses. We conclude that maternal glucocorticoid treatment increases the expression of specific TJ proteins in vivo, patterns of TJ protein expression vary after exposure to single and multiple glucocorticoid courses, and decreases in blood-brain barrier permeability are associated with increases in claudin-1, claudin-5, and ZO-2 expression and decreases in ZO-1 expression. In utero glucocorticoid exposure alters the molecular composition of the barrier and affects fetal blood-brain barrier function.

Displacement of tight junction proteins from detergent-resistant membrane domains by treatment with sodium caprate

We have investigated the effect of sodium salt of capric acid (C10) on major tight junction proteins such as claudin and occludin, and also examined the involvement of lipid rafts with C10-induced alterations on these proteins. We firstly examined the C10 effect on the barrier function of tight junctions by measuring transepithelial electrical resistance (TER) and the flux of FITC dextran 4400 (FD-4). As a result, the increase in the FD-4 flux and decrease in the TER value were observed by incubation with C10 (10mM) for 30min, suggesting loss of the barrier function. In addition, C10 incubation produced an increase in solubility to Triton X-100 for claudin 4, 5 and occludin but not for claudin 1, 2, 3. Since it has been reported lipid raft disruption causes an increase in Triton X-100 solubility, it is suggested that effect of C10 on these proteins are involved with lipid rafts. From the lipid raft isolation study, we clarified the distribution of these proteins in lipid rafts. These results strongly indicate the displacement of specific tight junction proteins, claudin 4, 5 and occludin, from lipid raft by the treatment with C10 and involvement of this displacement with the absorption enhancing mechanism of C10.

Tight Junction Regulation by Morphine and HIV-1 Tat Modulates Blood–Brain Barrier Permeability

Human immunodeficiency virus (HIV)-1 patients who abuse opiates are at a greater risk of developing neurological complications of AIDS. Alterations in blood-brain barrier (BBB) integrity are associated with cytoskeletal disorganization and disruption of tight junction (TJ) integrity. We hypothesize that opiates in combination with HIV-1 viral proteins can modulate TJ expression in primary brain microvascular endothelial cells (BMVEC), thereby compromising BBB integrity and exacerbating HIV-1 neuropathogenesis. We investigated the effect of morphine and/or tat on the expression of TJ proteins ZO-1, JAM-2, Occludin and P-glycoprotein and the functional effects of TJ modulation in BMVEC. Morphine and/or tat, via the activation of pro-inflammatory cytokines, intracellular Ca(2+) release, and activation of myosin light chain kinase, modulated TJ expression resulting in decreased transendothelial electric resistance and enhanced transendothelial migration across the BBB. These studies may lead to the development of novel anti-HIV-1 therapeutics that target specific TJ proteins, thus preventing TJ disruption in opiate using HIV-1 patients.

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2. The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors. Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4-70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling. Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.