Effect of estradiol and dihydrotestosterone on hypergravity-induced MAPK signaling and occludin expression in human umbilical vein endothelial cells

Female astronauts have been reported to have a higher incidence of post-flight orthostatic intolerance (POI) compared with that of their male counterparts. POI may result from increased permeability of the endothelial cell (EC) layer in the vasculature. The goal of this study has been to determine whether estradiol (E₂) and dihydrotesterone (DHT) alter human umbilical vein ECs (HUVECs) responses to short term (10 min) hypergravity (1–3 g) mimicking the g force experienced by astronauts during liftoff. E₂ and DHT rapidly (within 5 min) activated MAPK (mitogen-activated protein kinase) in HUVEC at 1 g in a receptor-dependent manner. Liftoff inhibited MAPK phosphorylation, and rapid E₂ and DHT activation of MAPK was blocked. Liftoff simulation or brief (5–90 min) treatment with E₂ or DHT at 1 g had no effect on the expression of the EC tight-junction protein occludin. However, 24-h pre-treatment of HUVECs with E₂ and DHT prior to liftoff simulation significantly increased occludin expression, and hypergravity exposure did not alter this increase. These data provide evidence for a possible protective effect of E₂ and DHT on EC function as indicated by increased occludin; this may help maintain the integrity of EC tight junction and could thus retard or reduce the incidence of POI.This work was supported by NASA grant NAG5-12874 to C.M.K. and R.S.K. and by American Heart Association Ohio Valley Affiliate grant 0555270B to C.M.K. The American Heart Association Ohio Valley supported W.K.S. with an affiliate post-doctoral fellowship (0425431B).     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Expression, mitogenic activity and regulation by growth hormone of growth hormone/insulin-like growth factor in Branchiostoma belcheri

Our aim has been to characterize the molecular mechanisms regulating the expression of the channel-forming tight-junctional protein claudin-2 in response to the pro-inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated, for example, in active Crohn’s disease. TNFα caused an 89% decrease of the paracellular resistance in colonic HT-29/B6 cells, whereas transcellular resistance was unaltered. The claudin-2 protein level was increased by TNFα without changes in subcellular tight-junctional protein localization as revealed by confocal laser scanning microscopy. Enhanced gene expression was identified as the source of this increase, since claudin-2-specific mRNA and promoter activity was elevated, whereas mRNA stability remained unaltered. Specific inhibitors and phospho-specific antibodies revealed that the increased gene expression of claudin-2 after TNFα treatment was mediated by the phosphatidylinositol-3-kinase pathway. Thus, the up-regulation of claudin-2 by TNFα is attributable to the regulation of the expression of the gene, as a result of which epithelial barrier function is disturbed, for example, during chronic intestinal inflammation. J. Mankertz and M. Amasheh contributed equally to this work. This work was supported by the Deutsche Forschungsgemeinschaft and the Sonnenfeld-Stiftung Berlin.     

Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung

In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7–11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, β-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, β-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-α from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the BMVg Grant E/B41G/1G302/1A402 and the 6th Framework program of the European Union, NanoBioPharmaceutics.     

TGF-β3 and TNFα perturb blood-testis barrier (BTB) dynamics by accelerating the clathrin-mediated endocytosis of integral membrane proteins: A new concept of BTB regulation during spermatogenesis

In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must “open” transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-β3 (TGF-β3) and tumor necrosis factor α (TNFα) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-β3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-β3 (3 ng/ml) and TNFα (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 μM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-β3-induced protein internalization. This inhibition of TGF-β3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-β3 on protein internalization was further confirmed by RNAi using specific TGF-β receptor I (TβR1) siRNA duplexes. When TβR1 was knocked down, the TGF-β3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-β3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-β3 and TNFα via an enhancement of protein endocytosis at the BTB.     

Loss of tight junction barrier function and its role in cancer metastasis

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.     

NHS-A isoform of the NHS gene is a novel interactor of ZO-1

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.     

Tight junctions as targets of infectious agents

The epithelial barrier is a critical border that segregates luminal material from entering tissues. Essential components of this epithelial fence are physical intercellular structures termed tight junctions. These junctions use a variety of transmembrane proteins coupled with cytoplasmic adaptors, and the actin cytoskeleton, to attach adjacent cells together thereby forming intercellular seals. Breaching of this barrier has profound effects on human health and disease, as barrier deficiencies have been linked with the onset of inflammation, diarrhea generation and pathogenic effects. Although tight junctions efficiently restrict most microbes from penetrating into deeper tissues and contain the microbiota, some pathogens have developed specific strategies to alter or disrupt these structures as part of their pathogenesis, resulting in either pathogen penetration, or other consequences such as diarrhea. Understanding the strategies that microorganisms use to commandeer the functions of tight junctions is an active area of research in microbial pathogenesis. In this review we highlight and overview the tactics bacteria and viruses use to alter tight junctions during disease. Additionally, these studies have identified novel tight junction protein functions by using pathogens and their virulence factors as tools to study the cell biology of junctional structures.     

Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-β-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an ∼80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-β-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-β-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-β-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-β-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and  48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.