Ginsenoside Re improves skin barrier function in HaCaT keratinocytes under normal growth conditions

Ginsenoside Re (Re), a major ginsenoside of ginseng, enhanced the cornified cell envelope (CE) formation in HaCaT keratinocytes under normal conditions. In HaCaT keratinocytes, Re was also able to upregulate filaggrin protein and caspase-14 activity in a concentration-dependent manner. These findings reasonably imply that Re possesses a desirable property of improving skin barrier function.     

Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-, -, and - (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC- inhibitor röttlerin, implicating a novel isozyme, likely PKC-. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC--dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex. protein kinase C; epithelial barrier function     

Ceramide biosynthesis in keratinocyte and its role in skin function

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1–CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.     

Uric acid modulates vascular endothelial function through the down regulation of nitric oxide production

Abstract

Endothelial dysfunction characterized by decreased nitric oxide (NO) bioavailability is the first stage of coronary artery disease. It is known that one of the factors associated with an increased risk of coronary artery disease is a high plasma level of uric acid. However, causative associations between hyperuricaemia and cardiovascular risk have not been definitely proved. In this work, we tested the effect of uric acid on endothelial NO bioavailability. Electrochemical measurement of NO production in acetylcholine-stimulated human umbilical endothelial cells (HUVECs) revealed that uric acid markedly decreases NO release. This finding was confirmed by organ bath experiments on mouse aortic segments. Uric acid dose-dependently reduced endothelium-dependent vasorelaxation. To reveal the mechanism of decreasing NO bioavailability we tested the effect of uric acid on reactive oxygen species production by HUVECs, on arginase activity, and on acetylcholine-induced endothelial NO synthase phosphorylation. It was found that uric acid increases arginase activity and reduces endothelial NO synthase phosphorylation. Interestingly, uric acid significantly increased intracellular superoxide formation. In conclusion, uric acid decreases NO bioavailability by means of multiple mechanisms. This finding supports the idea of a causal association between hyperuricaemia and cardiovascular risk.     

Restriction of sulfur-containing amino acids alters claudin composition and improves tight junction barrier function

Restriction of sulfur-containing amino acids (SCAA) has been shown to elicit a similar increase in life span and decrease in age-related morbidity as caloric restriction. The singular importance of epithelial barrier function in both physiological homeostasis and prevention of inflammation raised the issue of examining the effect of SCAA restriction on epithelial tight junction structure and permeability. Using a well-described in vitro, epithelial model, the LLC-PK(1) renal epithelial cell line, we studied the effects of SCAA restriction in culture medium. Reduction of methionine by 90%, cysteine by 50%, and total elimination of cystine resulted in dramatically lower intracellular pools of these amino acids and their metabolite, taurine, but the intracellular pools of the non-SCAA were all elevated. Cell growth and differentiation were maintained, and both confluent cell density and transepithelial short circuit current were unaffected. Certain tight junctional proteins, such as occludin and claudins-1 and -2 were not altered. However, claudins-3 and -7 were significantly decreased in abundance, whereas claudins-4 and -5 were markedly increased in abundance. The functional result of these structural changes was improved barrier function, as evidenced by increased transepithelial electrical resistance and decreased transepithelial (paracellular) diffusion of D-mannitol.     

A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn^−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers–Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn^−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn^−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X = 4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn^−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn^−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn^−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn^−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn^−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn^−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn^−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn^−/− CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn^−/− CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn^−/− mice and possibly Ehlers–Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.     

CXCR4 antagonist AMD3100 modulates claudin expression and intestinal barrier function in experimental colitis

Ulcerative colitis is a gastrointestinal disorder characterized by local inflammation and impaired epithelial barrier. Previous studies demonstrated that CXC chemokine receptor 4 (CXCR4) antagonists could reduce colonic inflammation and mucosal damage in dextran sulfate sodium (DSS)-induced colitis. Whether CXCR4 antagonist has action on intestinal barrier and the possible mechanism, is largely undefined. In the present study, the experimental colitis was induced by administration of 5% DSS for 7 days, and CXCR4 antagonist AMD3100 was administered intraperitoneally once daily during the study period. For in vitro study, HT-29/B6 colonic cells were treated with cytokines or AMD3100 for 24 h until assay. DSS-induced colitis was characterized by morphologic changes in mice. In AMD3100-treated mice, epithelial destruction, inflammatory infiltration, and submucosal edema were markedly reduced, and the disease activity index was also significantly decreased. Increased intestinal permeability in DSS-induced colitis was also significantly reduced by AMD3100. The expressions of colonic claudin-1, claudin-3, claudin-5, claudin-7 and claudin-8 were markedly decreased after DSS administration, whereas colonic claudin-2 expression was significantly decreased. Treatment with AMD3100 prevented all these changes. However, AMD3100 had no influence on claudin-3, claudin-5, claudin-7 and claudin-8 expression in HT-29/B6 cells. Cytokines as TNF-α, IL-6, and IFN-γ increased apoptosis and monolayer permeability, inhibited the wound-healing and the claudin-3, claudin-7 and claudin-8 expression in HT-29/B6 cells. We suggest that AMD3100 acted on colonic claudin expression and intestinal barrier function, at least partly, in a cytokine-dependent pathway.     

AhR activation protects intestinal epithelial barrier function through regulation of Par-6

The Par complex (Par-6/Par-3/aPKC) plays a key role in the maintenance of the intestinal barrier function through the regulation of epithelial junction formation. The aryl hydrocarbon receptor (AhR) has been shown to be an important regulator for intestinal homeostasis. In this study, we investigated the role of the AhR activation on the regulation of Par complex. AhR activation by 6-formylindolo (3,2-b) carbazole (FICZ) represses the abnormal expression of the Par complex in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In T84 cells, overexpression of Par-6 causes intestinal barrier dysfunction. Lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction and increase in Par-6 expression was prevented by AhR activation. However, FICZ did not alter the expression of Par-3 or aPKC. Furthermore, AhR activation alleviated LPS-induced increase of Par-6 through repressing the expression of activating protein-2γ (Ap-2γ). These results reveal the protective effects of AhR activation on LPS induced disruption of intestinal epithelial barrier function through suppressing the expression of Par-6 expression. Our findings provide novel insights into the protective role of AhR in intestinal barrier function.     

The epidermal barrier function is dependent on the serine protease CAP1/Prss8

Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival.     

Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor

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Rho GTPases in the regulation of pulmonary vascular barrier function

Pulmonary endothelial permeability is an important determinant of vascular adaptation to changes in oxygen tension, blood pressure, levels of growth factors or inflammatory cytokines. The Ras homologous (Rho) family of guanosine triphosphate phosphatases (Rho GTPases), key regulators of the actin cytoskeleton, regulate endothelial barrier function in response to a variety of environmental factors and signalling agents via the reorganization of the actin cytoskeleton, changes in receptor trafficking or the phosphorylation of junctional proteins. This review provides a brief summary of recent knowledge on Rho-GTPase-mediated effects on pulmonary endothelial barrier function and focuses in particular on their role in pulmonary vascular disorders, including pulmonary hypertension, chronic obstructive pulmonary disease, acute lung injury and acute respiratory distress syndrome.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease.

Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by Ecto-5'-nucleotidase/CD73-dependent mechanism

Extracellular purines are important signaling molecules that mediate both inflammatory (ATP, ADP) and anti-inflammatory (adenosine) effects in the vasculature. The duration and magnitude of purinergic signaling is governed by a network of purine-converting ectoenzymes, and endothelial and lymphoid cells are generally characterized by counteracting ATP-inactivating and ATP-regenerating/adenosine-eliminating, phenotypes, respectively. By using cultured human umbilical vein endothelial cells and normal or leukemic lymphocytes as an in vitro model of leukocyte-endothelial interactions, we have identified a link between the adhesion cascade and extracellular purine turnover. Upon adhesion, lymphocytes suppress endothelial purine metabolism via (i) inhibition of ecto-5'-nucleotidase/CD73-mediated AMP hydrolysis, (ii) rapid deamination of the remaining adenosine, and (iii) maintenance of the sustained pericellular ATP level through continuous nucleotide release and phosphotransfer reactions. Compensation of the loss of adenosine promotes vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran) and decreases transendothelial leukocyte migration. Together, these data show that adherent lymphocytes attempt to prevent adenosine formation in the endothelial environment that, as a consequence, may impair the vascular barrier function and facilitate the subsequent step of leukocyte transmigration into the tissue. These leukocyte adhesion-mediated shifts in the local nucleotide and nucleoside concentrations represent a previously unrecognized paracrine mechanism affecting the functional state of the targeted vascular endothelium and coordinately regulating lymphocyte trafficking between the blood and tissues.     

Involvement of the junctional adhesion molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function

Junctional adhesion molecule-1 (JAM1) is a tight junction-associated immunoglobulin superfamily protein implicated in the regulation of tight junctions and leukocyte transmigration. The structural basis for the function of JAM1 has yet to be determined. Here we provide evidence that JAM1 homodimer formation is important for its function in epithelial cells. Experiments were conducted to determine the effects of a panel of JAM1 monoclonal antibodies on epithelial barrier recovery after transient disruption by calcium switch. Two monoclonal antibodies were observed to inhibit barrier recovery in contrast to another monoclonal antibody that had no effect. Epitope mapping by phage display revealed that both inhibitory antibodies bind to a region of JAM1 located within the N-terminal Ig-like loop (residues 111-123). Competition experiments with synthetic peptides and site-directed mutagenesis confirmed the location of this epitope. Analysis of the crystal structure of JAM1 revealed that this epitope includes residues within the putative homodimer interface, and one of the two inhibitory antibodies was then shown to block JAM1 homodimer formation in vitro. Finally, mutations within the homodimer interface were shown to prevent enrichment of JAM1 at points of cell contact, presumably by interference with homophilic interactions. These findings suggest that homodimer formation may be important for localization of JAM1 at tight junctions and for regulation of epithelial barrier function.     

Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line,HaCaT

Interleukin 15 (IL-15) is a potent stimulator of proliferation and an inhibitor of apoptosis in lymphocytes. We attempted to elucidate the mechanism of IL-15 function in HaCaT keratinocytes. We found that 5-bromo-2(')-deoxyuridine incorporation increased in a dose-dependent manner with IL-15. This was blocked by MEK inhibitor U0126 or PI 3-K inhibitor LY294002. ERK1/2 and Akt phosphorylation by IL-15 were detected in a dose- and time-dependent manner. U0126 and LY294002 abolished ERK1/2 and Akt phosphorylation, respectively. DNA fragmentation and Annexin V binding accompanied by UVB-induced apoptosis were reduced by 30-50% with IL-15. Taken together, IL-15 induced cellular proliferation and had an anti-apoptotic effect on keratinocytes, in which ERK1/2 and Akt phosphorylation played crucial roles. The signal transduction pathways of IL-15 in keratinocytes were partially elucidated; they share a substantial part with growth signals induced by EGF. These results suggest a therapeutic approach to inflammatory skin diseases by controlling these signals.     

Role of microtubule cytoskeleton in regulation of endothelial barrier function

Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.