Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12 mM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50 mM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50 mM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24 h of exposure to 50 mM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10 nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins

Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin

Strains of Bacteroides fragilis associated with diarrheal disease (enterotoxigenic B. fragilis) produce a 20-kDa zinc-dependent metalloprotease toxin (B. fragilis enterotoxin; BFT) that reversibly stimulates chloride secretion and alters tight junctional function in polarized intestinal epithelial cells. BFT alters cellular morphology and physiology most potently and rapidly when placed on the basolateral membrane of epithelial cells, suggesting that the cellular substrate for BFT may be present on this membrane. Herein, we demonstrate that BFT specifically cleaves within 1 min the extracellular domain of the zonula adherens protein, E-cadherin. Cleavage of E-cadherin by BFT is ATP-independent and essential to the morphologic and physiologic activity of BFT. However, the morphologic changes occurring in response to BFT are dependent on target-cell ATP. E-cadherin is shown here to be a cellular substrate for a bacterial toxin and represents the identification of a mechanism of action, cell-surface proteolytic activity, for a bacterial toxin.

Toll-Interacting Protein Deficiency Leads to Increased Susceptibility to Acute and Chronic Colitis.

BACKGROUND: Sensing of bacterial products via Toll-like receptors is critical to maintain gut immune homeostasis. The Toll-Interacting Protein (Tollip) inhibits downstream signaling through the IL-1 receptor, TLR-2 and TLR-4. Here,we aimed to address the role of Tollip in acute and chronic inflammatory responses in the gut. MATERIAL AND METHODS: WT or Tollip-deficient mice were exposed to dextran sulfate sodium (DSS) 1.5% in the drinking water during 7 days. To generate bone-marrow chimeras, WT or Tollip deficient mice were 900-rads irradiated, transplanted with WT or Tollip deficient bone-marrow cells and challenged with DSS 2-3 months after transplantation. IL-10 deficient mice were bred with Tollip deficient mice and colitis was compared at various time points. RESULTS: Upon DSS exposure, Tollip-deficient mice had increased body weight loss and increased pro-inflammatory cytokine expression compared to WT controls. Challenge of bone-marrow chimeras showed that colitis susceptibility was also increased when Tollip deficiency was restricted to non-hematopoietic cells. DSS-exposure lead to a disorganized distribution of zona-occludens-1, a tight junction marker and increased number of apoptotic, cleaved caspase 3 positive, epithelial cells in Tollip-deficient compared to WT mice. Chronic colitis was also affected by Tollip deficiency as Tollip/IL-10 deficient mice had more severe histological stigmata of colitis and higher IL-17 expression than IL-10 deficient controls. CONCLUSION: Tollip in non-hematopoietic cells is critical for adequate response to a chemical-induced stress in the gut and to hamper chronic bacteria-driven colitis. Modulation of epithelial cell integrity via Tollip likely contributes to the observed defects.

MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus.

In the glomeruli of the granule cell layer of mammalian cerebellum, neuronal extensions are interconnected by numerous small, nearly isodiametric (diameters up to 0.1 micron), junctions previously classified as puncta adherentia related to the vinculin-containing, actin microfilament-anchoring junctions of the zonula adherens of epithelial and certain other cells. Using immunofluorescence and immunoelectron microscopy, we have found, however, that these junctions are negative for E- and VE-cadherin, for desmosomal cadherins, and also for vinculin, alpha-actinin, and desmoplakin, but they do contain, in addition to the protein plakoglobin common to all forms of adhering junctions, the plaque proteins alpha- and beta-catenin and the transmembrane glycoprotein M-cadherin previously found as a spread--i.e., not junction bound--plasma membrane protein in certain fetal and regenerating muscle cells and in satellite cells of adult skeletal muscle. We conclude that these M-cadherin-containing junctions of the granule cell layer represent a special type of adhering junction, for which we propose the term contactus adherens (from the Latin contactus, for touch, site of bordering upon, also influence), and we discuss the differences between the various adhering junctions on the basis of their molecular constituents.

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.