scholarly journals Dual Action of the PN159/KLAL/MAP Peptide: Increase of Drug Penetration across Caco-2 Intestinal Barrier Model by Modulation of Tight Junctions and Plasma Membrane Permeability

Pharmaceutics ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 73 ◽  
Author(s):  
Alexandra Bocsik ◽  
Ilona Gróf ◽  
Lóránd Kiss ◽  
Ferenc Ötvös ◽  
Ottó Zsíros ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Efflux Pump ◽  
Intestinal Barrier ◽  
Cell Penetrating Peptides ◽  
Intestinal Epithelial ◽  
Plasma Membrane Permeability ◽  
Cell Penetrating ◽  
Dual Action ◽  
Epithelial Barriers

The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood–brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S25-S25
Author(s):  
Li Zuo ◽  
Feng Cao ◽  
Wei-Ting Kuo ◽  
Jerrold Turner
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Barrier ◽  
Intestinal Epithelial ◽  
Binding Interaction ◽  
Chaperone Protein ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 > 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

scholarly journals Tight Junction in the Intestinal Epithelium: Its Association with Diseases and Regulation by Phytochemicals

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Bonggi Lee ◽  
Kyoung Mi Moon ◽  
Choon Young Kim
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Epithelium ◽  
Essential Role ◽  
Inflammatory Diseases ◽  
Intestinal Barrier ◽  
Nutritional Factors ◽  
Nutritional Interventions ◽  
Intestinal Epithelia ◽  
Pathological Conditions

The intestine plays an essential role in integrating immunity and nutrient digestion and absorption. Adjacent intestinal epithelia form tight junctions (TJs) that are essential to the function of the physical intestinal barrier, regulating the paracellular movement of various substances including ions, solutes, and water across the intestinal epithelium. Studies have shown that TJ dysfunction is highly associated with metabolic and inflammatory diseases. Thus, molecular and nutritional factors that improve TJ activity have gained attention in the pharmaceutical and medicinal fields. This review focuses on the association between TJ and diverse pathological conditions, as well as various molecular and nutritional interventions designed to boost TJ integrity.

scholarly journals Impaired Gut Epithelial Tight Junction Expression in Hemodialysis Patients Complicated with Intradialytic Hypotension

2018 ◽  
Vol 2018 ◽  
pp. 1-6
Author(s):  
Tsai-Kun Wu ◽  
Paik-Seong Lim ◽  
Jong-Shiaw Jin ◽  
Ming-Ying Wu ◽  
Chang-Hsu Chen
Keyword(s):  
Tight Junction ◽  
Intestinal Barrier ◽  
Intradialytic Hypotension ◽  
Intestinal Epithelial ◽  
Advanced Chronic Kidney Disease ◽  
Epithelial Tight Junction ◽  
Group 3 ◽  
Group 2 ◽  
The Impact ◽  
Group 1

Background. There is accumulating evidence pointing to uremia-induced impairment of the intestinal epithelial barrier structure in advanced chronic kidney disease (CKD) and hemodialysis (HD) patients. In this study, the impact of intradialytic hypotension on intestinal barrier integrity is being explored. Methods. Immunohistochemical staining was used to detect the expression of 4 types of tight junction (TJ) proteins such as occludin, zonula occludens-1 (ZO-1), claudin-1, and claudin-4, in colonic samples of a group of patients receiving segmental colectomy. Five patients with nondialysis CKD (group 2), 5 HD patients with intradialytic hypotension (group 3), and 5 non-CKD subjects (group 1) were examined. Results. Both patients’ groups 2 and 3 demonstrated significantly reduced expression of occludin as compared to group 1 (p<0.05 and p<0.01, resp.). Except for claudin-4, expression of all markers of TJ proteins was significantly reduced in patients’ group 3 as compared to control (p<0.01). In addition, decreased expressions of claudin-1 and ZO-1 were also more pronounced in group 3 when compared to group 2. Conclusions. This study extends the earlier finding by demonstrating that dialysis-related hypotension caused even marked depletion of the key protein constituents of the epithelial TJ.

Role of Epithelial Cells in Initiation and Propagation of Intestinal Inflammation. Eliminating the static: tight junction dynamics exposed

2006 ◽  
Vol 290 (4) ◽  
pp. G577-G582 ◽  
Author(s):  
Le Shen ◽  
Jerrold R. Turner
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Intestinal Inflammation ◽  
Intestinal Barrier ◽  
Barrier Properties ◽  
Structural Modifications ◽  
Mucosal Surfaces

Like all mucosal surfaces, the intestine forms a barrier that separates the external environment, i.e., the gut lumen, from the protected internal milieu. The intestinal barrier is formed by the epithelial cells that line the luminal surface. Plasma membranes of these cells prevent free passage of hydrophilic molecules across this barrier but do not seal the space between cells. This function is provided by the tight junction. Each cell is encircled at the apicolateral boundary by the tight junction, which seals the paracellular space. The tight junction does not form a completely impermeant seal, however, because that would prevent paracellular absorption of essential nutrients and ions; intestinal tight junctions are “leaky” and allow solutes to be transported paracellularly according to size and charge. Abundant data are available to demonstrate that barrier properties of tight junctions can be modulated in response to physiological, pharmacological, and pathophysiological stimuli, but the structural modifications responsible for these responses are poorly defined. Recent advances in understanding the role of tight junction dynamics in response to such stimuli are the focus of this review.

scholarly journals Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides

2020 ◽  
Vol 49 (7) ◽  
pp. 2323-2330
Author(s):  
Emilie Mathieu ◽  
Anne-Sophie Bernard ◽  
H. Y. Vincent Ching ◽  
Andrea Somogyi ◽  
Kadda Medjoubi ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Cell Penetrating Peptides ◽  
Inflammatory Activity ◽  
Intestinal Epithelial ◽  
Cell Penetrating ◽  
Anti Inflammatory ◽  
Superoxide Dismutase Mimic ◽  
Superoxide Dismutase Mimics

A superoxide dismutase mimic was functionalized with three peptides: -R9, -RRWWRRWRR or -Fx-r-Fx-K (MPP). They were studied in intestinal epithelial cells in an inorganic cellular chemistry approach: quantification, distribution and bio-activity.

New ways of thinking about (and teaching about) intestinal epithelial function

2008 ◽  
Vol 32 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Kim E. Barrett
Keyword(s):  
Ion Transport ◽  
Tight Junctions ◽  
Hydrogen Exchange ◽  
De Novo ◽  
Intestinal Barrier ◽  
Barrier Properties ◽  
Chloride Secretion ◽  
Sodium Absorption ◽  
Intestinal Epithelial ◽  
The Impact

This article summarizes a presentation made at the Teaching Refresher Course of the American Physiological Society, which was held at the Experimental Biology meeting in 2007. The intestinal epithelium has important ion transport and barrier functions that contribute pivotally to normal physiological functioning of the intestine and other body systems. These functions are also frequently the target of dysfunction that, in turn, results in specific digestive disease states, such as diarrheal illnesses. Three emerging concepts are discussed with respect to ion transport: the complex interplay of intracellular signals that both activate and inhibit chloride secretion; the role of multiprotein complexes in the regulation of ion transport, taking sodium/hydrogen exchange as an example; and acute and chronic regulation of colonic sodium absorption, involving both sodium channel internalization and de novo synthesis of new channels. Similarly, recently obtained information about the molecular components of epithelial tight junctions and the ways in which tight junctions are regulated both in health and disease are discussed to exemplify ways to teach about intestinal barrier properties. Finally, both genetically determined intestinal diseases and those arising as a result of infections and/or inflammation are described, and these can be used as the means to enhance the basic and clinical relevance of teaching about intestinal epithelial physiology as well as the impact that the understanding of such physiology has had on associated therapeutics. The article also indicates, where relevant, how different approaches may be used effectively to teach related concepts to graduate versus medical/professional student audiences.

scholarly journals Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

2008 ◽  
Vol 294 (4) ◽  
pp. G1060-G1069 ◽  
Author(s):  
A. Seth ◽  
Fang Yan ◽  
D. Brent Polk ◽  
R. K. Rao
Keyword(s):  
Hydrogen Peroxide ◽  
Tight Junction ◽  
Map Kinase ◽  
Tight Junctions ◽  
Barrier Function ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Cell Monolayers ◽  
Dependent Mechanism

Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and β-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCβI and PKCε. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

scholarly journals Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers

2017 ◽  
Vol 8 ◽  
pp. 1396-1406 ◽  
Author(s):  
Dong Ye ◽  
Mattia Bramini ◽  
Delyan R Hristov ◽  
Sha Wan ◽  
Anna Salvati ◽  
...  
Keyword(s):  
Silica Nanoparticles ◽  
Intestinal Epithelial Cells ◽  
Intestinal Barrier ◽  
Silica Nanoparticle ◽  
Intestinal Epithelial ◽  
Lung Epithelium ◽  
Imaging Flow Cytometry ◽  
Transport Studies ◽  
Epithelial Barriers

Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.

scholarly journals Exploring Effects of Chitosan Oligosaccharides on the DSS-Induced Intestinal Barrier Impairment In Vitro and In Vivo

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2199
Author(s):  
Yujie Wang ◽  
Rong Wen ◽  
Dongdong Liu ◽  
Chen Zhang ◽  
Zhuo A. Wang ◽  
...  
Keyword(s):  
Tight Junction ◽  
Structural Characteristics ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Chitosan Oligosaccharide ◽  
Intestinal Epithelial ◽  
Mucus Layer ◽  
Intestinal Mucus ◽  
Epithelial Tight Junction

Intestinal barrier dysfunction is an essential pathological change in inflammatory bowel disease (IBD). The mucus layer and the intestinal epithelial tight junction act together to maintain barrier integrity. Studies showed that chitosan oligosaccharide (COS) had a positive effect on gut health, effectively protecting the intestinal barrier in IBD. However, these studies usually focused on its impact on the intestinal epithelial tight junction. The influence of COS on the intestinal mucus layer is still poorly understood. In this study, we explored the effect of COS on intestinal mucus in vitro using human colonic mucus-secreted HT-29 cells. COS relieved DSS (dextran sulfate sodium)-induced mucus defects. Additionally, the structural characteristics of COS greatly influenced this activity. Finally, we evaluated the protective effect of COS on intestinal barrier function in mice with DSS-induced colitis. The results indicated that COS could manipulate intestinal mucus production, which likely contributed to its intestinal protective effects.

scholarly journals OP0245 MICROBIOTA-INDUCED INTESTINAL BARRIER DYSFUNCTION PRECEDES THE ONSET OF ARTHRITIS AND ALLOWS THE SHUTTLING OF IMMUNE CELLS FROM THE GUT THE JOINTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 154.2-154
Author(s):  
M. Zaiss ◽  
N. Taijc ◽  
K. Sarter ◽  
V. Azizov ◽  
L. Bucci ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Immune Cells ◽  
Barrier Function ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Barrier Dysfunction ◽  
Intestinal Barrier Dysfunction ◽  
Microbial Dysbiosis ◽  
Intestinal Dysbiosis

Background:While it is known that microbial dysbiosis is associated with the onset of rheumatoid arthritis, mechanistic insights how it facilitates the development of arthritis remained largely elusive to date. It is especially interesting how microbial dysbiosis affects the transition from asymptomatic autoimmunity to arthritis. We speculated that a breakdown of intestinal barrier function caused by microbial dysbiosis allows immune cells to shuttle from the gut to the joints.Objectives:To test whether intestinal barrier function is impaired before the onset of human RA and experimental arthritis and to seek for evidence that immune cells from the gut migrate to the joints.Methods:In a longitudinal cohort of RA-at risk individuals markers of disturbed intestinal barrier function, such as zonulin, were analysed and linked to RA onset. Furthermore, new-onset RA patients were assessed for gut leakiness and their intestinal biopsies for the expression of tight junction proteins and immune cell infiltration. In the murine model of collagen-induced arthritis, sequential analysis of intestinal dysbiosis, intestinal barrier function and arthritis onset was carried out. Additionally, barrier function was assessed on intestinal organoids exposed to faecal supernatants from eu- and dysbiotic mice with and without inhibition of zonulin. Furthermore, three types of interventions restoring intestinal barrier function were carried out for testing their effects on the inhibition of arthritis onset. Finally, photo- converted cells from the gut were traced in the joints to test for cellular trafficking from one to the other compartment.Results:Zonulin, a potent regulator for intestinal tight junctions, was elevated in autoimmune mice and men before the onset of arthritis and predicted the onset of human RA. Intestinal barrier functions as well as epithelial tight junctions were decreased before the onset of experimental arthritis and at onset of human RA. In mice, induction of autoimmunity was followed by rapid intestinal dysbiosis followed by gut leakiness before arthritis started. Faecal supernatants of arthritic mice induce epithelial barrier dysfunction in intestinal organoids in zonulin dependent manner. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate, CB1R agonist or zonulin antagonist larazotide inhibited the development of arthritis. Finally, using photoconvertible mice, gut-borne immune cells were identified that homed to the joints when barrier function was impaired.Conclusion:In summary, these data show the intestinal barrier dysfunction precedes the onset of RA and allows the trafficking of immune cells from the gut to the joints. Targeting of intestinal tight junction function may therefore allow preventing the onset of RA.Acknowledgments:Funded by the DFG-FOR2886 PANDORA, DFG–CRC118, Staedtler foundation, Johannes und Frieda Marohn-Stiftung, Else Kröner-Fresenius foundation, Interdisciplinary Centre for Clinical Research, Erlangen (IZKF), BMBF-MASCARA and the IMI funded projectRTCure.Disclosure of Interests:Mario Zaiss: None declared, Narges Taijc: None declared, Kerstin Sarter: None declared, Vugar Azizov: None declared, laura Bucci: None declared, Yubin Luo: None declared, Juan de Dios Cañete: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB

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