{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T14:06:54Z","timestamp":1787580414491,"version":"build-2736575974"},"reference-count":53,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2021,8,14]],"date-time":"2021-08-14T00:00:00Z","timestamp":1628899200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["AM141\/11-2"],"award-info":[{"award-number":["AM141\/11-2"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Tumor necrosis factor alpha (TNF\u03b1) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNF\u03b1 in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U\/mL TNF\u03b1 induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNF\u03b1 signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNF\u03b1 mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.<\/jats:p>","DOI":"10.3390\/ijms22168746","type":"journal-article","created":{"date-parts":[[2021,8,15]],"date-time":"2021-08-15T21:43:55Z","timestamp":1629063835000},"page":"8746","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0276-2773","authenticated-orcid":false,"given":"Linda","family":"Droessler","sequence":"first","affiliation":[{"name":"Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0516-3963","authenticated-orcid":false,"given":"Valeria","family":"Cornelius","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2867-044X","authenticated-orcid":false,"given":"Alexander G.","family":"Markov","sequence":"additional","affiliation":[{"name":"Department of General Physiology, Faculty of Biology, Saint Petersburg State University, Universitetskaya nab., 7\u20139, 199034 Saint Petersburg, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Salah","family":"Amasheh","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1038\/35067088","article-title":"Multifunctional strands in tight junctions","volume":"2","author":"Tsukita","year":"2001","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1083\/jcb.142.1.117","article-title":"Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration","volume":"142","author":"Lostaglio","year":"1998","journal-title":"J. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1016\/j.febslet.2011.01.028","article-title":"Predicted expansion of the claudin multigene family","volume":"585","author":"Mineta","year":"2011","journal-title":"FEBS Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1007\/s00360-009-0440-7","article-title":"Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine","volume":"180","author":"Markov","year":"2010","journal-title":"J. Comp. Physiol. B"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1111\/j.1749-6632.2009.04062.x","article-title":"Epithelial tight junctions in intestinal inflammation","volume":"1165","author":"Schulzke","year":"2009","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1038\/ncpgasthep0528","article-title":"Mechanisms of disease: Pathogenesis of Crohn\u2019s disease and ulcerative colitis","volume":"3","author":"Sartor","year":"2006","journal-title":"Nat. Clin. Pract. Gastroenterol. Hepatol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1136\/gut.40.4.443","article-title":"Tumour necrosis factor and Crohn\u2019s disease","volume":"40","year":"1997","journal-title":"Gut"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4145","DOI":"10.1242\/jcs.070896","article-title":"TNFalpha-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFkappaB signaling","volume":"123","author":"Amasheh","year":"2010","journal-title":"J. Cell Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1007\/s002329900333","article-title":"Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line","volume":"161","author":"Marano","year":"1998","journal-title":"J. Membr. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1080\/00365520903131973","article-title":"Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines","volume":"44","author":"Amasheh","year":"2009","journal-title":"Scand. J. Gastroenterol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1152\/physrev.00019.2012","article-title":"Claudins and the modulation of tight junction permeability","volume":"93","author":"Yu","year":"2013","journal-title":"Physiol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1083\/jcb.200902153","article-title":"Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo","volume":"189","author":"Marchiando","year":"2010","journal-title":"J. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1111\/j.1749-6632.2012.06526.x","article-title":"Myosin light chain kinase: Pulling the strings of epithelial tight junction function","volume":"1258","author":"Cunningham","year":"2012","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Jin, Y., and Blikslager, A.T. (2020). The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase\/Rho Kinases. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21103550"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"G422","DOI":"10.1152\/ajpgi.00412.2004","article-title":"Mechanism of TNF-\u03b1 modulation of Caco-2 intestinal epithelial tight junction barrier: Role of myosin light-chain kinase protein expression","volume":"288","author":"Ma","year":"2005","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"292","DOI":"10.3389\/fphar.2017.00292","article-title":"Myosin Light Chain Kinase: A Potential Target for Treatment of Inflammatory Diseases","volume":"23","author":"Xiong","year":"2017","journal-title":"Front. Pharmacol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"392","DOI":"10.3892\/mmr.2021.12031","article-title":"Phosphorylated-myosin light chain mediates the destruction of small intestinal epithelial tight junctions in mice with acute liver failure","volume":"23","author":"Wu","year":"2021","journal-title":"Mol. Med. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"He, W.-Q., Wang, J., Sheng, J.-Y., Zha, J.-M., Graham, W.V., and Turner, J.R. (2020). Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21030993"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1007\/s00418-005-0067-z","article-title":"Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine","volume":"125","author":"Schierack","year":"2006","journal-title":"Histochem. Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Zakrzewski, S.S., Richter, J.F., Krug, S.M., Jebautzke, B., Lee, I.F., Rieger, J., Sachtleben, M., Bondzio, A., Schulzke, J.D., and Fromm, M. (2013). Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0079643"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1177\/0300985811402846","article-title":"Swine as models in biomedical research and toxicology testing","volume":"49","author":"Swindle","year":"2012","journal-title":"Vet. Pathol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1017\/S0954422410000077","article-title":"Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?","volume":"23","author":"Guilloteau","year":"2010","journal-title":"Nutr. Res. Rev."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2802","DOI":"10.1007\/s10620-011-1688-9","article-title":"Increase in the tight junction protein claudin-1 in intestinal inflammation","volume":"56","author":"Poritz","year":"2011","journal-title":"Dig. Dis. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1590\/S0100-879X2010007500020","article-title":"Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells","volume":"43","author":"Cui","year":"2010","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"G367","DOI":"10.1152\/ajpgi.00173.2003","article-title":"TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation","volume":"286","author":"Ma","year":"2004","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1111\/j.1749-6632.2000.tb05242.x","article-title":"Epithelial barrier defects in HT-29\/B6 colonic cell monolayers induced by tumor necrosis factor-alpha","volume":"91","author":"Gitter","year":"2000","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1242\/jcs.112.1.137","article-title":"Tumor necrosis factor-alpha (TNF\u03b1) regulates the epithelial barrier in the human intestinal cell line HT-29\/B6","volume":"112 Pt 1","author":"Schmitz","year":"1999","journal-title":"J. Cell Sci."},{"key":"ref_28","first-page":"3217","article-title":"Integrins and epithelial cell polarity","volume":"127 Pt 15","author":"Lee","year":"2014","journal-title":"J. Cell Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/sj.cdd.4401189","article-title":"Tumor necrosis factor signaling","volume":"10","author":"Wajant","year":"2003","journal-title":"Cell Death Differ."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1192","DOI":"10.1038\/cdd.2009.51","article-title":"PUMA is directly activated by NF-kappaB and contributes to TNF-alpha-induced apoptosis","volume":"16","author":"Wang","year":"2009","journal-title":"Cell Death Differ."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1111\/j.1749-6632.2012.06502.x","article-title":"Barrier dysfunction and bacterial uptake in the follicle-associated epithelium of ileal Crohn\u2019s disease","volume":"1258","author":"Keita","year":"2012","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1111\/apha.12559","article-title":"Claudin expression in follicle-associated epithelium of rat Peyer\u2019s patches defines a major restriction of the paracellular pathway","volume":"216","author":"Markov","year":"2016","journal-title":"Acta Physiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1093\/rheumatology\/keq031","article-title":"Transmembrane TNF-alpha: Structure, function and interaction with anti-TNF agents","volume":"49","author":"Horiuchi","year":"2010","journal-title":"Rheumatology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1136\/gut.2006.094375","article-title":"Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn\u2019s disease","volume":"56","author":"Zeissig","year":"2007","journal-title":"Gut"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"705485","DOI":"10.3389\/fimmu.2021.705485","article-title":"The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation","volume":"12","author":"Richter","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"401","DOI":"10.3389\/fcell.2020.00401","article-title":"Selective Targeting of TNF Receptors as a Novel Therapeutic Approach","volume":"8","author":"Fischer","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1924","DOI":"10.1159\/000472367","article-title":"Rebeccamycin Attenuates TNF-alpha-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production","volume":"41","author":"Watari","year":"2017","journal-title":"Cell Physiol. Biochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1042\/bj20021535","article-title":"The specificities of protein kinase inhibitors: An update","volume":"371 Pt 1","author":"Bain","year":"2003","journal-title":"Biochem. J."},{"key":"ref_39","first-page":"6293","article-title":"ML-7 attenuates airway inflammation and remodeling via inhibiting the secretion of Th2 cytokines in mice model of asthma","volume":"17","author":"Huang","year":"2018","journal-title":"Mol. Med. Rep."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1111\/j.1749-6632.2012.06528.x","article-title":"Dynamic properties of the tight junction barrier","volume":"1257","author":"Weber","year":"2012","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1002\/cpz1.143","article-title":"Electrophysiologic Analysis of Tight Junction Size and Charge Selectivity","volume":"1","author":"Shashikanth","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1161\/CIRCRESAHA.107.161687","article-title":"Identification of cardiac-specific myosin light chain kinase","volume":"102","author":"Chan","year":"2008","journal-title":"Circ. Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"12580","DOI":"10.1038\/s41598-019-48884-w","article-title":"Cardiac MLC2 kinase is localized to the Z-disc and interacts with \u03b1-actinin2","volume":"9","author":"Cai","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1177\/1753425917690815","article-title":"TGF-beta1 protects intestinal integrity and influences Smads and MAPK signal pathways in IPEC-J2 after TNF-alpha challenge","volume":"23","author":"Xiao","year":"2017","journal-title":"Innate Immun."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/S0022-4804(03)00311-1","article-title":"Tumor necrosis factor alpha disrupts tight junction assembly","volume":"116","author":"Poritz","year":"2004","journal-title":"J. Surg. Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"C1191","DOI":"10.1152\/ajpcell.00144.2008","article-title":"Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line","volume":"295","author":"Baker","year":"2008","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1007\/s00441-009-0751-8","article-title":"TNFalpha up-regulates claudin-2 expression in epithelial HT-29\/B6 cells via phosphatidylinositol-3-kinase signaling","volume":"336","author":"Mankertz","year":"2009","journal-title":"Cell Tissue Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"C38","DOI":"10.1152\/ajpcell.00388.2014","article-title":"Tumor necrosis factor-alpha induces a biphasic change in claudin-2 expression in tubular epithelial cells: Role in barrier functions","volume":"309","author":"Amoozadeh","year":"2015","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Zhang, C., Yan, J., Xiao, Y., Shen, Y., Wang, J., Ge, W., and Chen, Y. (2017). Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-alpha Treated Cell Monolayers. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18010157"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1913","DOI":"10.1242\/jcs.060665","article-title":"Claudin-2, a component of the tight junction, forms a paracellular water channel","volume":"123","author":"Rosenthal","year":"2010","journal-title":"J. Cell Sci."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Radloff, J., Cornelius, V., Markov, A.G., and Amasheh, S. (2019). Caprate Modulates Intestinal Barrier Function in Porcine Peyer\u2019s Patch Follicle-Associated Epithelium. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20061418"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"579","DOI":"10.3389\/fphys.2017.00579","article-title":"Molecular Characterization of Barrier Properties in Follicle-Associated Epithelium of Porcine Peyer\u2019s Patches Reveals Major Sealing Function of Claudin-4","volume":"8","author":"Radloff","year":"2017","journal-title":"Front. Physiol."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Markov, A.G., Fedorova, A.A., Kravtsova, V.V., Bikmurzina, A.E., Okorokova, L.S., Matchkov, V.V., Cornelius, V., Amasheh, S., and Krivoi, I.I. (2020). Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21145067"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/16\/8746\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:46:13Z","timestamp":1760165173000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/16\/8746"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,14]]},"references-count":53,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["ijms22168746"],"URL":"https:\/\/doi.org\/10.3390\/ijms22168746","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,14]]}}}