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A possible role for α-dystrobrevin in the reorganization of tight junctions in epithelial cells
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alpha-dystrobrevin (α -DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex (DGC) in skeletal muscle cells. Isoforms of α -DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, α -DBs are assumed to be important in cell signalling and cell differentiation. We have assessed the role of a-DB in two epithelial cell lines (MDCK I and HT 29) that are in different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-α-DB antibody, we have investigated its localization and association with tight junction (TJ)-associated proteins before and after protein kinase C (PKC) activation with phorbol myristate acetate (PMA). In both cell lines, there was submembranous localization of α -DB both apically and basolaterally, as assessed with confocal imaging. PKC caused a reorganization of TJ, which was parallel with increased localization of α -DB to TJ areas, especially in MDCK I cells. Moreover, the TJ-associated protein Z0-1 co-immunoprecipitated with α -DB, as displayed with SDS-PAGE and immunoblotting. Thus, α -DBs not only take part in the regulation of the contact between intra- and extracellular proteins at basolateral membranes, but possibly also in the regulation of tight junctions via ZO-1 and PKC-activation.

Keyword [en]
dystrobrevin, tight junction, protein kinase C, epithelium, dystrophin-glycoprotein complex
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-97568OAI: oai:DiVA.org:liu-97568DiVA: diva2:648858
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2013-09-17
In thesis
1. Protein kinase C-dependent regulation of the epithelial barrier
Open this publication in new window or tab >>Protein kinase C-dependent regulation of the epithelial barrier
2004 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The intestinal epithelium is essential for the absorption of nutrients and for the defence against harmful substances in the gut. The permeability and barrier functions of epithelia are mainly controlled by the intercellular tight junctions. These are dynamic structures that are affected by diverse extracellular physiological and pathological stimuli. The regulatory mechanisms that control the function of tight junctions are under intense investigation and it is known that they involve classical second messengers and signalling pathways, for example protein kinase C (PKC).

In the present thesis, the effects of PKC activation in cell monolayers of the human colon cancer cell line HT 29 and the canine kidney cell line MDCK I were studied. The cells, having different origins display distinct barrier characteristics. Thus, HT 29 forms a leaky epithelium with poorly developed tight junctions, whereas MDCK I represents a very tight epithelium with fully developed tight junctions.

The results show, that activation of PKC correlates with galactose-induced differentiation of HT 29 to small intestine-like cells and formation of a permeability barrier. PKC activation further promotes establishment of the epithelial barrier in the cells, as was demonstrated by decreased permeability to sodium fluorescein and increased transepithelial electric resistance (TER). Confocal imaging revealed parallel redistribution of the tight junction proteins occludin and claudin-1 to the most apical intercellular contacts. By contrast, in MDCK I cells PKC activation caused increased permeability, decreased TER and disassembly of occludin and claudin-1 from the tight junctions. Interestingly, in both cell lines PKC activation resulted in a decrease in the threonine phosphorylation of occludin.

α-dystrobrevin was originally identified in muscle cells, but has also been found in other tissues, e.g. epithelia. Furthermore, α-dystrobrevin is suggested to be involved in granulocytic differentiation. In this study, it is shown that several isoforms of α-dystrobrevin, some of which are tyrosine-phosphorylated, are expressed in HT 29 and MDCK I cells displaying apical and basolateral submembranous localization. α-dystrobrevin co-immunoprecipitates with actin and the tight junction associated protein Z0-1 and concentrates at the sites of reorganizing tight junctions after activation of PKC in the cells.

In conclusion, the data show that PKC induced regulation of the epithelial barrier involves specific molecular mechanisms, and achieves distinct effects in the different cell types and at different developmental stages. The results also suggest a possible role for α-dystrobrevin in the cellular control of tight junctions.

Abstract [sv]

Vår tarmslemhinna har till uppgift att absorbera näringsämnen, men den fungerar också som skydd mot farHga ämnen, som kan finnas i tannen. Slemhinnans dubbla funktion som transportör och barriär kontrolleras huvudsakligen av s.k. täta fogar mellan cellerna. Fogarna är dynamiska strukturer som påverkas av olika fysiologiska och patologiska retningar från tarmkanalen. De regulatoriska mekanismerna som kontrollerar fogarnas funktion är inte helt klarlagda. Det är dock känt att fogarna regleras via klassiska cellsignaleringsvägar och signalmolekyler, t. ex. proteinkinas C (PKC).

Vi har undersökt effekterna av PKC-aktivering i epitelceller, dels liT 29-celler som härstammar från humana tjocktannscancerceller, dels MDCK I-celler som isolerats från hundnjureslemhinna. Cellerna som har olika ursprung och därmed olika funktioner, uppvisar också skillnader i barriäregenskaperna. HT 29-celler bildar ett otätt cellager (epitel) med fogar som inte är helt utvecklade, medan MDCK I-celler växer till ett mycket tätt cellager med väl fungerande fogar. Syftet med vår aktuella forskning var att utröna de molekylära mekanismer bakom regleringen av epitelcellsbarriären genom att studera effekten av PKC-aktivering i de två cellinjema.

Våra resultat visar, att aktiviteten hos PKC i HT 29-celler ökar parallellt med cellernas differentiering till tunntarmslika celler och bildning av enpermeabilitetsbarriär. PKC aktivering i redan differentierade celler förstärker barriärfunktionen, vilket ses som minskad genomsläpplighet för en fluorescent molekyl och ökad elektrisk resistans över cellagret. Med konfokal mikroskopi visar vi att proteinerna occludin och claudin-1, som ingår i fogkomplexet, omlokaliseras till den apikala delen av cellernas kontaktyta. Hos MDCK I celler däremot orsakar PKC-aktivering ökad genomsläpplighet och minskad elektrisk resistans över cellagret samt upplösning av de sammanhängande nätverken av ocdudin respektive claudin-1 längs de täta fogarna. En intressant upptäckt är att båda cellinjerna orsakar PKC-aktiveringen en minskning av treoninfosforyleringen hos occludin.

α-dystrobrevin är ett protein som har identifierats i muskelceller, och på senare tid även i andra celltyper, t.ex. epitelceller. Vi visar att flera former av adystrobrevin, varav några är tyrosinfosforylerade, finns i både HT 29 och MDCK I-celler och är lokaliserade till det apikala och basolaterala membranet. Proteinerna aktin och Z0-1, som är associerade till de täta fogarna, kan immunprecipiteras med α-dystrobrevin, och PKC-aktivering i båda celltyperna orsakar en koncentration av dystrobrevin vid de apikala cellkontakterna.

Vår slutsats är att PKC-inducerad reglering av den epiteliala barriären involverar specifika mekanismer och åstadkommer olika effekter beroende på celltyp och grad av differentiering. Våra resultat antyder också att α-dystrobrevin kan ha betydelse i regleringen av de täta fogarna.

Publisher
46 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 64
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22989 (URN)2363 (Local ID)91-7373-815-8 (ISBN)2363 (Archive number)2363 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2013-09-17

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Sjö, AnitaMagnusson, Karl-EricHolmgren Peterson, Kajsa

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