Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption.
Chitosan has been successfully used as an excipient for trans-epithelial drug delivery systems. It is known to transiently open intercellular tight junctions thus increasing the permeability of an epithelium. In order to investigate the possible role of protein kinases in trans-epithelial delivery, changes in trans-epithelial electrical resistance ('TEER') of epithelial (Caco-2) cell monolayers were assessed in response to chitosan glutamate treatment, in the presence and absence of specific protein kinase inhibitors. Changes in subcellular localisation of the tight junction protein ZO-1 observed by immunofluorescence and western blotting of cellular fractions were also assessed. Inhibition of protein kinase C (PKC), but not mitogen activated protein kinase (MAPK) was found to prevent the chitosan-mediated decrease in TEER, and changes in localisation of ZO-1. In order to determine which PKC isozymes were responsible for the chitosan-mediated tight junction disruption, the activation of the PKC isozymes alpha, beta and delta was investigated. A chitosan-mediated translocation of PKC alpha but not PKC beta or delta from the cytosol to the membrane fraction, indicative of PKC alpha activation was observed. Thus, treatment of Caco-2 cells with chitosan may result in the activation of PKC-dependent signal transduction pathways which affect tight junction integrity.
Smith
Jennifer M
JM
Smith and Nephew Group Research, York Science Park, Heslington, York YO10 5DF, UK.
Dornish
Michael
M
Wood
Edward J
EJ
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D048271:Chitosan / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D016503:Drug Delivery Systems; D004847:Epithelial Cells / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction; D013347:Subcellular Fractions; D019108:Tight Junctions / Q000378:metabolism*; D013997:Time Factors; D062826:Zonula Occludens-1 Protein
D001672:Biocompatible Materials; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D018698:Glutamic Acid; D017830:Octoxynol; D048271:Chitosan; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha