Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A.
Clostridium difficile toxin A increases paracellular permeability in colonic epithelial T84 cells by mechanisms involving RhoA glucosylation and actin depolymerization. However, we previously observed that toxin A-mediated decline in transepithelial electrical resistance preceded changes in cell morphology and tight junction ultrastructure (Hecht, G., Pothoulakis, C., LaMont, J. T., and Madara, J. L. (1988) J. Clin. Invest. 82, 1516-1524). Recent studies also showed that C. difficile toxins induce early cellular responses, including activation of mitogen-activated protein kinases, generation of reactive oxygen metabolites, and calcium influx. The aim of this study was to investigate whether toxin A-induced early cellular responses contribute to the permeability changes. We found that toxin A stimulated the activities of membrane and cytosolic protein kinase Calpha (PKCalpha) and cytosolic PKCbeta. A specific PKCalpha/beta antagonist (myristoylated PKCalpha/beta peptide) blocked toxin A-mediated RhoA glucosylation. Furthermore, decreased transepithelial electrical resistance and increased translocation of ZO-1 from tight junction occurred within 2-3 h of toxin A exposure and were also inhibited by PKCalpha/beta antagonist. During this time period, toxin exposure did not induce translocation of ZO-2, dephosphorylation or translocation of occludin, or cell rounding. Our data indicate that PKC signaling regulates toxin A-mediated paracellular permeability changes and ZO-1 translocation.
Chen
Ming L
ML
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Pothoulakis
Charalabos
C
LaMont
J Thomas
JT
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D016466:CHO Cells; D003106:Colon / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006224:Cricetinae; D004768:Enterotoxins / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021381:Protein Transport; D015398:Signal Transduction; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; C041348:tcdA protein, Clostridium difficile; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein