A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells.
We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.
Beau
Isabelle
I
Faculté de Pharmacie, INSERM Unit 510, Châtenay-Malabry, France 92296.
Cotte-Laffitte
Jacqueline
J
Amsellem
Raymonde
R
Servin
Alain L
AL
D000818:Animals; D018938:Caco-2 Cells; D002227:Carbazoles / Q000494:pharmacology; D002458:Cell Fractionation; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007422:Intestines / Q000166:cytology / Q000201:enzymology / Q000821:virology*; D007546:Isoquinolines / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D062793:Occludin; D017830:Octoxynol / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012401:Rotavirus / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D019108:Tight Junctions / Q000737:chemistry / Q000201:enzymology / Q000821:virology*; D014158:Transcription, Genetic; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D002227:Carbazoles; D007211:Indoles; D007546:Isoquinolines; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D012333:RNA, Messenger; D013449:Sulfonamides; C569042:TJP1 protein, human; C569046:TJP2 protein, human; D013873:Thionucleotides; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C016957:adenosine-3',5'-cyclic phosphorothioate; C057416:KT 5720; D017830:Octoxynol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide