title: Protein kinase Calpha-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability.
abstract: Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCalpha and PKCzeta, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCalpha-DN and PKCzeta-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCalpha, and PKCzeta activities were knocked down using dominant negative mutants (T17Rho, PKCalpha-DN, and PKCzeta-DN, respectively). PKCalpha and Rho, but not PKCzeta and Rho, interacted at the level of Rho, with PKCalpha being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCalpha signal pathway interactions.
authors.affiliation: Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Stamatovic
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Dimitrijevic
authors.forename: Richard F
authors.initials: RF
authors.lastname: Keep
authors.forename: Anuska V
authors.initials: AV
authors.lastname: Andjelkovic
mesh_terms: D000818:Animals; D001921:Brain / Q000098:blood supply; D018929:Cell Culture Techniques; D002458:Cell Fractionation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000494:pharmacology*; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019793:Fluorescein; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008954:Models, Biological; D009154:Mutation; D010539:Permeability / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D051571:Protein Kinase C-alpha / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects; D054460:rho-Associated Kinases
chemical_list: D018932:Chemokine CCL2; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D051571:Protein Kinase C-alpha; D019793:Fluorescein