Protein Kinase Cα-RhoA Cross-talk in CCL2-induced Alterations in Brain Endothelial Permeability

• Published in: The Journal of biological chemistry 2006-03, Vol.281 (13), p.8379-8388
• Main Authors: Stamatovic, Svetlana M., Dimitrijevic, Oliver B., Keep, Richard F., Andjelkovic, Anuska V.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M513122200

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, PKCα and PKCζ, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCα-DN and PKCζ-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, PKCα, and PKCζ activities were knocked down using dominant negative mutants (T17Rho, PKCα-DN, and PKCζ-DN, respectively). PKCα and Rho, but not PKCζ and Rho, interacted at the level of Rho, with PKCα 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/PKCα signal pathway interactions.