Protein kinase Cα-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability

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