Caveolae-mediated internalization of occludin and claudin-5 during CCL2-induced tight junction remodeling in brain endothelial cells.
Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.
Departments of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Svetlana M
SM
Stamatovic
Richard F
RF
Keep
Michael M
MM
Wang
Ivana
I
Jankovic
Anuska V
AV
Andjelkovic
D000818:Animals; D019921:Biotinylation; D001921:Brain / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000737:chemistry*; D018925:Chemokines / Q000378:metabolism; D062507:Claudin-5; D011992:Endosomes / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D007249:Inflammation; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D062793:Occludin; D019108:Tight Junctions / Q000378:metabolism*
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D018925:Chemokines; D062507:Claudin-5; C491511:Cldn5 protein, mouse; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse