TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress.
Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxia-induced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events.
Hicks
Kali
K
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030, USA.
O'Neil
Roger G
RG
Dubinsky
William S
WS
Brown
Rachel C
RC
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D013312:Stress, Physiological; D050052:TRPC Cation Channels / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D001381:Azepines; D002121:Calcium Channel Blockers; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D010750:Phosphoproteins; D050052:TRPC Cation Channels; D000098662:Vasodilator-Stimulated Phosphoprotein; C070571:ML 7; C071058:moesin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium