FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl.
Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human pulmonary endothelium. Permeability of cultured human pulmonary endothelial cells was assessed using transendothelial electrical resistance and dextran transwell assays. Junctional complex formation was assessed using membrane fractionation and immunofluorescence. Pharmacological inhibitors and small interfering (si)RNA were utilised to determine the effects of individual components on permeability. Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. β-catenin, occludin, claudin-5 or zona occludens protein (ZO)-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced focal adhesion kinase (FAK) phosphorylation and focal adhesion formation, with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, protein kinase (PK)A, PKG, PKC or protein phosphatase 2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity and c-Abl siRNA attenuated peak barrier enhancement after FTY720. FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signalling.
Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.
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L
Wang
E T
ET
Chiang
J T
JT
Simmons
J G N
JG
Garcia
S M
SM
Dudek
D022005:Adherens Junctions / Q000473:pathology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D000068876:Fingolimod Hydrochloride; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation; D008168:Lung / Q000187:drug effects*; D008246:Lysophospholipids / Q000378:metabolism; D010539:Permeability; D010766:Phosphorylation; D011409:Propylene Glycols / Q000494:pharmacology*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D019108:Tight Junctions / Q000473:pathology
D008246:Lysophospholipids; D011409:Propylene Glycols; D034741:RNA, Small Interfering; C060506:sphingosine 1-phosphate; D016315:Proto-Oncogene Proteins c-abl; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine