Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins

• Published in: American Journal of Physiology: Cell Physiology Vol. 297; no. 4; pp. C945 - C954
• Main Authors: Sarai, Kei, Shikata, Kenichi, Shikata, Yasushi, Omori, Kazuyoshi, Watanabe, Naomi, Sasaki, Motofumi, Nishishita, Shingo, Wada, Jun, Goda, Noriko, Kataoka, Noriyuki, Makino, Hirofumi
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.10.2009
• Subjects: Actins - metabolism; Adherens Junctions - drug effects; Adherens Junctions - physiology; Biochemistry; Cadherins - physiology; Cell Adhesion Molecules - metabolism; Cells; Cells, Cultured; Electric Impedance; Endothelial Cells - drug effects; Endothelial Cells - physiology; Endothelium, Vascular - drug effects; Endothelium, Vascular - physiology; Fingolimod Hydrochloride; Focal Adhesion Protein-Tyrosine Kinases - physiology; Humans; Hyperglycemia - metabolism; Immunosuppressive Agents - pharmacology; Kinases; Lysophospholipids - metabolism; Microvessels - cytology; Monomeric GTP-Binding Proteins - physiology; Phosphates; Phosphorylation; Propylene Glycols - pharmacology; Proteins; rac GTP-Binding Proteins - physiology; Receptors, Lysosphingolipid - physiology; rho GTP-Binding Proteins - physiology; Ribonucleic acid; RNA; Sphingosine - analogs & derivatives; Sphingosine - metabolism; Sphingosine - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00606.2008

1 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2 Faculty of Health Sciences, Okayama University Medical School, 3 Department of Medical Engineering, Kawasaki Medical School, Okayama, Japan Submitted 24 November 2008 ; accepted in final form 4 August 2009 Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting endothelial barrier promotion by FTY720. To elucidate whether FTY720 induces signaling events related to endothelial barrier enhancement under hyperglycemic conditions, human microvascular endothelial cells (HMVECs) preincubated with hyperglycemic (30 mM) medium were treated with 100 nM FTY720 for 3 h. Immunofluorescent microscopy and coprecipitation study revealed FTY720-induced focal adhesion kinase (FAK)-associated adherens junction (AJ) assembly at cell-cell contacts coincident with formation of a prominent cortical actin ring. FTY720 also induced transmonolayer electrical resistance (TER) augmentation in HMVEC monolayers in both normoglycemic and hyperglycemic conditions, implying endothelial barrier enhancement. Similar to S1P, site-specific FAK tyrosine phosphorylation analysis revealed FTY720-induced FAK [Y 576 ] phosphorylation without phosphorylation of FAK [Y 397 /Y 925 ]. Furthermore, FTY720 conditioned the phosphorylation profile of FAK [Y 397 /Y 576 /Y 925 ] in hyperglycemic medium to the same pattern observed in normoglycemic medium. FTY720 challenge resulted in small GTPase Rac activation under hyperglycemic conditions, whereas increased Rho activity in hyperglycemic medium was restored to the basal level. Rac protein depletion by small interfering RNA (siRNA) technique completely abolished FTY720-induced FAK [Y 576 ] phosphorylation. These findings strongly suggest the barrier protective effect of FTY720 on HMVEC monolayers in hyperglycemic medium via S1P signaling, further implying the possibility of FTY720 as a therapeutic agent of diabetic vascular disorder. endothelial barrier function; human microvascular endothelial cell; diabetes mellitus; transmonolayer electrical resistance; cytoskeleton Address for reprint requests and other correspondence: K. Shikata, Dept. of Medicine and Clinical Science, Okayama Univ. Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-0858, Japan (e-mail: shikata{at}md.okayama-u.ac.jp ).