title: eNOS and Hsp90 interaction directly correlates with cord formation in human lymphatic endothelial cells.
abstract: Endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90) have been reported to contribute to angiogenesis and lymphangiogenesis. However, the functions of these proteins during lymphangiogenesis are unclear. In the present study, we first observed the cord formation pattern of human dermal microvascular lymphatic endothelial cells (HMVEC-dLy) on Matrigel over 2 to 8 h. The length of cord formation increased, peaked at 4 h, and then started to decline after 6 to 8 h of incubation. siRNA-targeted NOS3 significantly reduced the cord formation ability of HMVEC-dLy cells by 27% relative to control. This result confirmed the importance of eNOS in cord formation by human lymphatic endothelial cells. In addition, immunoprecipitation and Western blotting indicated that the interaction between eNOS and Hsp90 was maximal at 4 h, and then the proteins dissociated. This interaction correlated with the observation of cord formation of human lymphatic endothelial cells on Matrigel. Moreover, we found that the eNOS level decreased as the eNOS and Hsp90 complex disassociated during the late stage of cord formation. An Hsp90 inhibitor, 17-DMAG, was able to inhibit the eNOS and Hsp90 interaction, decrease the level of eNOS, and significantly inhibit cord formation to 38% of the level observed in the control. For the first time, we report that the interaction between eNOS and Hsp90 plays an important role in determining eNOS levels and in regulating cord formation of human lymphatic endothelial cells in vitro.
authors.affiliation: Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.
authors.forename: Orawin
authors.initials: O
authors.lastname: Prangsaengtong
authors.forename: Keiichi
authors.initials: K
authors.lastname: Koizumi
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Senda
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sakurai
authors.forename: Ikuo
authors.initials: I
authors.lastname: Saiki
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; D052250:Nitric Oxide Synthase Type III