Fibulin-5 Regulates Angiopoietin-1/Tie-2 Receptor Signaling in Endothelial Cells.
BACKGROUND
Fibulin-5 is an extracellular matrix glycoprotein that plays critical roles in vasculogenesis and embryonic development. Deletion of Fibulin-5 in mice results in enhanced skin vascularization and upregulation of the angiogenesis factor angiopoietin-1 (Ang-1), suggesting that Fibulin-5 functions as an angiogenesis inhibitor. In this study, we investigate the inhibitory effects of Fibulin-5 on Ang-1/TIE-2 receptor pathway signaling and cell survival in human endothelial cells.


RESULTS
Recombinant wild-type and RGE-mutant Fibulin-5 proteins were generated through stable transfection of HEK293 and CHO cells, respectively. In vitro solid phase binding assays using pure proteins revealed that wild-type Fibulin-5 does not bind to Ang-1 or TIE-2 proteins but strongly binds to heparin. Binding assays using human umbilical vein endothelial cells (HUVECs) indicated that wild-type Fibulin-5 strongly binds to cells but RGE-mutant Fibulin-5, which is incapable of binding to integrins, does not. Pre-incubation of HUVECs for 1 hr with Fibulin-5 significantly increased caspase 3/7 activity, ERK1/2 phosphorylation, and expressions of the transcription factor early growth response 1 (EGR1) and the dual-specificity phosphatase 5 (DUSP5). Fibulin-5 also strongly attenuated Ang-1-induced TIE-2 and AKT phosphorylation, decreased Ang-1-induced expressions of the transcription factors Inhibitor of DNA Binding 1 (ID1) and Kruppel-like Factor 2 (KLF2), and reversed the inhibitory effect of Ang-1 on serum deprivation-induced cytotoxicity and caspase 3/7 activity.


CONCLUSIONS
We conclude that Fibulin-5 strongly binds to the endothelial cell surface through heparin-sulfate proteoglycans and possibly integrins and that it exerts strong anti-angiogenic effects by reducing endothelial cell viability and interfering with the signaling pathways of the Ang-1/TIE-2 receptor axis.
Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada.
Wilson
W
Chan
Translational Research in Respiratory Diseases, McGill University Health Centre, and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, QC, Canada.
Hodan
H
Ismail
Translational Research in Respiratory Diseases, McGill University Health Centre, and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, QC, Canada.
Dominique
D
Mayaki
Translational Research in Respiratory Diseases, McGill University Health Centre, and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, QC, Canada.
Veronica
V
Sanchez
Faculty of Dentistry, McGill University and Shriners Hospital for Children, Montréal, QC, Canada.
Kerstin
K
Tiedemann
Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada.
Elaine C
EC
Davis
Translational Research in Respiratory Diseases, McGill University Health Centre, and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, QC, Canada.
Sabah N A
SN
Hussain
D042683:Angiopoietin-1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D051766:Early Growth Response Protein 1 / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D042787:Receptor, TIE-2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D042683:Angiopoietin-1; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D016326:Extracellular Matrix Proteins; C492373:FBLN5 protein, human; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; C096161:KLF2 protein, human; D051741:Kruppel-Like Transcription Factors; D011994:Recombinant Proteins; D042787:Receptor, TIE-2