title: Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the retinal capillary endothelium.
abstract: OBJECTIVE
  To investigate the causal relationship between VEGF and tissue factor (TF) expression, and its intracellular signaling in the retinal capillary endothelium both in vitro and in vivo.
  
  
  METHODS
  TF mRNA and protein expression in cultured bovine retinal capillary endothelial cells (BRECs) were detected by RT-PCR and western blotting. The expression and subcellular localization of Egr-1 were analyzed by immunocytochemistry and western blotting. Involvement of p44/p42 MAPK pathway in this signaling was assessed using PD98059. Electrophoretic mobility shift assay (EMSA) was performed using human TF Egr-1/Sp-1 overlapping promoter region (-85 to -70). Decoy oligonucleotide was transfected into BRECs to clarify the critical transcription factor mediating VEGF-induced TF gene expression. To evaluate the importance of GC rich region in VEGF-induced TF protein expression in rat retinas, Mithramycin was intraperitoneally administered.
  
  
  RESULTS
  VEGF stimulated TF mRNA and protein expression in cultured BRECs, reaching maximal effect after 4 h and 10 h, respectively. VEGF activated transcription factor Egr-1 within 60 min. Inactivation of Egr-1 by PD98059 resulted in the prohibition of VEGF-induced TF gene expression. EMSA revealed the increment of Egr-1 binding with TF promoter region by displacing Sp1 after treatment with VEGF. Transfection of the Egr-1/Sp-1 overlapping decoy into BRECs inhibited VEGF-dependent TF gene expression. Mithramycin almost completely suppressed VEGF-induced TF protein expression in retinal capillary system in vivo (80%, p<0.01).
  
  
  CONCLUSIONS
  Transcription factor Egr-1, which lies downstream of p44/p42 MAPK, critically mediates VEGF-dependent TF expression in the retinal capillary endothelium.
authors.affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sassa
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hata
authors.forename: Toshinori
authors.initials: T
authors.lastname: Murata
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamanaka
authors.forename: Masae
authors.initials: M
authors.lastname: Honda
authors.forename: Toshio
authors.initials: T
authors.lastname: Hisatomi
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Fujisawa
authors.forename: Taiji
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Kubota
authors.forename: Kazunori
authors.initials: K
authors.lastname: Nakagawa
authors.forename: Katsuo
authors.initials: K
authors.lastname: Sueishi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Ishibashi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002196:Capillaries; D002417:Cattle; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D051766:Early Growth Response Protein 1; D024202:Electrophoretic Mobility Shift Assay; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D017874:Immediate-Early Proteins; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008926:Plicamycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011914:Rats, Inbred BN; D012171:Retinal Vessels / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000502:physiology*; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D016228:Endothelial Growth Factors; D005419:Flavonoids; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D013925:Thromboplastin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008926:Plicamycin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one