Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the retinal capillary endothelium.
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.
Sassa
Yukio
Y
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
Hata
Yasuaki
Y
Murata
Toshinori
T
Yamanaka
Ichiro
I
Honda
Masae
M
Hisatomi
Toshio
T
Fujisawa
Kimihiko
K
Sakamoto
Taiji
T
Kubota
Toshiaki
T
Nakagawa
Kazunori
K
Sueishi
Katsuo
K
Ishibashi
Tatsuro
T
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
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