Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the retinal capillary endothelium

Autor: Yasuaki Hata, Yukio Sassa, Toshinori Murata, Taiji Sakamoto, Toshio Hisatomi, Ichiro Yamanaka, Toshiaki Kubota, Kazunori Nakagawa, Tatsuro Ishibashi, Kimihiko Fujisawa, Masae Honda, Katsuo Sueishi
Rok vydání: 2002
Předmět:
Male
Vascular Endothelial Growth Factor A
MAPK/ERK pathway
medicine.medical_treatment
Blotting
Western
Electrophoretic Mobility Shift Assay
Endothelial Growth Factors
Biology
Immediate-Early Proteins
Thromboplastin
Cellular and Molecular Neuroscience
Tissue factor
Rats
Inbred BN
Gene expression
medicine
Animals
Electrophoretic mobility shift assay
RNA
Messenger
Transcription factor
Cells
Cultured
Early Growth Response Protein 1
Flavonoids
Mitogen-Activated Protein Kinase 1
Lymphokines
Messenger RNA
Mitogen-Activated Protein Kinase 3
Reverse Transcriptase Polymerase Chain Reaction
Vascular Endothelial Growth Factors
Growth factor
Retinal Vessels
Promoter
Plicamycin
Immunohistochemistry
Molecular biology
Sensory Systems
Capillaries
Rats
Up-Regulation
DNA-Binding Proteins
Ophthalmology
Intercellular Signaling Peptides and Proteins
Cattle
Endothelium
Vascular
Mitogen-Activated Protein Kinases
hormones
hormone substitutes
and hormone antagonists
Signal Transduction
Transcription Factors
Zdroj: Graefe's Archive for Clinical and Experimental Ophthalmology. 240:1003-1010
ISSN: 0721-832X
DOI: 10.1007/s00417-002-0576-6
Popis: Purpose. 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
Databáze: OpenAIRE
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