Novel angiogenic inhibitor DN-9693 that inhibits post-transcriptional induction of connective tissue growth factor (CTGF/CCN2) by vascular endothelial growth factor in human endothelial cells.
Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)-mediated induction of the ctgf/ccn2 gene was a post-transcriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.
Kondo
Seiji
S
Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8525, Japan.
Tanaka
Noriko
N
Kubota
Satoshi
S
Mukudai
Yoshiki
Y
Yosimichi
Gen
G
Sugahara
Toshio
T
Takigawa
Masaharu
M
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004730:Endothelium, Vascular; D005786:Gene Expression Regulation / Q000187:drug effects; D006207:Half-Life; D006801:Humans; D017874:Immediate-Early Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000187:drug effects; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011799:Quinazolines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D055513:Connective Tissue Growth Factor; C049662:1,5-dihydro-7-(1-piperidinyl)-imidazo(2,1-b)quinazolin-2(3H)-one