Thrombin induces endothelial arginase through AP-1 activation.
Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction.
Zhu
Weifei
W
Department of Cell Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA.
Chandrasekharan
Unni M
UM
Bandyopadhyay
Smarajit
S
Morris
Sidney M
SM
DiCorleto
Paul E
PE
Kashyap
Vikram S
VS
D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D001119:Arginase / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*
D051698:Activating Transcription Factor 2; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; D001119:Arginase