Regulation of PDGF-B in endothelial cells exposed to cyclic strain.
The present study was designed to examine the regulation by cyclic strain of endothelial cell (EC) platelet-derived growth factor-B chain (PDGF-B) expression. We demonstrate in this study that bovine aortic ECs subjected to 10% (but not 6%) average strain resulted in a 2.6-fold increase in PDGF-B steady state mRNA and immunoreactive protein. Nuclear runoff transcription assays confirmed the induction of PDGF-B transcripts. To address the regulation of PDGF-B gene expression by cyclic strain, we transfected bovine aortic ECs with a construct containing 450 bp of human PDGF-B promoter sequence coupled to chloramphenicol acetyltransferase (CAT), and found that subjecting these cells to 10% average strain resulted in a twofold increase in CAT activity by 4 hours. Analysis of nested 5' deletions of the promoter transfected into ECs demonstrated a 55% drop-off in activity between position -313 and -153, with no induction of activity with the - 101-bp minimal promoter. Since a shear stress response element (SSRE) is located at position -125, we tested the hypothesis that the SSRE site was necessary and/or sufficient for induction of PDGF-B activity with strain. Electromobility shift assays revealed that nuclear proteins from ECs exposed to strain for short intervals (30 minutes) bound to the PDGF-B SSRE. However, transfection of ECs with hybrid promoter constructs containing the SV40 sequence promoter downstream of the SSRE or the -153 PDGF-B promoter sequence bearing a mutation in the SSRE demonstrated that the SSRE was not necessary for inducible reporter gene expression in ECs exposed to cyclic strain.
Sumpio
B E
BE
Department of Surgery (Vascular), Yale University School of Medicine, New Haven, Conn 06510, USA. bauer.sumpio@yale.edu
Du
W
W
Galagher
G
G
Wang
X
X
Khachigian
L M
LM
Collins
T
T
Gimbrone
M A
MA
Resnick
N
N
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D013314:Stress, Mechanical; D014158:Transcription, Genetic / Q000502:physiology
D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis