Fluid Shear Stress Induction of the Tissue Factor Promoter In Vitro and In Vivo Is Mediated by Egr-1

Abstract

Abstract-Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m 2 shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow-restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo. Key Words: fluid shear stress Ⅲ tissue factor promoter Ⅲ Egr-1 transcription factor Ⅲ vascular endothelial cells E ndothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to blood flow. Locally disturbed flow at arterial curvatures or bifurcations is characterized by both low and high oscillatory wall shear stresses that are conveyed to the cell as both a change in pressure and a change in the stretch capacity of the EC lining. 1-3 As a consequence of the local perturbation in laminar shear stress, a number of genes have been identified whose promoter activity is positively or negatively regulated by shear stress (reviewed in References 4 to 8). Several cis-acting elements have been implicated in mediating shear modulation of gene expression, and the term shear stress responsive element (SSRE) was first proposed after the identification of the sequence GAGACC. 9 This SSRE was demonstrated to confer shear stress responsiveness on the platelet-derived growth factor (PDGF) B promoter, 9 a heterologous SV40 promoter, 10 and has since been located in a number of other promoters that have been shown to be shear stress responsive in ECs. 11 The GAGACC sequence binds nuclear factor B (NF-B) p50 -p65 heterodimers, and consistent with this observation, the wild-type HIV-1 LTR, but not an LTR lacking the NF-B binding site, was also shown to be responsive to shear stress. 5 Other transcription factors that have been shown to mediate shear stress activation of a promoter are AP1, 12,13 Sp1, 14 and Egr-1. 15 Recently, the transcription fa...

Shear Stress Decreases Endothelial Cell Tissue Factor Activity by Augmenting Secretion of Tissue Factor Pathway Inhibitor

“…Lin et al 21 and Houston et al 22 both reported that shear stress alone (no cytokine exposure) transiently induced TF mRNA over periods of 1 to 3 hours. Transcription factors likely involved in these shear stress-induced changes in TF mRNA include Sp1, whose increased activity is associated with hyperphosphorylation, 21 and/or Egr-1, 22-24 but not nuclear factor-B.…”

“…4 In those studies, either human umbilical vein ECs were subjected to 12 dyne/cm 2 for 1 to 2 hours 21 or human aortic ECs were exposed to 15 dyne/cm 2 for 1 to 3 hours. 22 Lin et al found that TF mRNA by Northern blotting was essentially no longer present after 6 hours; Houston et al, who performed RT-PCR, did not look beyond 3 hours. Although the present work does not address this issue, we did find that TF antigen at 3 hours increased with increasing shear stress.…”

Tissue Factor and Cardiovascular Disease: Quo Vadis?

“…89,90 Hypertension increases shear stress leading to activation of NFκB and Egr-1, and subsequently to upregulation of TF gene expression. 91,92 Angiotensin-convertingenzyem (ACE) inhibitors downregulate TF expression in vivo, 93 while angiotensin II upregulates it in vascular cells via the angiotensin II type 1 receptor. 94 …”

The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells

“…[18][19][20][21][22][23] Egr-1, which encodes a serum-inducible zinc finger nuclear phosphoprotein, is rapidly induced in cultured cells by a wide variety of mitogenic and nonmitogenic stimuli. In endothelial cells, Egr-1 has been shown to be activated by acidic fibroblast growth factor, 24 basic fibroblast growth factor, 25,26 vascular endothelial growth factor (VEGF), 27 epidermal growth factor (EGF), 28,29 shear stress, [30][31][32] cyclical strain, 33 and hypoxia. 34,35 In turn, the Egr-1 transcription factor may induce the expression of a wide range of target genes including PDGF-A, 36 PDGF-B, tissue factor (TF), 27 Flt-1, 37 transforming growth factor (TGF)-␤, tumor necrosis factor (TNF)-␣, urokinasetype plasminogen activator (u-PA), and metalloproteinases.…”