Elsevier

Gene

Volume 315, 2 October 2003, Pages 33-41
Gene

Egr-1 target genes in human endothelial cells identified by microarray analysis

https://doi.org/10.1016/S0378-1119(03)00730-3Get rights and content

Abstract

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by ≥3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFα-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

Introduction

Vascular endothelium is a non-thrombogenic surface of normally quiescent cells that lies at the inner surface of blood vessels. The integrity of this endothelium is fundamental for maintaining the homeostasis of vasculature. Injury of this lining results in dramatic changes in the functional characteristics of the endothelium, rendering it adhesive and prothrombotic. These initial events are well correlated with subsequent inflammation and proliferation in vasculature, which is associated with the development of vascular diseases such as atherosclerosis and restenosis (Ross, 1999).

Early growth response factor-1 (Egr-1) was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli, including growth factors, cytokines, hypoxia, physical forces, and injury, all of which are implicated in the progress of vascular diseases (Silverman and Collins, 1999). As a transcription factor, Egr-1 can induce expression of a set of vasculature genes, such as PDGF-A and B chain, bFGF, TGFβ, TNFα, and intracellular adhesion molecule-1 (Silverman and Collins, 1999). Recent studies demonstrated that Egr-1 expression is elevated in both atherosclerotic lesions and neointima after vascular injury McCaffrey et al., 2000, Santiago et al., 1999a. Interestingly, reduction of Egr-1 expression by the Egr-1 antisense technology decreased intimal hyperplasia in balloon-injured carotid through inhibiting smooth muscle cell migration and proliferation Santiago et al., 1999b, Fahmy and Khachigian, 2002. Taken together, these findings suggest that Egr-1 is the key mediator in orchestrating the functional characteristics of the vessel wall after injury.

To further understand the role of Egr-1 in vasculature, we used commercially available oligonucleotide-based microarrays from Affymetrix (U95Av2 GeneChips, Santa Clara, California) to quantitate the Egr-1-mediated changes in expression of more than 12,000 mRNAs in human vascular endothelial cells.

Section snippets

Cell culture and adenovirus infection

Human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) were purchased from Bio-Whittaker (San Diego, California). HUVECs were cultured in endothelial cell growth medium-2 (EGM-2, Bio-Whittaker) containing 5% FBS, human basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), human epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), 50 μg/ml gentamicin, 50 ng/ml amphotericin-B, hydrocortisone, and ascorbic acid. HASMCs

Overexpression of Egr-1 in HUVEC

To overexpress Egr-1 in HUVEC, we utilized a recombinant adenovirus (AdEgr-1*) that co-expresses the green fluorescent protein (GFP) and the constitutively active Egr-1*, which is resistant to repression by endogenous NAB transcriptional corepressors. After infection with AdEgr-1* or the control virus AdGFP at a titer of ∼5 pfu/per cell for 24 h, almost all of cells (>99%) were GFP-positive (Fig. 1A). No apoptosis was observed in HUVEC infected with adenovirus at up to 10 pfu/cell in this

Discussion

To further explore the role of Egr-1 in vasculature, we have employed adenovirus-mediated Egr-1 overexpression in human vascular endothelial cells (HUVEC) to identify novel Egr-1 target genes using Affymetrix microarray analyses. In the present study, 229 up-regulated and 75 down-regulated genes were identified with ≥3-fold changes in HUVEC. Among them, several genes have been previously identified as Egr-1 target genes. However, most of them are novel Egr-1 target genes. These results will

Acknowledgements

This work was partially supported by the NIH (grants HL068878, HL03676, and S06GM08248) and the Swiss National Science Foundation (grant 31-57125.99 to M.U.E.). M.F. and Y.L. are supported by the fellowships from the American Heart Association Southeast Affiliate.

References (32)

  • F.S. Santiago et al.

    Early growth response factor-1 induction by injury is triggered by release and paracrine activation by fibroblast growth factor-2

    Am. J. Pathol.

    (1999)
  • E.S. Silverman et al.

    Pathways of Egr-1-mediated gene transcription in vascular biology

    Am. J. Pathol.

    (1999)
  • J. Svaren et al.

    EGR1 target genes in prostate carcinoma cells identified by microarray analysis

    J. Biol. Chem.

    (2000)
  • A.P. Vicari et al.

    TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development

    Immunity

    (1997)
  • S.K. Bae et al.

    Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia

    Cancer Res.

    (1999)
  • C. Boer et al.

    RhoA/Rho kinase and nitric oxide modulate the agonist-induced pulmonary artery diameter response time

    Am. J. Physiol. Heart Circ. Physiol.

    (2002)
  • Cited by (0)

    1

    Equally contributed to this paper.

    View full text