Thrombin-induced Autoinhibitory Factor, Down Syndrome Critical Region-1, Attenuates NFAT-dependent Vascular Cell Adhesion Molecule-1 Expression and Inflammation in the Endothelium

Tatsuhiko Kodama,Takashi Minami,William C. Aird,Mai Miura

doi:10.1074/jbc.m513112200

Abstract

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.

Highlights

The endothelium is highly malleable cell layer constantly responding to changes within the extracellular environment and responding in ways that are usually beneficial but at times harmful to the organism
We have recently demonstrated that vascular endothelial growth factor (VEGF)3- and thrombin-mediated activation of calcineurin-NFAT signaling in endothelial cells is autoinhibited by the induction of the Down syndrome critical region (DSCR)-1 gene [4]
Identification of NFAT and NF-␬B-dependent Pathways in Thrombin-treated Human Primary Endothelial Cells—In a previous study we demonstrated that thrombin and VEGF treatment of Human umbilical vein endothelial cells (HUVEC) resulted in marked and rapid up-regulation of the DSCR-1 gene and secondary inhibition of NFAT signaling [4]

Summary

Introduction

The endothelium is highly malleable cell layer constantly responding to changes within the extracellular environment and responding in ways that are usually beneficial but at times harmful to the organism. The DSCR-1 gene ( known as calcipressin 1, MCIP-1, and Adapt 78) consists of 7 exons, of which exons 1– 4 can be alternatively spliced, resulting in a number of different mRNA isoforms [5, 6] These isoforms have different expression patterns and are regulated by distinct transcriptional mechanisms. PAR-1 is linked to a number of signal intermediates (including mitogenactivated protein kinase, protein kinases C and A, phosphatidylinositol 3-kinase, and calcineurin), transcription factors (such as Egr-1, NF-␬B, NFAT, SP-1, and AP-1), and downstream target genes (e.g. vascular adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, fractalkine, and monocyte chemoattractant protein-1, endothelial-specific molecule-1, bone morphogenetic protein-2, and HB-EGF) [4, 11,12,13]. The results suggest that DSCR-1-mediated inhibition of NFAT signaling may be leveraged for therapeutic gain in inflammatory states

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Conclusion