Abstract 129: Inflammation-mediated Down Syndrome Critical Region (DSCR)-1 Promoter Activation Shows Vascular-bed Specific Patterns in Hprt-locus targeted transgenic mice
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 (VEGF) result in dramatic upregulation of Down Syndrome Critical Region (DSCR)-1 encoding exons 4–7 (DSCR-1s), an auto-inhibitory factor of calcineurin-NFAT signaling, in endothelial cells (ECs). Constitutive expression of DSCR-1s in activated ECs abolished nuclear localization of NFAT, attenuated EC proliferation, tube formation, tumor growth and inflammatory response. The goal of the current study was to evaluate the transcriptional regulation of the DSCR-1s promoter in vivo. To that end, the 1.7-kb 5′-flanking region of the human DSCR-1 gene was coupled to LacZ and the resulting construct introduced into the Hprt locus of mice by homologous recombination. Transgenic embryos displayed strong, specific and uniform expression within the endothelium. Reporter gene activity was significantly downregulated after birth, with expression limited to cardiac myocytes, a subset of neurons, and blood vessels of the brain and diaphragm. Systemic administration of lipopolysaccharide or VEGF resulted in upregulation of the transgene (as well as endogenous DSCR-1s mRNA) in ECs and smooth muscle cells of the aorta, and in microvascular ECs of the lung, brain, and kidney, but not of the skeletal muscle, thymus, spleen, or liver. The 1.7 kb promoter also directed expression in the microvascular endothelium of B16-melanoma and Lewis lung carcinoma (LLC) tumor xenografts, an effect that was abrogated by local injection of the NFAT inhibitor, cyclosporin A. In cultured ECs from dermal microvascular, DSCR-1 promoter activities are marked upregulated with conditioned media from B16-melanoma and LLC, but not from normal skin fibroblast or hypoxia. Stimulated DSCR-1 activities are downregulated in the presence of siRNAs against NFATc1, c2, and c3. Together, these findings suggest that the upstream promoter of human DSCR-1 contains information for environmentally responsive NFAT-dependent gene expression. The Hprt-locus targeted mouse provides a novel tool for studying the mechanisms of NFAT-activation in inflammation and neo-angiogenesis.