244 GATA-1 PLAYS A CRITICAL ROLE IN ENDOTHELIAL CELL-SPECIFIC EXPRESSION OF THE 1.1-kb CD55 PROMOTER REGION

Abstract

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue. Some of the cell surface regulators, also known as membrane complement regulatory proteins (CRPs), are the membrane cofactor protein (CD46), the decay-accelerating factor (CD55), and protectin (CD59). The CD55 is a 70 kDa glycolipid-anchored membrane-bound protein that has regulatory activity by preventing C3 convertase formation and is reported to be expressed in blood and vascular endothelial cells. Its high expression in leukocytes and endothelium is thought to safeguard against locally augmented C3 activation that can occur with inflammation. In this study, we originally cloned the 1.1-kb promoter region of the CD55 gene and constructed a luciferase reporter plasmid, pGL3-1.1CD55. The pGL3-1.1CD55 plasmid and its control plasmid, pGL3-control (SV40 promoter), were transfected into endothelial (MS-1 and BAEC) and epithelial (HaCaT and HEK293) cells, and the soluble fraction of the cell lysates was assayed for luciferase activity. Its promoter activity was compared with that of the promoters of endothelial-specific genes such as MCP, Flk-1, ICAM-2, and throbomodulin. Luciferase activity in each sample was normalized to the ²-galactosidase activity of the same sample and the data were analyzed by Sigma Plot program (P < 0.01 versus control). Our results showed that the 1.1 kb CD55 promoter was the strongest among the endothelial cell-specific promoters. To define the important region for the strong expression, the deletion constructs containing 0.96, 0.86, and 0.74, CD55 promoter regions were prepared. Relative to the activity of the control SV40 promoter, decreased luciferase activity was obtained with these deletion constructs, suggesting that the about 0.2 kb 52-flanking region (between -1125 and -967) of the 1.1 kb CD55 promoter region was important for the strong gene expression. Interestingly, on the 52-end of the 0.2 kb region, we could detect two GATA-1 binding sites (from -1122 to -1118 and from -1111 to -1107); the deletion of the sequences between -1125 and -1100 (ΔGATA-1) significantly reduced the promoter activity. Furthermore, ectopic expression of GATA-1 dose-dependently induced the 1.1 kb CD55 promoter activity, but not the ΔGATA-1 activity, strongly implying that the GATA-1 binding site is important for the strong expression. Finally, we confirmed the binding of GATA-1 on the CD55 promoter region with the electrophoretic mobility shift assay (EMSA) using the 28 base pair oligonucleotide probes corresponding to the GATA-1 binding site and chromatin immunoprecipitation (Ch-IP) using anti-GATA-1 antibody. Taken together, these results strongly suggested that GATA-1 plays a critical role in endothelial cell-specific expression of the 1.1 kb CD55 promoter region.

This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea, and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-070-C00095).