215 COMPARISON OF STRONG ENDOTHELIAL CELL-SPECIFIC PROMOTERS FOR EXPRESSION OF HUMAN COMPLEMENT REGULATORY PROTEINS IN PORCINE XENOGRAFT ENDOTHELIAL CELLS

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 cells. Activation of complement pathways results in the formation of the membrane attack complex (MAC). Several cell surface regulators known as membrane complement regulatory proteins (mCRPs) prevent the formation of MAC. For blocking the formation of MAC in the xenotransplanted cells, we tried to overexpress mCRPs in the endothelial cells to prevent the hyperacute rejection (HAR), because one of the major obstacles to xenotransplantation of vascularized organs was reported to be HAR. For this purpose, we isolated human MCP (membrane cofactor protein, CD46) (1.5 kb) and DAF (decay-accelerating factor, CD55) (1.1 kb) promoter regions from human genomic DNA isolated from the human embryonic kidney cell line HEK293, and cloned into pGL3 plasmid for luciferase reporter assay. We also inserted other known endothelial cell-specific promoters, such as Flk-1 (fetal liver kinase-1) and ICAM-2 (intercellular adhesion molecule-2), thrombomodulin promoters, into pGL3 vector to compare their promoter activities. We transfected the plasmids into several endothelial cell lines such as bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC), and mouse pancreatic microvascular endothelial cells (MS1). We also used two epithelial cell lines, human embryonic kidney epithelial cells (HEK293) and human epidermal keratinocytes (HaCaT). The endothelial specific expression of the promoters were compared with those of SV40, CMV, and EF-2a (elongation factor) promoters which are generally used in mammalian gene expression. Luciferase assays showed that among the endothelial cell-specific promoters, the 1.1 kb DAF promoter was the strongest, followed by the Flk-1 promoter. The promoter activity of 1.1 kb DAF was about 2-fold that of the Flk-1 promoter. Moreover, the 1.1 kb DAF and Flk-1 promoters were chosen as the best endothelial specific promoters, as both showed about 5 times more luciferase activity in the endothelial cells compared to that in the epithelial cell lines. We also found that the 5′-flanking region between −1126 and −968 of the 1.1 kb DAF promoter was very important for the endothelial-specific strong gene expression. On the 0.2 kb region of DAF promoter, we could detect several conserved nucleotide sequences interacting with the specific transcription factors, such as GATA-1, USF, CdxA, SRY, Sox-5, and Sp1. Interestingly, deletion of the GATA-1 motif between −1113 and −1104 reduced the promoter activity of the DAF promoter by about 25%. We conclude that the 1.1 kb DAF promoter is a suitable candidate promoter for strong endothelial cell-specific expression of mCRP genes.