FUNCTIONAL-CHARACTERIZATION OF BOVINE VON-WILLEBRAND-FACTOR GENE PROMOTER IN BOVINE ENDOTHELIAL-CELLS DEMONSTRATES SPECIES-SPECIFIC PROPERTIES

Von Willebrand factor (vWF), a protein necessary for platelet adhesion and thrombus formation, is specifically synthesized in endothelial ce lls and in platelet precursors (megakaryocytes). We previously demonst rated that the sequences localized either in the 5'-flanking region or in the first exon of human (hu) vWF gene (VWF), which regulate the ce ll-specific transcription, are not conserved in the bovine counterpart . In order to look for cis-acting elements involved in the endothelial expression of bovine (be) VWF, fragments including 113 base pairs (bp ) of a sequence 5'-flanking the transcription start point (tsp, + 1) a nd various deletions of the first 233 bp exon were linked in plasmids to the bacterial chloramphenicol (Cm) acetyltransferase gene (cat). Th ese constructs were analyzed by transient transfection in calf pulmona ry artery endothelial (CPAE), human epithelial (HeLa) from cervix and green monkey fibroblasts from kidney (COS) cells. The longest fragment , containing 229 bp of the first exon, was the most active, with ident ical cat expression in the three cell types. The CAT activity was equi valent to that measured by transfection of the same cells with the bas al promoter (from bp -89 to +19) of hu VWF. Addition of upstream bo VW F sequences from bp -113 to -1362 resulted in progressive reduction of the activity of the -113/+229 fragment. The upstream negative regulat ory domains between -1362 and -278 also repressed the heterologous thy midine kinase (tk) promoter in CPAE and HeLa cells. Comparison of resu lts with those previously obtained by transfection of hu vWF promoter in bovine endothelial cells demonstrates that the cis-acting elements do not behave identically in bo vWF promoter. In particular, positive tissue-specific elements able to overcome the negative regulation in e ndothelial cells could not be found in bo VWF between bp -1362 and + 2 29. (C) 1997 Elsevier Science B.V.