Cytomegalovirus (CMV) infection of cndothelium with resultant enhanced expression of ICAM-1 has been implicated in the pathophysiology of cardiac allpgraft vasculopathy. Infection of human umbilical vein endothelial cells (HUVECs) with the CMV strain, VHL/E, resulted in a 4-fold increase in ICAM-1 mRNA at 2 hours postinfection, which declined to a 1.5-fold increase 24 hours post-infection. Because of the rapid rise in mRNA levels following infection, we hypothesized that the CMV immediate early (IE) proteins, known to be transcriptional transactivators of certain heterologous promoters, may regulate the ICAM-1 promoter-regulatory region. HUVECs were transfected with plasmid constructs containing consecutive 5' upstream end deletions from -1165 to -277 bp of a cloned ICAM-1 gene promoter-regulatory region and upstream flanking sequences fused to the chloramphenicol acetyl transferase (CAT) gene, with or without a plasmid construct containing the CMV major IE promoter upstream of the CMV IE1, IE2, or IE1,IE2 coding regions. Cells were harvested 48 hours after transfection, and the acetylation of 14C-chloramphenicol determined by thin layer chromatography and autoradiography. Using this transient expression assay, the following results were obtained: 1) CMV e2 upregulated expression of the ICAM-1 gene, with IE1 and IE2 acting in a synergistic manner, and 2) only the 5' region between -370 bp and -277 bp was critical for upregulation by the IE gene products. These results show that the IE gene products synergistically transactivate the heterologous ICAM-1 promoter in human endothelial cells, and that a 100 bp region in the promoter is necessary for activation by IE proteins. The molecular mechanism(s) involved in the transactivation of the ICAM-1 promoter within this critical region are under investigation.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|