on July 31, 2003
Published online before print July 31, 2003, doi: 10.1161/01.ATV.0000089326.63053.9A
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Submitted on February 27, 2003
Accepted on July 5, 2003
Ets Motifs Are Necessary for Endothelial Cell-Specific Expression of a 723-bp Tie-2 Promoter/Enhancer in Hprt Targeted Transgenic Mice
Takashi Minami *;
From the Research Center for Advanced Science and Technology (T.M., T.K.), University of Tokyo, Japan; Department of Molecular Medicine (T.M., W.C.A., R.D.R.), Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Mass; and Department of Biology (J.A.K., V.E., R.D.R.), Massachusetts Institute of Technology, Cambridge, Mass.
* To whom correspondence should be addressed. E-mail: minami{at}med.rcast.u-tokyo.ac.jp.
Objective--Tie-2 is an endothelial cell-specific receptor tyrosine kinase that is involved in the remodeling of blood vessels and angiogenesis. Our goal was to characterize Tie-2 promoter function as a means of providing insight into the mechanisms of endothelial cell-specific gene regulation.
Methods and Results--When targeted to the Hprt locus of mice, a small Tie-2 promoter fragment (containing a 300-bp intronic enhancer coupled upstream to a 423-bp core promoter) (T-short) directed widespread endothelial cell expression in vivo. The T-short promoter contains 2 clusters of Ets sites, one in the first exon, the other in the intronic enhancer. In cultured endothelial cells, a combined mutation of the Ets motifs resulted in a significant reduction in promoter activity. Consistent with these results, the same Ets mutations resulted in a loss of detectable expression of the T-short promoter in all vascular beds with the notable exception of the brain.
Conclusions--These results suggest that the T-short promoter contains information for widespread expression in the vascular tree, Ets sites are necessary for in vivo promoter activity, and the shorter Tie-2 fragment may be useful as a tool to direct heterologous gene expression within the intact endothelium.
Key words: Tie-2 • endothelial cells • transgenic mice • gene regulation • Ets motifs
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