Ets-1 is a critical regulator of
Ang II-mediated vascular
inflammation and remodeling
J. Clin. Invest. Yumei Zhan, et al. 115:2508 doi:10.1172/JCI24403 [
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Figure 6Regulation of the human
MCP-1 gene by Ets-1. (
A) Schematic of the human
MCP-1 promoter with putative Ets-1 binding sites (filled circles). The transcription start site is indicated by the right arrow, and MCP-1 deletion constructs are designated A, B, and C. Regions used for PCR analysis in the ChIP assay (ChIP1–4) are shown with expected size of PCR fragments. Locations of Ets-1–binding site mutations are shown as M1 (–2,253) and M2 (–402). (
B) Transactivation of the
MCP-1 promoter (construct A) by a panel of Ets factors in RASMCs shown as fold induction compared with control pCI expression plasmid. (
C) ChIP assay of
MCP-1 promoter using HASMCs after Ang II stimulation (100 nM) for 2 and 4 hours. An Ets-1 polyclonal antibody was used for precipitation. PCR analysis of the input (control), in the absence of antibody (–Ab), and after immunoprecipitation with antibody (+Ab) using primers corresponding to 4 regions of the
MCP-1 promoter. Molecular weight markers are shown on the left. (
D) Gel mobility shift assay using oligonucleotide probe encoding the –2,253
MCP-1 promoter Ets-1 site. HASMCs were collected after stimulation with Ang II for 0 (Control), 2, 4, and 16 hours. The 4-hour lysate was incubated with the wild-type oligonucleotide probe, in the presence of either 10 ng of unlabeled (Cold) wild-type or mutant oligonucleotide or 4 μg of an Ets-1 polyclonal antibody or IgG isotype-matched control. (
E) Ang II–induced (100 nM) transactivation of deletion constructs A, B, and C and site-directed mutants of the full-length
MCP-1 promoter constructs containing the M1, M2, and M1 + M2 mutations in RASMCs are shown as fold induction compared with unstimulated control. (
F) Transactivation of deletion constructs A, B, and C and site-directed mutants M1, M2, and M1 + M2 by Ets-1 in RASMCs are shown as fold induction compared with contransfection with empty mammalian expression plasmid.
