IGFBP-3 inhibits Egr-1 transcription by inactivating Erk-Elk1 and Elk1 binding to SRE sites in the Egr-1 promoter. (A-B) IGFBP-3's effects on Egr-1 promoter activity. H460 cells were transiently cotransfected with Egr-1 Luc constructs (A) or an Egr-1 Luc construct carrying mutations in the 3 5′-SRE sites (SRE 3, 4, and 5; B), along with an empty vector (pEV) or pBP-3. The important genetic elements in the Egr-1 regulatory region are shown, including the SRE sites, CRE sites, GC, and TATA boxes. The data are the mean ± SD from 3 independent experiments, with 4 replications per experiment. *P < .05, **P < .01, ***P < .001 compared with pEV-transfected cells. (C) IGFBP-3 reduces in vivo binding of Elk-1 to the Egr-1 promoter. H460 cells, treated with rBP-3 or bovine serum albumin or infected with Ad-BP-3 or Ad-EV, cross-linked and immunoprecipitated with antibodies specific for IGFBP-3, Elk-1, SRF, or a normal serum control antibody (immunoglobulin). The second primer denotes PCR samples using a pair of negative control primers corresponding to the exon 1 sequence of the Egr-1 gene. (D-E) Western blot analysis for the indicated proteins in H460 cells (D) and R⁻ and R⁺ cells (E) treated with the indicated concentrations of rBP-3 for 2 days and stimulated with IGF-1 for 15 minutes.