Transcriptional activation of CXCR4 by PGE₂. (A) HMECs were stimulated with physiologic PGE₂ levels for 1 hour. Thereafter, CXCR4 mRNA expression was determined. (B) HMECs were stimulated with 10^–9 M of PGE₂ at indicated time points and CXCR4 mRNA expression was analyzed. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as control. A representative experiment of 2 is shown. (C) A schematic representation of reporter gene constructs containing the CXCR4 promoter region –691 bp up to +87 bp is provided, which shows the presence of consensus DNA-binding elements for several transcription factors. Controls consisted of the basic PGL3 vector containing the appropriate construct transfected into primary HMECs, in the presence or absence of PGE₂, VEGF, or bFGF. The promoter activity for each construct after different stimulus is shown as normalized dual luciferase activity. The largest construct (pGL3-777) exhibited very little activity in response to different stimuli. The construct –191 to +87 (pGL3 279) yielded a 4-to 5-fold increase in activity after stimulation with VEGF, bFGF, and PGE₂. (D) Sp1-binding sites are essential for induction of CXCR4 by VEGF, bFGF, and PGE₂. Deletion studies were performed using the pGL3 279 construct as template; a construct with the NF-κB DNA-binding element deleted (pGL3 248) but containing the 3 Sp1-binding sites exhibited very similar responses to the construct pGl3 279. The smallest construct –50 to +83 (pGL3 142), which lacks the 3 Sp1-binding sites, showed virtually no response to any stimuli. The experiments depicted in this figure were performed several times. The bars represent SEM of 3 experiments, in which samples were run in triplicate.