HSF1-Dependent COX-2 Induction by Heat was detected in cells transfected with Del1, Del2 or Del3 constructs, suggesting that the region spanning between 23120 to 22221 may contain the element/elements responsible for heat- induced COX-2 transcriptional activity. Differently from heat stress, TPA was able to induce significant reporter activity in cells transfected with all four constructs. These results are consistent with the findings of others who identified the elements responsive to TPA in the first 550 bp upstream the TSS [22]. To identify the enhancer elements responsible for HS-induced COX-2 gene expression, the identified region spanning between 23120 to 22221 was analyzed by TFSEARCH version 1.3 (Yutaka Akiyama, TFSEARCH: Searching Transcription Factor Binding Sites, http://www.cbrc.jp/research/db/FSEARCH. html) [24]. Using a cut-off threshold score of 85%, TFSEARCH prediction analysis identified several transcription factors that may bind to this region. Among these factors, one putative HSF1- binding element located at position 22495 (HSE) and one putative GATA-binding element (GATA-RE) located at 22570 from the transcription start site were detected (Fig. 4A). Since HSEs represent the target for HSF1-mediated heat shock response regulation and GATA response elements were reported to be able to transactivate the HSP70 promoter [25], we focused our Figure 4. Characterization of distal cis-acting HSE regulatory elements in the human COX-2 gene. (A) Nucleotide sequence of human COX-2 promoter region between 23120 and 22221, as indicated in Fig. 3A. Location of putative binding sites for HSF1 and GATA transcription factors in the COX-2 promoter identified by TFSEARCH are indicated. (B) Schematic representation of the wild type COX-2-PGL3 construct (WT) and the D-HSE-D-GATA, D-HSE and D-GATA PGL3 constructs. (C,D) WT, D-HSE-D-GATA, D-HSE and D-GATA deletion constructs (shown in B) were transfected in HUVEC (C) and HCT116 (D) cells. After 24 h cells were subjected to heat shock (43uC for 40 min) (+ HS), or left untreated (- HS). Whole- cell extracts were analyzed for luciferase activity after 6 h recovery at 37uC. The data, expressed as fold induction of untreated control, represent the mean of quadruplicate samples from two independent experiments. Error bars indicate 6 S.D. * = P,0.05. doi:10.1371/journal.pone.0031304.g004 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31304