HUVECs were treated with the COX-2-selective inhibitor NS398 or DMSO vehicle either 1 hour before or during HS at 43uC for 40 min. After a 2 hour recovery period at 37uC, PGE2 production was determined by ELISA. As shown in Fig. 2F, treatment with NS398 resulted in a complete block of PGE2 production, demonstrating that heat-induced COX-2 is responsible for prostanoid synthesis during heat stress. Both mRNA de novo transcription and increased mRNA stability have been reported to be involved in the induction of COX-2 [21,22,23]. To determine whether gene transcription was required for COX-2 induction by heat, HUVECs were pretreated with actinomycin D (5 mg/ml) for 45 min and then subjected to HS at 43uC for 40 min. At 1,5 hours during the recovery period at 37uC, total RNA was extracted and levels of COX-2 and b-actin mRNA were analyzed by real-time PCR. As shown in Fig. 2G, the increases in COX-2 mRNA level was abolished by actinomycin D treatment, indicating that COX-2 mRNA induction by heat involves de novo gene transcription. Identification of the promoter elements responsible for COX-2 gene regulation by heat The regulation of COX-2 gene (Ptgs2) transcription is very complex, involving distinct signaling pathways and different controlling mechanisms depending on the specific stimulus and the cell type. Sequence analysis of the 59-flanking region of the HSF1-Dependent COX-2 Induction by Heat human COX-2 gene has identified several potential transcription- al cis-acting regulatory elements, located in the proximal region of the COX-promoter within the first 550 bp upstream of the transcriptional start site (TSS), with the exception of a peroxisome proliferator response element (PPRE), that is located at 23721 to 23707 upstream of TSS [22]. To identify novel heat-regulated elements in the COX-2 promoter, we performed a computer- based sequence analysis of the 59-flanking region. As shown in Fig. 3A, three regions highly conserved in human and mouse were identified between positions 23120 and 21396 upstream of the TSS. To investigate the role of these regions in COX-2 regulation by heat, a series of 59-deletion constructs including all three (FL- COX2-PGL3, FL), two (-2221-COX2-PGL3, Del1), one (-1787- COX2-PGL3, Del2) or none (-1396-COX2-PGL3, Del3) of the highly conserved regions were subcloned into the pGL3 reporter vector and used for functional analysis (Fig. 3B). HUVECs and HCT116 cells were transiently transfected with the different constructs; after 24 h, transfected cells were either subjected to heat stress at 43uC for 40 min or stimulated with the COX-2 inducer TPA (12-O-tetradecanoylphorbol 13-acetate) as positive control. Luciferase activity was determined after a 6 h recovery period at 37uC. As shown in Fig. 3C and D, heat stress induced reporter activity in both HUVEC or HCT116 cells transfected with the FL construct, while no significant transcriptional activity Figure 3. Identification of the promoter region responsible for COX-2 gene regulation by heat. (A) Schematic representation of human- mouse conserved sequences in the upstream COX-2 gene promoter region identified by using ECR Evolutionary Conserved Regions (ECR) browser (http://ecrbrowser.dcode.org/). X-axis represents base position from transcription start site (+1), and y-axis indicates percentage identity in a window of 100 nucleotides centered on that position. Black areas represent conserved regions that meet ECR default criteria (ECR similarity $70%, ECR length $100 nucleotide). The arrows indicate the nucleotide coordinates of the full length and deletion constructs (Del1, Del2 and Del3) represented in B. (B) Schematic representation of the full-length (FL) and three deletion constructs containing different portions of the 59-flanking sequences of the human COX-2 promoter region (Del1, Del2 and Del3). The negative numbers (-) noted in each construct indicate the distance from the transcription start site. (C,D) Full-length and deletion constructs subcloned into the PGL3-Basic Vector upstream from the luciferase gene were transfected in HUVEC (C) and HCT116 (D) cells. After 24 h cells were subjected to heat shock (43uC for 40 min) (filled bars), treated with 25 ng/ml TPA (dashed bars) or left untreated (empty bars). 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.g003 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31304