HSF1-Dependent COX-2 Induction by Heat Figure 6. Time-course of COX-2 expression at febrile temperatures in endothelial cells. HUVECs were either kept at 37uC (control) or were incubated at 40 and 41uC as described in Material and Methods. At different times total RNA was extracted and levels of COX-2 (A) and HSP70 (B) were analyzed by real-time PCR. Relative quantities of COX-2 and HSP70 RNAs were normalized to b-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate 6 S.D. Fold induction was calculated by comparing the induction of the indicated genes in the treated samples to the relative control, which was arbitrarily set to 1. doi:10.1371/journal.pone.0031304.g006 Therefore, COX-2 protein is typically present for only a few hours after its synthesis. Depending on the cell type, COX-2 expression can be rapidly induced by a large number of stimuli, which include proin- flammatory cytokines such as IL-1, IL-2 and TNFa, bacterial endotoxin (LPS), growth factors, oncogenes, and tumor promoters (TPA) [22]. The regulation of COX-2 gene transcription is very complex, involving distinct signaling pathways and different controlling mechanisms depending on the specific stimulus and the cell type. The promoter region of the COX-2 gene presents sequences of typical immediate early genes with potential transcriptional regulatory cis-elements mainly located in the 550 nucleotide region upstream of the transcriptional start site (TSS); these include two cyclic AMP response elements (CRE), a sterol response element (SRE), two nuclear factor kappa B (NF-kB) sites, an SP1 site, a CAAT enhancer binding protein C/EBP, two AP-1 sites, an E-box, and a TATA box [22]. A distal peroxisome proliferator response element (PPRE) has also been identified at position 23721 from the TSS [34]. The relative contribution of each cis-acting promoter element and its interaction with transcription factors activated by multiple signaling pathways depends on the cell type, the stimulus and the time following the stimulus [22]. We have now identified a new putative distal cis-acting heat shock element (HSE) located at position 22495 from the TSS, responsible for binding the heat-regulated factor HSF1. We provide evidence that heat stress promotes COX-2 expression in human cells, and that this effect is mediated by HSF1. Following heat stress at 43uC for 40 min, COX-2 mRNA levels start to accumulate at the end of heat treatment, reaching maximal levels at 1,5 hour during the recovery period. Similarly to the effect described for other COX-2 inducers, heat-induced COX-2 expression is transient, and COX-2 levels return back to basal 6 hours after heat exposure. No change in COX-1 mRNA levels are observed at all times after HS. Parallel with COX-2 mRNA, COX-2 protein levels were also found to increase at the end of heat treatment, reaching maximal levels at 1,5–3 hours during the recovery period at 37uC. Differently from the heat shock protein HSP70, COX-2 protein returned to basal levels after 6 hours at physiological temperature, possibly due to proteolysis by the 26 S proteasome. Both mRNA de novo transcription and increased mRNA stability have been reported to be involved in the induction of COX-2 [22,23]. We have shown that heat-induced COX-2 expression is regulated at the transcriptional level and have identified, by in vitro reporter gene activity assay and deletion mutant constructs analysis, the promoter region responsive to heat induction and the position of the HSE involved. As indicated above, the new putative cis-acting heat shock element identified was located in a distal region of the COX-2 promoter at position 22495 from the transcription start site. In vitro and in vivo binding of HSF1 to the HSE identified in the COX-2 promoter was demonstrated by gel shift analysis and chromatin immunoprecipitation analysis, respectively. Deletion of the HSE sequence resulted in abrogation of heat-induced COX-2 transcription, indicating that HSF1 plays a central role in the transcriptional control of this gene during heat-stress. The presence of a distal HSE sequence located at 22495 from the TSS in the COX-2 promoter adds a new element in the complex regulation of COX-2 expression and also represents a novel finding in heat shock regulation of human genes. In fact the functional HSEs characterized so far in the promoters of classical and non-classical heat-shock human genes are usually restricted to the promoter region proximal (,1000 bp) to the PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31304