maintained as long as HSF1 trimers remain bound to the HSE; when either the stress signal is removed or damaged proteins are no longer generated, the HSR attenuates rapidly, with subsequent conversion of HSF1 back to the monomeric state [7]. Inducible acetylation has also been recently shown to negatively regulate DNA binding activity [8]. Although HSF1 was originally identified as the master regulator of HSP genes, recent studies have indicated an expanding role of this factor, providing evidence that, in some cases, HSF1 may also control the expression of genes with non-chaperone function, some of which participate in the regulation of inflammatory and immune responses [9,10,11]. Based on these observations, we investigated the effect of heat stress on the expression of cyclooxygenase 2 (COX-2), a key enzyme in the regulation of the inflammatory response, catalyzing the rate-limiting step in the synthesis of prostanoids and thromboxanes [12,13]. Herein we report that in human cells heat stress induces the expression of COX-2 at the transcriptional level, by a mechanism involving HSF1-binding to a distal cis-acting HSE regulatory sequence located in the promoter at position 22495 from the transcription start site. Results Heat stress induces the expression of the COX-2 gene in human cells To investigate the effect of heat stress (HS) on COX-2 expression, primary blood monocytes from healthy donors, human umbilical vein endothelial cells (HUVECs) and two human cancer cell lines, HCT116 colorectal adenocarcinoma and lymphoblas- toid Jurkat cells, known to express low levels of COX-2 mRNA and protein [14,15,16,17], were incubated at 43uC for 40 min and allowed to recover at 37uC for 90 min. At this time, total RNA was extracted and levels of COX-2, COX-1 and b-actin mRNA were analyzed by RT-PCR and real-time PCR. As shown in Fig. 1A and B, heat treatment resulted in an increase of COX-2 mRNA levels in all cells analyzed; however, HUVECs and HCT116 cells were found to be more sensitive to COX-2 induction by heat as compared to human monocytes and Jurkat cells. Differently from COX-2, no changes in COX-1 levels were detected in the same samples (Fig. 1A). As expected HS also caused an increase in HSP70 mRNA levels in all cell types (data not shown). The elevated COX-2 mRNA levels found in heat-stressed endothelial cells, together with the recent observation that thermal stress may influence endothelium functions [18], prompted us to analyze the mechanism of COX-2 induction by heat in the endothelium. Induction of COX-2 expression and PGE production by heat stress in endothelial cells To gain further insight into the regulation of COX-2 induction in response to heat stress we initially performed kinetics studies on COX-2 mRNA and protein expression in endothelial cells. HUVECs were either kept at 37uC or were subjected to HS at 43uC for 40 min. At the end of heat treatment, or at different times during the recovery period at 37uC, total RNA was analyzed for COX-2, COX-1, HSP70 and b-actin mRNA levels by RT- PCR and real-time PCR. In parallel samples whole-cell extracts were analyzed for levels of COX-2, HSP70 and b-actin proteins by Western blot. As shown in Fig. 2A and B, COX-2 mRNA levels started to accumulate at the end of heat treatment, reaching maximal levels at 1,5 hour during the recovery period, and went back to control levels after 6 hours. No change in COX-1 mRNA levels were observed at all times, while HSP70 mRNA started to accumulate at the end of heat treatment and went back to control HSF1-Dependent COX-2 Induction by Heat Figure 1. Heat stress induces COX-2 mRNA expression in human cells. Human endothelial cells (HUVEC), peripheral blood monocytes (MW), colon carcinoma cells (HCT116) and T lymphoblastoid cells (Jurkat) were either kept at 37uC (- HS) or incubated at 43uC for 40 min (+ HS) and allowed to recover at 37uC for 90 min. At this time, total RNA was extracted and levels of COX-2, COX-1 and b-actin mRNA were analyzed by RT-PCR (A) and real-time PCR (B). For real-time PCR, relative quantities of COX-2 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. * = P,0.05. doi:10.1371/journal.pone.0031304.g001 levels after 8 hours of recovery at 37uC. Parallel with COX-2 mRNA, COX-2 protein levels started to increase at the end of heat treatment, reached maximal levels at 1,5-3 hours during the recovery period and went back to control levels after 6 hours at 37uC. This is expected since COX-2 protein is typically present for only a few hours after its synthesis, due to the presence of an instability element located upstream of the C-terminus that targets the protein to the ER-associated degradation system and the proteasome [19]. HSP70 protein started to accumulate at the end of heat treatment, but, differently from COX-2, continued to accumulate for at least 8 hours after HS. The subcellular localization of COX-2 in heat-stressed endothelial cells was examined by confocal microscopy. HUVECs growing on coverslips were subjected to heat stress as described above and allow to recover at 37u for 3 h. As shown in Fig. 2D, under normal conditions COX-2 protein was found to be expressed at low levels and to be localized mainly within the nucleus of HUVECs, as previously reported [20]. In heat-stressed cells, a strong nuclear COX-2 immunofluorescence signal was detected at 3 h after treatment; in addition, increased COX-2 levels were found to be diffusely distributed throughout the cytoplasm (Fig. 2D). To determine whether COX-2 up-regulation resulted in an enhancement of COX-2 metabolites generation, the production of prostaglandin E2 (PGE2), a major COX-2 metabolite, was determined. HUVECs were heat stressed as described above and, at different times during the recovery period, whole-cell extracts were analyzed for levels of COX-2 by Western blot, and PGE2 production was determined in the culture supernatants by ELISA. As positive control, HUVECs were stimulated with IL-1b and COX-2 levels and PGE2 production were determined 6 hours PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31304