attention on these two elements by performing fine structure deletion analysis. A series of constructs deleted for the binding elements for HSF1 (D-HSE), GATA1 (D-GATA), and for both HSF1 and GATA1 (D-HSE-D-GATA) were therefore generated (Fig. 4B). The D-HSE-D-GATA, D-HSE and D-GATA or the wild-type (WT) constructs were transiently transfected in HUVEC and HCT116 cells. At 24 h after transfection, cells were subjected to heat stress, and the promoter activity was determined as described above. As shown in Fig. 4C and D, the deletion of both HSE and GATA response elements resulted in an almost complete block of heat-induced transcription. Deletion of the GATA element alone did not alter the ability of the COX-2 promoter to respond to heat; on the other hand, deletion of the HSE element prevented transcriptional activation, identifying the HSE sequence present in the COX-2 promoter as a critical element for heat-induced COX- 2 transcription. HSF1 binds directly to the COX-2 promoter in vitro and in vivo The identification of a functional HSE element in the COX-2 promoter suggested that HSF1 might participate directly in the regulation of COX-2 expression. Heat stress is known to activate HSF1 as early as few minutes. To establish the role of HSF1 in COX-2 up-regulation by heat, kinetics experiments were performed to determine HSF1 DNA- binding activity to the COX-2 promoter in vitro and in vivo. HUVECs were subjected to a 40 min heat shock at 43uC and then allowed to recover at 37uC. At different times during heat treatment or the recovery period, whole-cell extracts were analyzed for HSF1 DNA-binding activity by EMSA using a large DNA fragment of 100 bp (from 22579 to 22480) of the COX-2 promoter region comprising binding sites for the GATA and HSF1 transcription factors (Fig. 5A). In parallel, the same samples were analyzed for HSF1 DNA-binding activity by EMSA using a 30 bp oligonucleotide containing an ideal HSE from the HSP70 promoter [26]. A specific slower-migrating mobility complex, subsequently identified as HSF1 (see below), was found to appear as early as 20 min in the heat-shocked samples using the COX-2 promoter derived 100 bp sequence (Fig. 5B, upper panel). The slower- migrating mobility complex persisted up to 40 min of the recovery period, after which time it started to attenuate. This kinetics is consistent with the kinetics of HSF1 phosphorylation/dephos- phorylation, which is known to be strictly associated with HSF1- DNA binding activity (Fig. 5B middle panel) [27]. A similar DNA- binding pattern was obtained when the ideal HSE probe was used (Fig. S1). Similarly to the DNA-binding analysis using the ideal HSE element, two faster migrating bands present in both heat- treated and control samples representing the non-specific (NS) and the constitutive HSE binding activity (CHBA) respectively [26], were detected also using the COX-2 promoter derived sequence. To determine whether HSF1 was involved in the formation of the slower-migrating mobility complex observed in Fig. 5B, a super-shift analysis was performed using specific polyclonal anti- HSF1 antibodies. As shown in Fig. 5C, the complex was selectively super-shifted in the presence of anti-HSF1 antibodies, indicating that HSF1 is the primary component of the heat-induced DNA binding activity observed. To investigate whether HSF1 is recruited to the COX-2 gene promoter, HUVECs were subjected to a 40 min heat shock at 43uC and then allowed to recover at 37uC. At different times during heat treatment and the recovery period HSF1 recruitment to the COX-2 and HSP70 promoters was analyzed by ChIP HSF1-Dependent COX-2 Induction by Heat analysis. HSF1-coprecipitating DNA was analyzed by PCR (Fig. 5D) and real-time PCR (Fig. 5E) with promoter-specific primers amplifying the COX-2 and HSP70 promoters respective- ly, and the rate of amplification was verified using cross-linked, not immunoprecipitated chromatin. The specificity of chromatin immunoprecipitation was determined by using a control unrelated antibody. Consistently with the DNA-binding activity revealed by EMSA (Fig. 5B, upper panel), HSF1 was recruited to the COX-2 promoter during heat treatment and for at least 90 min of the recovery period at 37uC (Fig. 5D,E upper panels). A similar kinetics of HSF1 recruitment was observed also for the HSP70 promoter (Fig. 5D,E lower panels). An identical set of experiments was performed in HCT116 cells. As shown in Fig. S2, the results obtained were comparable to the ones described above for HUVECs. In addition, heat-induced COX-2 mRNA expression was greatly reduced in HCT116 cells in which HSF1 expression was stably suppressed by RNA interference (Fig. S3), confirming an important role of this factor in heat-regulated COX-2 transcription. COX-2 expression is induced at febrile temperatures in endothelial cells Partial activation of HSF1 has been described during exposure to febrile, sub-heat shock temperatures [28,29,30,31]. To investigate whether COX-2 expression may be induced at febrile temperatures in endothelial cells, HUVECs were either kept at 37uC or were incubated at 40 and 41uC for 8 hours. At different times after treatment total RNA was extracted and levels of COX- 2, HSP70 and b-actin as control were analyzed by real-time PCR. As shown in Fig. 6A, COX-2 expression was increased, reaching maximal levels at 8 hours of continuous exposure to physiological fever temperature. At this time a modest increase in COX-2 protein level and PGE2 production was also observed (Fig. S4); however, the effect of febrile temperatures on the kinetics of different COX-2 metabolites production needs to be further investigated. Interestingly, the kinetics of heat-induced COX-2 expression differed from HSP70 mRNA accumulation, that, as expected, reached a pick at 2–3 hours after exposure (Fig. 6B). Discussion Cyclooxygenases-1 and -2 (COX-1 and COX-2), also known as prostaglandin (PG) endoperoxide synthase, are heme-containing ER membrane-bound N-glycoproteins that catalyze the commit- ted step in prostaglandin formation. COX-1 and 22 convert arachidonic acid, hydrolyzed from cell membrane phospholipids by a phospholipase A2 (PLA-2), to prostaglandin endoperoxide H2 (PGH2), the precursor of thromboxanes, prostacyclin and different prostaglandins [23,32]. COX-1 is constitutively expressed in almost all mammalian tissues under basal conditions, while COX-2 expression is highly restricted (except for kidneys, seminal vesicles and certain areas of the brain), but can be dramatically upregulated following the appropriate stimuli [22]. In addition, differences in regulation of the COX enzymes at the post- transcriptional and post-translational levels also contribute signif- icantly to their distinct patterns of expression. COX-1 mRNA and COX-1 protein are very stable, whereas both COX-2 mRNA and COX-2 protein have short half-lives. The 39-UTR of the human COX-2 gene contains 23 copies of the ‘ATTTA’ RNA instability element that participates in post-transcriptional regulation of COX-2 expression [33]. On the other hand, a unique 27 amino acid instability element located upstream of the C-terminus of COX-2 targets this isoform to the ER-associated degradation system and proteolysis by the cytosolic 26 S proteasome [19]. PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31304