TSS [35]. In the case of the lactate dehydrogenase promoter HSF1 binding to a distal HSE located at 23819 from the TSS was described following ERB2 overexpression [36]. However, HSF1 recruitment to distal HSEs leading to transcriptional activation under heat stress conditions has never been described to our knowledge. As indicated above, COX-2 transcription is dependent on the specific stimulus and the cell type. In response to heat we also observed different levels of COX-2 expression in different human primary and tumor cell lines. COX-2 levels appear to be enhanced considerably (3–4 fold as compared to control) in human endothelial cells and HCT116 colon carcinoma cells, and to a lesser extent in primary monocytes and lymphoblastoid cells (Fig. 1), but not in several breast cancer cell lines (data not shown). This effect could be due to the presence of other cell-type specific transcription factors needed to cooperate with HSF1 in COX-2 induction. On the other hand, it can be speculated that the differences observed in heat-induced COX-2 expression in different cell types may reflect differences in the chromatin modification signature associated with highly cell-type specific histone modification patterns, which in turn can affect HSF1 binding and/or its transcriptional potential [37,38]. It has been in fact recently demonstrated in Drosophila that the chromatin landscape before heat shock can dictate HSF binding to target DNA elements [39]. In the case of classical heat shock genes, however, because of the ubiquitous and conserved nature of the HSR, functional HSE binding sites are likely to be evolutionary constrained at the sequence level and actively maintained in the accessible state at the level of chromatin organization in all cell types [39]. The possibility of interplay of different HSFs in the regulation of COX-2 should be also considered. As indicated in the Introduc- tion, in human cells three different HSF family members exist, which exhibit differential specificity for different types of heat shock elements [5,40]. This, together with cell type-specific expression of HSFs [5,40], may be important in determining the target genes of each heat shock factor. COX-2 up-regulation resulted in an enhancement of COX-2 metabolites generation, as demonstrated by the production of PGE2. It should be noted that, despite the moderate increase in COX-2 mRNA level (3–4 fold) as compared to a strong induction of classical heat shock genes like HSP70 (50–80 fold), a potent biological response is achieved. In fact, as early as 2 hours after heat stress, the increase in PGE2 production was comparable to that reached in cells stimulated with the COX-2 inducer IL-1b, and PGE2 levels continued to rise until 4– 6 hours after stress. It should be noted that an increase in arachidonic acid metabolites production, associated to stimula- tion of a phospholipase A2 (PLA-2) activity, following heat shock has been previously reported in mammalian cells [41]. In the case of endothelial cells, the fact that treatment with the selective COX-2 inhibitor NS398 resulted in a complete block of PGE2 production, demonstrates that PGE2 production is dependent on de-novo synthesis of heat-induced COX-2 and is not merely the consequence of increased prostanoid secretion. This observation, together with the previously described increase of phospholipase A2 activity by heat, suggests the possibility that heat stress may coordinate the entire cascade of prostanoid synthesis by simultaneously inducing COX-2 protein expression and stimulating PLA-2 activity to increase COX-2 substrate bioavailability. Increased COX-2 levels have been previously observed at elevated temperatures in vivo in murine models [42,43]. The possibility that also in human cells COX-2 expression may be HSF1-Dependent COX-2 Induction by Heat induced in vivo at high temperatures, as in the case of burn injuries or hyperthermic treatment of cancer, should be considered. In the case of burns, that are known to cause the release of arachidonic acid metabolites [44], since thermal insults activate an inflamma- tory cascade which culminates in the infiltration of specialized cells into injury sites, it could be hypothesized that HSF1-induced COX-2 expression may participate in these events during the initial phase of inflammation. Elevated temperatures are also reached during hyperthermic treatment of tumors [45]. In particular, in the case of whole-body hyperthermia, a procedure used for metastatic cancer treatment, temperatures up to 42uC are reached and maintained for about 1 hour in the entire body [46]. In this case, vascular endothelium is subjected to heat stress conditions, which in vitro induce COX-2 expression and prosta- glandins production. The possibility that COX-2 expression and production of high levels of arachidonic acid metabolites may be induced under these conditions should be taken into consideration, especially in view of treatment with non-steroidal anti-inflamma- tory drugs. Interestingly, COX-2 expression was also induced at mild (40 and 41uC) temperatures in endothelial cells, reaching maximal levels after 8 hours of exposure, suggesting the possibility that COX-2 expression could be increased during fever. This phenomenon could contribute to the increase in blood prosta- glandin level that has been documented previously in the late phase of fever [47]. The role of HSF1, as well as HSF2, which has been recently shown to be a primary factor required for maintaining protein homeostasis against febrile range thermal stress in murine cells [48], on COX-2 regulation at febrile temperatures needs to be further investigated. Apart of the possible physiological implications, the identi- fication of COX-2 as an HSF1-target gene opens new perspectives on the role of this factor. HSF1 was originally identified as the master regulator of the transcription of heat shock genes, leading to the rapid expression of cytoprotective heat shock proteins. In mammalian organisms during stress conditions different HSP act in concert constituting the cell ‘‘protein repair machinery’’ preventing protein denaturation and aggregation that are detrimental to cells, and promoting degradation of irreversibly denaturated cytotoxic proteins [3,49,50]. Under normal conditions, HSP also function as chaperones that assist in protein folding [51]. Whereas it is well established that HSF1 regulates inducible HSP expression, recently it has become evident that the regulation of the mammalian HSR is a more complex phenomenon than previously thought, and in some cases HSF1 may also control the expression of genes with non-chaperone function, some of which participate in the regulation of the inflammatory and immune responses, including interleukins 1b (IL-1b), 6 (IL6), tumor necrosis factor a (TNFa) and different chemokines [11,52,53,54]. The fact that HSF1 also regulates the expression of an important mediator of inflammation as COX-2 reinforces these previous observations, and brings the attention to this less known and still poorly understood role of HSF1 during thermal stress. It could be hypothesized that if, on one side, HSF1 activates a fundamental and highly conserved defense response through the robust expression of classical heat shock genes with chaperone functions to protect individual cells, on the other it may also coordinate a different type of response to thermal injury, through a more finely tuned, cell-type specific induction of non-chaperone heat-responsive genes, among which COX-2. The understanding of this different aspect of HSF1 function may indicate new strategies in the treatment of immune and inflammatory disorders. PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31304