Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells Antonio Rossi1., Marta Coccia1,2., Edoardo Trotta1, Mara Angelini2, M. Gabriella Santoro1,2* 1 Institute of Translational Pharmacology, CNR, Rome, Italy, 2 Department of Biology, University of Rome Tor Vergata, Rome, Italy Abstract The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat- shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non- canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E2 in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion- mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position 22495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function. Citation: Rossi A, Coccia M, Trotta E, Angelini M, Santoro MG (2012) Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells. PLoS ONE 7(2): e31304. doi:10.1371/journal.pone.0031304 Editor: Harm Kampinga, University Medical Center - University of Groningen, The Netherlands Received November 3, 2011; Accepted January 6, 2012; Published February 8, 2012 Copyright: 2012 Rossi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Italian Ministry of Health, ISS projects ‘Ricerca Oncologica’ [N. 7OCF/7] and ‘Alleanza contro il Cancro’ [N. ACC12], and the EU-EICOSANOX project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: santoro@bio.uniroma2.it . These authors contributed equally to this work. Introduction The heat shock response (HSR) is a finely regulated and highly conserved mechanism, which protects living cells against proteo- toxic stress induced by different types of environmental and pathological conditions, initiating a regulatory cascade for recovery and adaptation [1]. This occurs by a nearly instantaneous induction of a set of genes, known as heat shock (HS) genes, leading to expression of cytoprotective heat shock proteins (HSP), which is proportional to the intensity and duration of stress [2,3]. Studies on the heat shock response have revealed insights on the stress-sensing cellular devices and on the role of heat shock proteins in repairing protein damage. The HSR is regulated by a family of heat shock transcription factors (HSFs) that are expressed and maintained in an inactive state under non-stress conditions. Mammalian genomes encode three homologues of HSF (HSF1, HSF2 and HSF4) regulating HSP expression. Among these HSF1 is considered to be the paralog responsible for regulating the heat-induced transcriptional response [4]. HSF2 has also been reported to contribute to inducible expression of heat shock genes through interplay with HSF1 [5]. HSF1 is generally found in the cytoplasm as an inert monomer lacking transcriptional activity; both DNA-binding and transcrip- tional transactivation domains are repressed through intramolec- ular interactions and constitutive serine phosphorylation [6]. Upon exposure to heat shock and other types of stresses, which cause protein damage, HSF1 is derepressed in a stepwise process that involves oligomerization of HSF1 monomers to a trimeric state, localization to the nucleus, inducible phosphorylation and sumoylation, and binding of nuclear-localized trimers to DNA sequences known as heat shock elements (HSE). Functional HSE sequences are characterized by an array of inverted repeats of the pentameric motif nGAAn and are usually located in the proximal region of HSF1-responsive gene promoters. Binding of HSF1 to HSE sequences is followed by a rapid shift in the transcriptional program resulting in the high rates of transcription of cytoprotective heat shock genes, which include molecular chaperones of the HSP70 and HSP90 families, HSP27 and other proteins of the network. High rates of transcription are PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31304