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Regulation of cyclooxygenase-2 expression by heat: a novel aspect of heat shock factor 1 function in human cells.

Rossi A, Coccia M, Trotta E, Angelini M, Santoro MG - PLoS ONE (2012)

Bottom Line: 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 -2495 from the transcription start site.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Pharmacology, CNR, 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-E(2) 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 -2495 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.

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Time-course of COX-2 expression at febrile temperatures in endothelial cells.HUVECs were either kept at 37°C (control) or were incubated at 40 and 41°C 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 β-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate ± 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.
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pone-0031304-g006: Time-course of COX-2 expression at febrile temperatures in endothelial cells.HUVECs were either kept at 37°C (control) or were incubated at 40 and 41°C 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 β-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate ± 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.

Mentions: 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 37°C or were incubated at 40 and 41°C for 8 hours. At different times after treatment total RNA was extracted and levels of COX-2, HSP70 and β-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).


Regulation of cyclooxygenase-2 expression by heat: a novel aspect of heat shock factor 1 function in human cells.

Rossi A, Coccia M, Trotta E, Angelini M, Santoro MG - PLoS ONE (2012)

Time-course of COX-2 expression at febrile temperatures in endothelial cells.HUVECs were either kept at 37°C (control) or were incubated at 40 and 41°C 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 β-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate ± 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275557&req=5

pone-0031304-g006: Time-course of COX-2 expression at febrile temperatures in endothelial cells.HUVECs were either kept at 37°C (control) or were incubated at 40 and 41°C 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 β-actin. All reactions were made in duplicates using samples derived from at least three biological repeats. Error bars indicate ± 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.
Mentions: 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 37°C or were incubated at 40 and 41°C for 8 hours. At different times after treatment total RNA was extracted and levels of COX-2, HSP70 and β-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).

Bottom Line: 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 -2495 from the transcription start site.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Pharmacology, CNR, 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-E(2) 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 -2495 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.

Show MeSH
Related in: MedlinePlus