Oncogene (2010) 29, 4216–4224 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability S Lecomte1, F Desmots1, F Le Masson2, P Le Goff1, D Michel1, ES Christians2 and Y Le Dre´an1 1Home´ostasie Intracellulaire des Prote´ines, UMR CNRS 6026, Interactions Cellulaires et Mole´culaires, IFR 140–Ge´nomique Fonctionnelle Agronomie et Sante´-, Universite´ de Rennes1, Rennes, France and 2Centre de Biologie du De´veloppement, UMR CNRS 5547, Universite´ Toulouse 3, Toulouse, France A single heat shock factor (HSF), mediating the heat and Morimoto, 1994; Yamamoto et al., 2009), suggesting shock response, exists from yeast to Drosophila, whereas that both factors control different set of genes. HSF1 several related HSFs have been found in mammals. This represents the archetypal stress transcription factor, raises the question of the specific or redundant functions of rapidly activated by a large variety of proteotoxic the different members of the HSF family and in particular stimuli. It regulates the stress-inducible expression of of HSF1 and HSF2, which are both ubiquitously heat shock proteins (HSPs) but also controls numerous expressed. Using immortalized mouse embryonic fibro- other genes as shown by the transcriptome analysis blasts (iMEFs) derived from wild-type, Hsf1À/À, Hsf2À/À (Trinklein et al., 2004). Recently, it was shown that or double-mutant mice, we observed the distinctive HSF1, by favoring cellular adaptation and survival in behaviors of these mutants with respect to proteasome response to environmental stress, also enhances onco- inhibition. This proteotoxic stress reduces to the same genic transformation (Dai et al., 2007). In comparison extent the viability of Hsf1À/À- and Hsf2À/À-deficient cells, with HSF1, HSF2 is a less efficient transcriptional but through different underlying mechanisms. Contrary to activator and appears to be differently responsive to Hsf2À/À cells, Hsf1À/À cells are unable to induce pro- stimuli (Sistonen et al., 1992). However, inhibition of the survival heat shock protein expression. Conversely, proteasome activates both HSF1 and HSF2 (Kawazoe proteasome activity is lower in Hsf2À/À cells and the et al., 1998). Proteasome inhibition leads to the expression of some proteasome subunits, such as Psmb5 accumulation of misfolded proteins and consequently and gankyrin, is decreased. As gankyrin is an oncoprotein induces expression of all the major HSPs (Bush et al., involved in p53 degradation, we analyzed the status of p53 1997), but Pirkkala et al. (2000) clearly showed that in HSF-deficient iMEFs and observed that it was strongly HSF1, but not HSF2, has a key role in this induction. stabilized in Hsf2À/À cells. This study points a new role for Nevertheless, to add to the complexity of this proteo- HSF2 in the regulation of protein degradation and toxic response, our group showed that an HSF1/HSF2 suggests that pan-HSF inhibitors could be valuable tools heterocomplex was present on the promoter of the to reduce chemoresistance to proteasome inhibition chaperone gene clusterin, following proteasome inhibi- observed in cancer therapy. tion (Loison et al., 2006). The existence of the HSF1/ Oncogene (2010) 29, 4216–4224; doi:10.1038/onc.2010.171; HSF2 heterotrimer was recently confirmed (Sandqvist published online 24 May 2010 et al., 2009) and it was shown that HSF2 can act as a modulator of HSF1 activity in response to proteotoxic Keywords: heat shock factor; MG132; proteasome; insults (Ostling et al., 2007). Hence, the respective roles gankyrin; p53 of HSF1 and HSF2 remain unclear when cells are exposed to proteasome inhibitors. The proteasome/ubiquitin system constitutes the Introduction major pathway for the regulated degradation of intracellular proteins. The proteasome holoenzyme is Vertebrate genome contains 3–5 members of the heat a large multi-subunit complex composed of a 20S shock transcription factor (HSF) family (for reviews, see proteolytic core particle associated with one or two Pirkkala et al., 2001; Akerfelt et al., 2007). Within this 19S regulatory particles (Pickart and Cohen, 2004). family, HSF1 and HSF2 possess closely related DNA- The 20S core particle is a cylindrical structure made up binding domains, but they exhibit some slight differ- of four heteromeric rings. The two outer rings are ences in heat shock element (HSE) recognition (Kroeger composed of seven different a subunits (named a1toa7), whereas the two inner rings are composed of seven different b subunits (b1–b7) and form the catalytic Correspondence: Dr Y Le Dre´an, Home´ostasie Intracellulaire des chamber. Although these subunits are evolutionary Prote´ines, UMR CNRS 6026, Universite´de Rennes 1, Bat. 13, campus related and similarly conserved, only b1, b2andb5 de Beaulieu, Rennes Cedex 35 042, France. E-mail: [email protected] possess proteolytic properties. The 19S regulatory Received 10 June 2009; revised 23 February 2010; accepted 9 April 2010; particle comprises approximately 18 distinct subunits published online 24 May 2010 that form a lid controlling the access to the catalytic core HSF2-dependent expression of proteasome subunits S Lecomte et al 4217 particle. Proteins destined to be degraded by proteasome analysis of cellular viability. The WT or HSF-deficient must be conjugated to multi-ubiquitin chain for iMEFs were treated for 16 h with 1 mM of MG132, and recognition by the 19S regulatory particle. Dysfunction then apoptotic levels were determined by staining with of the proteasome pathway can lead to many disorders, annexin V-fluorescein isothiocyanate and 7-aminoacti- including cancers and neurodegenerative diseases (Paul, nomycin D (Figure 1d and Supplementary Figure S1). 2008). Proteasome is crucial for the regulation of cell Mortality of all the cell lines was increased in MG132- cycle and apoptosis and its specific inhibition by treated samples, and apoptosis level in HSF-deficient molecules has emerged as a promising strategy to treat cells was found to be twofold higher compared with WT cancers (Nencioni et al., 2007). For example, borte- cells. These data revealed that both factors, HSF1 and zomib (also known as PS-341 or Velcade) is the first HSF2, are critical for cell survival when proteasome is proteasome inhibitor used in the treatment of multiple inhibited. myeloma (Twombly, 2003). In addition, proteasome inhibition may modulate many other transcriptional HSP induction is differently affected events (Muratani and Tansey, 2003) and the activity in Hsf1À/À and Hsf2À/À iMEFs of the stress-related transcription factors, such as p53 HSPs are important chaperones induced by proteotoxic and HSFs. stress which contribute to cell death resistance (Beere, To determine the contribution of HSF2 versus HSF1 2005). Thus we analyzed the inducible expression of two in cell response to proteasome inhibitors, we used HSPs: HSP70 and HSP25. Messenger RNA levels of immortalized mouse embryonic fibroblasts (iMEFs) Hsp25 and Hsp70 in WT, HSF1 and/or HSF2-deficient derived from wild-type (WT), Hsf1 and/or Hsf2 knock- iMEFs were compared in the absence or presence of out mice (McMillan et al., 1998, 2002). We found that MG132 (Figures 2a and b). After MG132 exposure, both factors are essential for the cell viability but have only iMEFs expressing HSF1 (either WT or Hsf2À/À) distinct actions: as previously shown, HSF1 is required were able to exhibit a strong induced expression of both in the induction of pro-survival HSP expression and, in chaperones, whereas iMEFs deficient in HSF1 (Hsf1À/À contrast, HSF2 is involved in the regulation of protea- and Hsf1À/À & Hsf2À/À) did not. Those data indicated some subunit expression. that expression of inducible Hsp70 and Hsp25 is highly dependent on the presence of HSF1. These results were confirmed by immunoblot (Figure 2c): HSP25 and HSP70 are strongly increased in WT cells but not in Results HSF1-depleted cells (Hsf1À/À and Hsf1À/À & Hsf2À/À). Moreover, HSF2-depleted cells show higher levels of Proteasome inhibition is severely toxic for HSF1- and/or HSP25 and HSP70 than those observed in WT iMEFs, HSF2-deficient cells which could be explained by a lower level of protein To determine the specific contributions of HSF1 and degradation and thus an increase in the protein’s half- HSF2 to the cellular response to proteasome inhibition, life in those Hsf2À/À cell lines. Hence, it was unlikely that we first examined the cytotoxic effect of two proteasome the high sensitivity of Hsf2À/À cells to proteasome inhibitors: the peptide aldehyde MG132, a reversible inhibition could be explained by the lack of HSPs. This inhibitor, and the peptide epoxyketone epoxomicin, an suggested that another mechanism should be involved. irreversible inhibitor. To assess the viability of iMEF cells, we used the quantitative colorimetric 1-(4,5- dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) test Proteasome activity is decreased in Hsf2À/À iMEFs as cytotoxicity assay and established concentration– To test the possibility that proteasome function would effect curves after a 16 h treatment. With high concen- be natively affected, an in vitro assay based on the trations of MG132 (830 nM) or epoxomicin (250 nM), measurement of the chymotrypsin-like activity of Hsf1À/À, Hsf2À/À, or Hsf1À/À & Hsf2À/À iMEFs were proteasome present in crude protein extract was first significantly more sensitive to these proteasome inhibi- used. Second, the proteasome activity at the cellular tors than WT cells (Student’s t-test, Po0.05 and level was evaluated, by setting up an assay based on the Po0.001, respectively) (Figures 1a and b, respectively). degradation of a proteasome-targeted protein. By comparison, we treated cells with thapsigargin that A fluorescent proteasome substrate (Z-LLVY-AMC) induces a proteotoxic stress specific to the endoplasmic was incubated with cellular extracts from the different reticulum without affecting proteasome activity and iMEF cell lines.