Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro Mara A. Bonelli, Roberta R. Alfieri, Maura Poli, Pier Giorgio Petronini, and Angelo F. Borghetti 1 Sezione di Patologia Molecolare e Immunologia, Dipartimento di Medicina Sperimentale, Universita ` degli Studi di Parma, 43100 Parma, Italy The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expres- sion of the heat shock-inducible HSP70. When late pas- sage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20 –32, 1999). When late passage cells were serum-starved overnight, we ob- served a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37°C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum- starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incu- bated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsi- ble for HSF1 degradation. © 2001 Academic Press Key Words: HSP70; HSF1; HSP70 mRNA; MG132; pro- teasome; aging; human fibroblast; heat shock; serum deprivation. INTRODUCTION Cell protection from stress is mainly attributed to the induction of a family of proteins, the heat shock proteins (HSPs). As molecular chaperones, HSPs have the ability to prevent aggregation and/or to repair dam- aged proteins. It is well known that cells aged in cul- ture or isolated from aged organisms accumulate an increased level of altered proteins and have impaired responses and adaptation to stresses. The concomi- tance of these aspects has been investigated by study- ing the relationship between cell aging and expression of the heat-inducible HSP70, the prominent HSP. A reduction in the synthesis and accumulation of induc- ible HSP70 with age has been shown to occur in a variety of mammalian tissues and cells [1–5]. Several authors have investigated, at the molecular level, the attenuated expression of inducible HSP70 during in vitro aging of diploid human fibroblasts (HF). To date, the prevailing opinion is that the reduced synthesis of HSP70 observed in late passage HF exposed to heat shock is due to a decreased transcription of the HSP70 gene. In IMR-90 HF, the age-associated dysfunction in the signaling mechanism of the heat shock response has been assumed to occur at the gene level, since induction of the HSP70 mRNA level was significantly lower in late passage fibroblasts than in early passage fibroblasts [6]. This was subsequently corroborated by the observation of an age-dependent reduction in the heat-inducible binding of transcription factor HSF1 to the HSE-specific sequence [7]. A reduction in heat shock gene expression accompanied by reduced accu- mulation of the heat shock transcripts has also been reported in senescent WI-38 HF [8]. Recently, the de- cline in HSP70 expression during cellular senescence in vitro and in cells derived from old human subjects was found to be paralleled by a decrease in the levels of HSF1 [9]. Recently, we showed that in late passage WI-38 HF the presence or absence of serum during the last period of culturing before heat shock changes the step at which occurs the molecular lesion responsible for the attenuated HSP70 gene expression [1]. A care- ful analysis of the experimental culturing conditions used by the above-mentioned authors has indicated that some of them had deprived cells of serum for 12–24 h in order to ensure comparable quiescence be- tween early and late passage cells. Under serum-de- prived culture conditions, reduced expression of the 1 To whom correspondence and reprint requests should be ad- dressed at Sezione di Patologia Molecolare e Immunologia, Diparti- mento di Medicina Sperimentale, Universita ` degli Studi di Parma, Via Volturno 39, 43100 Parma, Italy. Fax: +39-0521-903742. E-mail: angelo.borghetti@unipr.it. 0014-4827/01 $35.00 165 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 267, 165–172 (2001) doi:10.1006/excr.2001.5237, available online at http://www.idealibrary.com on