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Accumulation of abnormal proteins is associated with neurodegenerative syndromes such as Alzheimer disease, Parkinson disease and Huntington disease, all age-related diseases. The existence of these diseases em- phasizes that defects impairing the body’s ability to remove or repair dam- aged macromolecules effectively accelerates the aging process. Cells are equipped with several different categories of repair and surveillance: DNA surveillance and repair systems, RNA surveillance systems, anti-oxidant systems and systems like the heat shock response (HSR) and the unfolded protein response (UPR) that deal with proteotoxic stress. Defi ciency of any of these cellular defence systems might tip the balance from repair to per- manent damage. Impairment of any of these repair and surveillance systems will eventually lead to an increase in proteotoxic stress. Maintaining proteos- tasis during aging is expected to prevent or at least ameliorate age-related protein folding and infl ammatory disease [1, 2].
Cells respond to cytoplasmic proteotoxic stress by producing additional chaperones. This response is called the heat shock response (HSR) and is mainly regulated at the level of transcription by heat shock factor 1 (HSF1). The activity of HSF1 declines with age. The protein is still present but can no longer be activated. One possible approach to prevent the decline in HSF1 activity during aging is either by targeting HSF1 directly or by targeting lon- gevity related factors which control HSF1 activity [3]. One potential drawback of maintaining or increasing HSF1 activity is that HSF1 also increases the risk of cancer, also often an age-related disease [4]. The other known way to upregulate the stress system is to cause cellular stress, which ultimately may be deleterious and in aging cells this method will be less effective than in normal cells because of the lower activity of HSF1. An alternative is to main- tain the capacity of the chaperoning network by boosting the expression of a single (co-)chaperone. To fi nd ways to boost the defence and repair system without the deleterious effects, we need to know more about the system, its critical nodes and rate limiting steps.
HSF1 targets in stressed and non-stressed cells.
To understand the role of HSF1, and the consequences of the loss of activity thereof, better, we used cellular model systems based on transformed hu- man embryonic kidney (HEK293) cells in which the transcriptional activity of HSF1 was inhibited by overexpression of HSF1 mutants. Dominant negative (dn)HSF1 lacks the transactivation domain but can still bind DNA. DnHSF1 is predicted to occupy the HSF1 binding sites and to repress transcription di- rected by those binding sites. HSF1-K80Q has lysine 80 in the DNA binding region replaced by glutamine, which inhibits DNA binding [3]. Expression of HSF1-K80Q should free the HSF1 binding sites (for an overview see fi gure 1). The rate of transcription of HSF1 target genes that are activated by HSF1 will decrease, while that of genes that are repressed by HSF1 will increase. The transcriptome changes as a result of exogenous expression of these
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HSF1 mutants have been measured using microarrays (Chapter 4 and 7). Inhibiting HSF1 activity in the absence of stress by overexpressing dnHSF1 resulted in a downregulation of 10 genes, about half of which are canonical HSF1 target genes such as DNAJB1; no genes were upregulated. Inhibiting HSF1 activity in the absence of stress by overexpressing HSF1-K80Q result- ed in a downregulation of 17 genes, while 11 genes were upregulated. There was no overlap between the 10 downregulated transcripts by dnHSF1 and the down- or upregulated transcripts in the HSF1-K80Q expressing cells. A comparison of our HSF1-K80Q microarray results with the published data using HeLa cells lacking HSF1 (siRNA) [5] showed only a single gene of which the transcript level changed signifi cantly in non-stressed cells express- ing HSF1-K80Q and in cells treated with siHSF1. The difference between the HEK293 and HeLa data could partly be explained by the fact that depletion of HSF1 by siRNA would free the chaperones which are usually complexed with HSF1 while the HSF1-K80Q mutant could capture more chaperones. Alternatively, HSF1 could participate in gene regulatory circuits for which the DNA binding activity is not required. To distinguish between these possibili- ties, the effect of lack of the HSF1 protein or just lack of HSF1 binding activ- ity needs to be compared in the same cells. Still, the HSF1 dependency in the absence of stress of most of the genes found in our microarray analysis is probably tissue specifi c.
One of the strongest downregulated transcript in dnHSF1 expressing cells was PMVK (see Chapter 6). Further analysis of the PMVK promoter region showed the presence of an HSF1 binding site in the region encoding the 5’UTR. HSF1 is not required to maintain transcription of PMVK in either non-stressed or stressed cells as PMVK mRNA levels were not decreased in cells overexpressing HSF1-K80Q. These data suggest that HSF1 can regulate the PMVK promoter, but under which conditions HSF1 does so is still unknown.
Loss of regulation by HSF1 does have consequences for cellular robustness. The heat induced expression of several chaperones, like HSPA1A, DNAJB1 and HSPB1, is completely blocked in presence of dnHSF1 or HSF1-K80Q. A non-functional HSR prevents complete recovery from heat stress. Therefore, dnHSF1 or HSF1-K80Q expressing cells show a higher stress state 24 hours after heat stress than normal cells do. For example, 24 hours after heat shock the GADD34 and GADD45B mRNA levels are higher in cells express- ing dnHSF1 or HSF1-K80Q compared with control cells. Evaluating the tran- scriptome changes in cells overexpressing the HSF1 mutants 24 hours after heat shock by microarray analysis might yield further insight in which stress pathways remain activated.