Dietary restriction (DR) is the only experimental manipulation known to retard aging in mammals; however, the mechanism responsible for the life prolonging action of DR is unknown at the present time. One attractive explanation is that the effect of DR arises at least partially through changes in gene expression. Using the heat shock gene hsp70 as a model system to elucidate the mechanism by which aging and DR alter gene transcription, we have found that the changes in hsp70 transcription that occur with age and DR are correlated to changes in a specific transcription factor: HSF (heat shock transcription factor). In higher eukaryotes, HSF is found in non-stressed cells in an inactive (i.e., non- DNA binding) monomeric form in the cytoplasm. An increase in temperature, or other stress, results in the activation of HSF to an oligomer that binds the heat shock element (HSE) in the promoter of the hsp70 gene. We have shown that age and DR alter the activation of HSF. The objective of the research described in this proposal is to elucidate the molecular mechanism whereby aging and DR alter the activation of HSF.

Specific Aims:

1. To determine if the changes in HSF activation are due to an alteration in HSF expression. The protein and mRNA levels of HSF will be measured as a function of age and DR.

2. To determine if the changes in HSF activation arise from alterations in the oligomerization of HSF. The oligomerization, phosphorylation, and translocation of HSF will be measured in cell extracts obtained from rats of various ages fed ad libitum and a DR diet.

3. To determine if the changes in HSF activation can be correlated to alterations in the physiochemical properties of HSF. The hydrodynamic properties of HSF, the affinity of HSF for the HSE, and the interaction of HSF with the hsp70 promoter will be studied as a function of age and DR.

4. To determine if the changes in the activation of HSF with age and DR arise from changes in the levels/activities of factors that inhibit the activation of HSF. A novel system has been designed for screening an expression cDNA library from the liver of old rats for the presence of cDNAs coding for inhibitors of HSF activation.

5. To demonstrate that HSF is responsible for the changes in the induction of hsp7o transcription. The ability of recombinant HSF to reverse the changes in the in vitro transcription of an hsp70-promoter/reporter template will be studied, and transgenic mice will be used to determine if the overexpression of HSF can reverse the age-related decline in hsp 70 transcription.

R01AG001548

1979-07-01

1999-03-31