Repression of the HSP70B promoter by NFIL6, Ku70, and MAPK involves three complementary mechanisms

We have studied mechanisms of HSP70 gene regulation at 37 degrees C by the cellular factors NF-IL6 and Ku70. As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression. However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3. We then examined whether NF-IL6 repressed HSP70B by a mechanism resembling Ku proteins. However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation. In addition, activation of the HSP 70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells. In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE. These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms.     

Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells

Phenylarsine oxide (PAO)forms a stable ring complex with vicinal dithiols that can be reversedwith 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or2-mercaptoethanol (2-ME). PAO at 2 µM or higher inhibited heat shockprotein 70 (HSP70) induction within minutes in cultured guinea piggastric mucosal cells exposed to heat (43°C) for 30 min. PAO did notaffect the nuclear translocation and phosphorylation of heat shockfactor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading tothe block of expression of HSP70 mRNA and accumulation of HSP70 in thecells. These inhibitions were completely reversed with 2 µM DMP butnot with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactionsbetween PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx)reversed the inhibition of the binding activity of HSF1 in whole cellextracts prepared from PAO-treated, heat-stressed cells. Our resultssuggest that PAO may react with vicinal-containing molecules includingTrx and specifically block the interaction between HSF1 and HSE.

     

Functional HSF1 requires aromatic-participant interactions in protecting mouse embryonic fibroblasts against apoptosis via G2 cell cycle arrest

The present study highlighted the aromatic-participant interactions in in vivo trimerization of HSF1 and got an insight into the process of HSF1 protecting against apoptosis. In mouse embryonic fibroblasts (MEFs), mutations of mouse HSF1 (W37A, Y60A and F104A) resulted in a loss of trimerization activity, impaired binding of the heat shock element (HSE) and lack of heat shock protein 70 (HSP70) expression after a heat shock. Under UV irradiation, wild-type mouse HSF1 protected the MEFs from UV-induced apoptosis, but none of the mutants offered protection. We found that normal expression of HSF1 was essential to the cell arrest in G2 phase, assisting with the cell cycle checkpoint. The cells that lack normal HSF1 failed to arrest in the G2 phase, resulting in the process of cell apoptosis. We conclude that the treatment with UV or heat shock stresses appears to induce the approach of HSF1 monomers directly via aromatic-participant interactions, followed by the formation of a HSF1 trimer. HSF1 protects the MEFs from the stresses through the expression of HSPs and a G2 cell cycle arrest.