Co-chaperones are limiting in a depleted chaperone network

Abstract

To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1.

Fig. 1

The HSF1 mutants HSF379 and HSF448 have different effects on basal and heat shock-induced Hsp70 expression. Parental Flp-In HEK293 cells and HEK293 cells carrying a stably integrated copy of the pcDNA5-HSF379 (HEK-HSF379) or pcDNA5-HSF448 (HEK-HSF448) plasmid were cultured in the absence or presence of doxycycline. Cells were exposed to a heat shock (30′, 45°C), harvested at the indicated time point (h) after heat shock, and subjected to western blot analysis using an anti-Hsp70 antibody

Fig. 2

The effects of dnHSF on basal and heat shock-induced activity of an Hsp70 promoter HEK293 cells carrying a stably integrated copy of the HSF379 (dnHSF1) were cultured in the absence (−) or presence (+) of doxycycline. Cells were transfected with a mixture of the Drosophila melanogaster Hsp70-luciferase reporter (pHL) and the Renilla Luciferase control plasmid pCMV-RL. At 48 h after transfection, cells were exposed to a heat shock of 30′ at 45°C (HS) or left at 37°C (37°C). When heat shocked, cells were allowed to recover for 6 h and harvested. Hsp70 promoter activities were determined by dividing firefly luciferase values by the corresponding renilla luciferase (experiments using the HSF448 line) or β-galactosidase (experiments using the dnHSF1 line) values to correct for varying transfection efficiencies. The relative luciferase activity in cells cultured at 37°C in absence of the various HSF1 mutants was set at 1. The results are the average of three independent transfections (standard deviations are indicated by error bars)

Fig. 3

Left panel the decay of heat shock protein levels during expression of dnHSF1. HEK-HSF379 cells were treated with doxycyclin for the time indicated and harvested. Right panel the level of heat shock proteins in MEF wild-type cells (+/+) and MEF cells lacking HSF1 (−/−) either before (−HS) or after heat shock and recovery (+HS). Cell lysates were subjected to SDS-PAGE and western blot analysis using the indicated antibodies

Fig. 4

Inhibition of promoter activity by dnHSF1. Control HEK-cDNA5 cells and HEK-HSF379 cells were treated with doxycyclin. After 3 days, cells were transfected with the indicated promoter reporter constructs (see also “Materials and methods”) and a βactin-βgal reporter. At 48 h after transfection, cells were harvested and assayed for reporter gene activities. Promoter activities were determined by dividing luciferase values by the corresponding β-galactosidase values to correct for varying transfection efficiencies. The bars correspond to the % activity of the promoter in the HEK-HSF379 cells compared with the control HEK-cDNA5 cells. The results are the average of three independent transfections (standard deviations are indicated by error bars)

Fig. 5

The effect of exogenous expression of dnHSF1 on eIF2α phosphorylation. HEK-cDNA5 cells and HEK-HSF379 cells were treated with doxycyclin for 48 h. Cells were then exposed to a heat shock of 30′ at 45°C (HS) or left at 37°C (37°C). When heat shocked, cells were allowed to recover for the indicated time before harvesting. Cell lysates were subjected to SDS-PAGE and western blot analysis using the indicated antibodies

Fig. 6

Exogenous expression of dnHSF1 reduces the glucocorticoid response. Control HEK-cDNA5 cells and HEK-HSF379 cells were treated with doxycyclin. After 3 days, cells were transfected with a glucocorticoid-responsive luciferase reporter (pGRE-Luc) and a βactin-βgal reporter. At 24 h after transfection, cells were either left untreated or exposed to the indicated concentrations of dexamethasone. At 48 h after transfection, cells were harvested and assayed for reporter gene activities. Promoter activities were determined by dividing luciferase values by the corresponding β-galactosidase values to correct for varying transfection efficiencies. The bars correspond to the activity of the glucocorticoid-responsive promoter in the presence of dexamethasone compared to the activity in untreated cells, which was set at 100%. Gray bars show the results for control HEK-cDNA5 cells, black bars those for HEK-HSF379 cells. The results are the average of three independent transfections (standard deviations are indicated by error bars)

Fig. 7

Effect of overexpression of (co-)chaperones on glucocorticoid signaling in HEK-cDNA5 and HEK-dnHSF1 cells. Control HEK-cDNA5 cells (light gray bars) and HEK-HSF379 cells (black bars) were treated with doxycyclin. After 3 days, cells were transfected with a mixture (4:1:5) of glucocorticoid-responsive luciferase reporter (pGRE-Luc), a βactin-βgal reporter, and the expression construct indicated. At 24 h after transfection, cells were either left untreated or exposed to the indicated concentrations of dexamethasone. At 48 h after transfection, cells were harvested and assayed for reporter gene activities. Relative luciferase activities and -fold induction were determined as described in the legend to Fig. 6. Standard deviations are indicated by the error bars

Fig. 8

Levels of exogenous expression of (co-)chaperones. Expression plasmids for the (co-)chaperones indicated on the left were transfected into either HEK-cDNA cells (control) or HEK-HSF379 cells (+DNA) and expression was induced by adding doxycyclin (+Dox), except for HSP90AA1, of which expression is constitutive. Protein levels were determined by western blotting and staining with the corresponding antibody (see “Materials and methods”). The arrowhead indicates HSPB8. Note that, in the case of DNAJA1, DNAJB6 and DNAJB8 antibody to the V5-tag carried by the exogenous proteins was used; the endogenous protein is thus not detected. β-actin was used as a loading control