Activation of heat shock factor 1 plays a role in pyrrolidine dithiocarbamate-mediated expression of the co-chaperone BAG3

Abstract

Adaptive responses to physical and inflammatory stressors are mediated by transcription factors and molecular chaperones. The transcription factor heat shock factor 1 (HSF1) has been implicated in extending lifespan in part by increasing expression of heat shock response genes. Pyrrolidine dithiocarbamate (PDTC) is a small thiol compound that exerts in vivo and in vitro anti-inflammatory properties through mechanisms that remain unclear. Here we report that PDTC induced the release of monomeric HSF1 from the molecular chaperone heat shock protein 90 (Hsp90), with concomitant increase in HSF1 trimer formation, translocation to the nucleus, and binding to promoter of target genes in human HepG2 cells. siRNA-mediated silencing of HSF1 blocked BAG3 gene expression by PDTC. The protein levels of the co-chaperone BAG3 and its interaction partner Hsp72 were stimulated by PDTC in a dose-dependent fashion, peaking at 6h. Inhibition of Hsp90 function by geldanamycin derivatives and novobiocin elicited a pattern of HSF1 activation and BAG3 expression that was similar to PDTC. Chromatin immunoprecipitation studies showed that PDTC and the inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin enhanced the binding of HSF1 to the promoter of several target genes, including BAG3, HSPA1A, HSPA1B, FKBP4, STIP1 and UBB. Cell treatment with PDTC increased significantly the level of Hsp90α thiol oxidation, a posttranslational modification known to inhibit its chaperone function. These results unravel a previously unrecognized mechanism by which PDTC and related compounds could confer cellular protection against inflammation through HSF1-induced expression of heat shock response genes.

Fig. 1

Cellular activation of HSF1 by PDTC. (A) Following exposure of HepG2 cells to PDTC (50 μM) for 0-60 min, cells were lysed and the HSF1 immunoprecipitates were analyzed by Western blot for the cosedimentation of Hsp90α (upper panel) and HSF1 (bottom panel). Rel. Units, ratio of Hsp90 over HSF1. (B) HepG2 cells were exposed to 50 μM PDTC for 1 or 3 h, after which cells were lysed and Hsp90α immunoprecipitates were analyzed by Western blot for the cosedimentation of HSF1 (upper panel) and Hsp90α (lower panel). IgG, isotype-specific mouse IgG used for control immunoprecipitation. Rel. Units, ratio of HSF1 over Hsp90. (C) Serum-starved HepG2 and M2 cells were exposed to PDTC (50 μM) for 1 h, washed and subjected to crosslinking with EGS as outlined in “Materials and Methods”. Total cell extracts were separated on 6% SDS-PAGE gels under reducing conditions and analyzed by Western blot for HSF1 (upper and middle panels) and Hsp90α, which was included as a loading control (bottom panel). The position of the molecular weight makers (in kDa) is shown on the left. ** and *** denote the formation of HSF1 oligomers. (D) The level of HSF1 in the nuclear (N) and cytosolic (C) fractions of HepG2 cells incubated with PDTC (50 μM) for 5, 15, 30 and 60 min was assessed by Western blot analysis (top panel). The membranes were reprobed for BRG1 and p65Rel to demonstrate the quality of our nuclear fractionation. Percent, ratio of [nuclear HSF1 over cytosol+nuclear pool of HSF1] x 100. (E) Serum-starved HepG2 cells were exposed to vehicle or PDTC (50 μM) for 1 h, washed and subjected to crosslinking with EGS. The nuclear (N) and cytosolic (C) fractions were prepared and analyzed by Western blot for HSF1, BRG1 and the cytosolic marker, GAPDH. ***, trimers of HSF1. For each experiment depicted in A-E, similar results were obtained in 2–3 independent experiments.

Fig. 2

Effect of PDTC on BAG3 and Hsp72 (HSPA1A) expression. Total RNA was extracted from HepG2 cells treated either with 50 μM PDTC for 1, 2, 4 and 8 h (panel A) or with the indicated concentrations of PDTC for 4 h (panel B) and then analyzed by real time PCR. Data are represented as fold increase relative to vehicle-treated groups. Error bars indicate standard deviation and may be smaller than the symbols. These experiments were repeated three times with comparable results. (C) Real time PCR analysis of the two HSF1 target genes in human PANC-1 cells treated with 50 μM PDTC for periods up to 4 h. Relative mRNA levels for HSPA1A (●) and BAG3 (❍) were normalized to GAPDH mRNA expression. Data are representative of two independent experiments, each performed in duplicate dishes. (D) Western blot analysis was performed using lysates from HepG2 cells treated with 50 μM PDTC for the indicated times. GAPDH was included as a loading control (bottom panel). Graphical representation for protein blots (BAG3 and Hsp72 over GAPDH). *P<0.05 and **P<0.01 vs. control. n =4. (E) HepG2 cells treated with 0–50 μM PDTC for 4 h were lysed, and immunoblotting was performed using antibodies to BAG3 and Hsp72. GAPDH was included as a loading control. Graphical representation for protein blots (BAG3 and Hsp72 over GAPDH). **P<0.01 vs. control. n =3.

Fig. 3

Effect of HSF1 siRNA on PDTC-induced expression of HSPA1A and BAG3. HepG2 cells were transfected with either negative control (❍) or HSF1 (●) siRNA for 72 h, after which they were serum-starved and then incubated with PDTC (50 μM) for 0, 2 and 4 h. Total RNA was extracted and analyzed by real time PCR. Data represent relative mRNA expression (normalized to GAPDH) and expressed as mean values ± S.D. of triplicates. Similar results were obtained in a second independent experiment. Error bars indicate standard deviation and may be smaller than the symbols

Fig. 4

Hsp90-binding drugs mimic PDTC effects on HSF1 activation. (A) Serum-starved HepG2 cells were incubated with 17-AAG (5 μM) for 0-3 h, followed by crosslinking reaction with EGS as outlined in the legend of Fig. 1C. Western blot analysis using total cell lysates was performed with anti-HSF1 antibody. Note the time-dependent increase in the formation of EGS-stabilized HSF1 trimers (upper panel) with concomitant reduction in HSF1 monomers (bottom panel). (B) Serum-starved 1321N1 cells were treated with vehicle, PDTC (50 μM) or 17-AAG (5 μM) for 1 h followed by EGS crosslinking reaction. (C) Serum-starved HepG2 cells were incubated for 1 h with novobiocin (0.2 and 0.6 mM), an Hsp90 inhibitor structurally unrelated to geldanamycin. Total cell lysates were analyzed by Western blot using antibodies to HSF1 and Hsp90α. ** denotes HSF1 trimers. (D) Serum-starved HepG2 cells were treated with vehicle (Veh.), PDTC (50 μM), DMAG (5 μM), or novobiocin (0.6 mM) for 1 h followed by the preparation of nuclear (N) and cytosolic (C) extracts. HSF1 levels were detected by Western blot analysis (upper panel). The membranes were reprobed with BRG1 (middle panel) and IκBα (bottom panel) antibodies. (E) Total RNA from HepG2 cells treated with vehicle or DMAG (5 μM) for 4 h was extracted and analyzed by real time PCR. Data represent relative HSF1, HSPA1A and BAG3 mRNA levels (normalized to GAPDH) and expressed as mean value ± S.D. of four replicates. (F) HepG2 cells treated with 5 μM DMAG for 0–24 h were lysed, and immunoblotting was performed using antibodies to BAG3 and Hsp72. GAPDH was included as a loading control (bottom panel). Graphical representation for protein blots (BAG3 and Hsp72 over GAPDH). For each experiment depicted in A-F, similar results were obtained in 2-3 independent experiments.

Fig. 5

Binding of HSF1 to HSE elements in target gene promoters. (A) All loci are shown at the same scale, from 1000 bases upstream to 500 bases downstream of the annotated transcriptional start site. Open circles indicate predicted HSEs, gray rectangles indicated transcribed regions, white rectangles indicate PCR products for ChIP, tick marks are every 200 bp. The gray box upstream of the HSPA1A is the HSPA1L pseudogene. (B) ChIP assays using agarose-bound HSF1 antibody were performed in serum-starved HepG2 cells treated either with vehicle (open bars), PDTC (50 μM, filled bars) or DMAG (5 μM, hatched bars) for 60 min. HSF1 binding to HSPA1A promoter in PDTC-treated cells was set at 100. Bars indicate mean values ± SEM from 3-5 independent experiments.

Fig. 6

Hsp90α contains cysteine residues that are S-thiolation targets. Serum-starved HepG2 cells were treated with vehicle or PDTC (50 μM, 2 h) after which cells were homogeneized in RIPA buffer supplemented with MBB (200 μM) for 30 min on ice. The alkylation reaction was quenched with excess L-cysteine. (A) The clarified cell lysates were subjected to immunoprecipitation with Hsp90 antibody and immunoblotted either with streptavidin-conjugated HRP (upper panel) or Hsp90 to confirm equal loading (bottom panel). (B) The clarified cell lysates were incubated with captavidin-linked agarose for 1 h at 4 ºC, and the immobilized proteins were eluted with biotin, followed by Western blot analysis with anti-Hsp90 (upper panel). Input controls are shown in the bottom panel. Similar results were obtained in a second independent experiment.