Arsenite stabilizes HIF-1α protein through p85α-mediated up-regulation of inducible Hsp70 protein expression

Abstract

Hypoxia-inducible factor-1α (HIF-1α) has been reported to regulate over 100 gene expressions in response to hypoxia and other stress conditions. In the present study, we found that arsenite could induce HIF-1α protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85α, a regulatory subunit of PI-3K, in MEFs (p85α(-/-)) dramatically decreased the arsenite-induced HIF-1α accumulation, indicating that p85α is crucial for arsenite effects on the stabilization of HIF-1α protein. Our further studies suggest that arsenite could induce inducible Hsp70 expression, and transfection of inducible Hsp70 into p85α(-/-) MEFs could restore HIF-1α protein accumulation. Moreover, the results using EMSA and Supershift assays indicate that p85α is crucial for arsenite-induced activation of the heat-shock transcription factor 1 (HSF-1), which is responsible for transcription of inducible Hsp70. Taken together, p85α-mediated HIF-1α stabilization upon arsenite exposure is specifically through HSF-1 activation and subsequent up-regulation of the inducible Hsp70 expression.

Fig. 1

Arsenite induced HIF-1α accumulation in both Cl41 cells and p85α^+/+ MEFs, but not in p85α^−/− MEFs. Cl41 cells, p85α^+/+ and p85α^−/− MEFs were exposed to 20 μM of arsenite as indicated. HIF-1α protein levels in whole cellular extracts were determined by Western blotting in Cl41 cells (a) and p85α^+/+ MEFs (b). Identification of p85α deficiency in p85α^−/− MEFs in comparison to that in p85α^+/+ MEFs (c). Comparison of HIF-1α protein induction due to arsenite (20 μM) exposure between p85α^+/+ and p85α^−/− MEFs (d). hif-1α mRNA levels were detected by RT-PCR (e) or quantitative PCR in both p85α^+/+ and p85α^−/− MEFs after 20 μM arsenite treatment (f)

Fig. 2

p85α attenuated the degradation of HIF-1α protein in arsenite-treated MEFs. a, b p85α^+/+ and p85α^−/− MEFs were pretreated with MG132 (10 μM) and arsenite (20 μM) for 6 h and then exposed to CHX (10 μM) for the indicated time points after the removal of MG132 and arsenite. Levels of HIF-1α were determined by Western blotting (a), and normalized to the internal control of β-actin (b). c–e p85α^+/+ and p85α^−/− MEFs were treated with arsenite (20 μM), MG132 (10 μM), or a combination of these two reagents for 6 h. The whole-cell extracts were probed with anti-HIF-1α, Akt, and phosphorylation of Akt at Ser473 antibodies (c); the ubiquitin levels of HIF-1α protein in precipitants were subjected to Western blotting assay using an anti-ubiquitin antibody (d); and hif-1α mRNA was assessed by RT-PCR (e)

Fig. 3

The blockage of HIF-1α degradation by p85α depended on activation of PI-3K/Akt in both Cl41 and MEF cells. a The phosphorylation levels of Akt at Serine 473 were measured in p85α^+/+ and p85α^−/− MEFs after treatment of cells with arsenite (20 μM) for the indicated time points; b, c Cl41 cells stable transfected with dominant negative mutants of p85α (Δp85α) and Akt (DN-Akt) or vector control (AP-1 mass1) were treated with arsenite (20 μM) for the indicated time spans. The induction of HIF-1α and phosphorylation of Akt at Serine 473 and Tyrosine 308 were detected as indicated

Fig. 4

Inducible Hsp70, but not constitutive Hsp70 or Hsp90, was up-regulated by arsenite in a PI-3K/Akt-dependent manner. a, b The induction of overall Hsp70 was evaluated in Cl41 cells (a) and p85α^+/+ and p85α^−/− MEFs (b) upon 20 μM of arsenite treatment. c The induction of overall Hsp70 was determined in p85α^+/+ and p85α^−/− MEFs upon treatment of cells for 6 h with arsenite (20 μM) or MG132 (10 μM), or combination of both; d p85α^+/+ and p85α^−/− MEFs were subject to pretreatment with 200 nM of Wortmannin for 30 min, and then exposed to 20 μM of arsenite for additional 6 h. Overall Hsp70 and inducible Hsp70 were detected by Western blotting. e, f The induction of overall Hsp70 and inducible Hsp70 was assessed in Cl41 cells stable transfected with Δp85α (e) or DN-Akt (f) in comparison to that in vector control transfectant

Fig. 5

PI-3K/Akt pathway regulated both Hsp70 nuclear translocation and the level of inducible hsp70 mRNA. a Immunofluorescence staining was conducted as described in ‘Materials and methods’ on both p85α^+/+ and p85α^−/− MEFs with or without 20 μM of arsenite treatment. The representative DAPI, inducible Hsp70 staining and merged pictures are shown, respectively. b The cytoplasm and nucleus fractions were isolated from p85α^+/+ MEFs with arsenite treatment for different time spans (0–6 h). HIF-1α and overall Hsp70 were detected with specific antibodies in each fraction as well as in whole cellular extract (input). GADPH was used here as an indicator of isolation efficacy. c, d RT-PCR was employed to detect the induction of inducible Hsp70 in both p85α^+/+ and p85α^−/− MEFs, under the conditions of 20 μM arsenite treatment for indicated time points (c), or under the treatment of cells with arsenite (20 μM), MG132 (10 μM) or combination of both for 6 h (d)

Fig. 6

HSF-1 plays an indispensable role in transcriptional up-regulation of inducible Hsp70. a Hsp70 promoter-luciferase reporter was transiently transfected into p85α^+/+ and p85α^−/− MEFs, and were then subject to luciferase assay as described in the Materials and methods section. The relative intensities of fluorescence were shown proportionally in comparison between p85α^+/+ and p85α^−/− transfectants. b The cytoplasm and nucleus isolation assay was conducted in both p85α^+/+ and p85α^−/− MEFs with or without 20 μM of arsenite treatment for 6 h. The levels of HSF-1, HIF-1α, overall Hsp70, and inducible Hsp70 were evaluated, respectively. Lamin B and GAPDH were used as indicators of isolation efficacy. c The binding efficiency of HSF-1 on the promoter region of the hsp70 gene was determined by EMSA and super gel shift assay, as described in the Materials and methods section. Briefly, the sequences of HSF-1 binding sites on the Hsp70 promoter region were designed and incubated with nuclear extract from p85α^+/+ or p85α^−/− MEFs with or without 20 μM of arsenite treatment; each sample was subjected to PAGE gel. d, e HSF-1^+/+ and HSF-1^−/− MEFs were exposed to arsenite as indicated. The levels of overall and inducible Hsp70 were determined by Western blotting (d), and the levels of hsp70 mRNA were detected by RT-PCR (e)

Fig. 7

Inducible Hsp70-specific stabilized HIF-1α protein upon arsenite exposure. a The construct expressing inducible Hsp70 and vector construct (as control) were introduced separately into p85α^−/− MEFs, and the corresponding stable transfectant was established. The induction of HIF-1α due to arsenite exposure was determined as indicated in p85α^−/− (Vector), p85α^−/− (Induc Hsp70), and p85α^+/+ MEFs. The expression levels of inducible Hsp70 were verified by Western blotting. b p85α^−/− MEFs were stably transfected with constitutive Hsp70 (Hsc70) expressing construct or the control vector. The success of transfection was confirmed by Western blotting (left panel). The arsenite-induced HIF-1α was detected in both transfectants (right panel). c WT MEFs were stably transfected with nonsense shRNA or two specific shRNAs targeting inducible Hsp70. The efficiency of knockdown was identified by Western blotting and the arsenite-induced HIF-1α protein expression was determined as indicated