Hsp72 mediates TAp73α anti-apoptotic effects in small cell lung carcinoma cells

Abstract

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. Due to alternative promoters and carboxy-terminal splicing, the P73 gene gives rise to a range of different isoforms. Interestingly, a particular increase in expression of the TAp73α isoform has been reported in various tumours. In addition, TAp73α has been shown to inhibit Bax activation and mitochondrial dysfunctions and thereby to confer small cell lung carcinoma (SCLC) cells resistance to drug-induced apoptosis. However, the precise mechanism by which TAp73α exerts its pro-survival effect is yet unclear. Here we report that TAp73α, but not TAp73β, regulates the expression of inducible Hsp72/HSPA1A. Hsp72 proved to be required for the survival effects of TAp73α as antisense knockdown of Hsp72 resulted in an abolishment of the anti-apoptotic effect of TAp73α in SCLC cells upon Etoposide treatment. Importantly, depletion of Hsp72 allowed activation of Bax, loss of mitochondrial membrane potential and lysosomal membrane permeabilization in SCLC cells even in the presence of TAp73α. Finally, we revealed that TAp73β counteracts the anti-apoptotic effect of TAp73α by preventing Hsp72 induction. Our results thus provide additional evidence for the potential oncogenic role of TAp73α, and extend the understanding of the mechanism for its anti-apoptotic effect.

Fig 1

TAp73α induces Hsp72 expression. (A) H82 and HEK-293 cells were co-transfected with a HSP72 promoter-luciferase vector, a β-galactosidase reporter vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α), as indicated. Cells were harvested after 24 hrs, and cell extracts assayed for luciferase and β-galactosidase activity. Relative luciferase units were compared after normalization to β-galactosidase activities. H82 (B) and HEK-293 (B, D) were transfected with expression vectors encoding TAp73α, TAp73β, ΔNp73α. (B) Total RNA was extracted from cells, followed by cDNA synthesis and PCR amplification of the indicated genes. Quantification of DNA band-intensity was made using ImageJ software. (C) H82 cells were co-transfected with EGFP and p73 expression vectors (TAp73α, TAp73β) (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (D) Total protein cell extracts were analysed by immunoblotting for the presence of Hsp72 and p73. G3PDH was used as protein loading control. (E) Total protein cell extracts were analysed by immunoblotting for the expression of p73, HSF1, Hsc70 and Hsp90 (bands marked * is due to previous staining with antibody against p73 and hence depict TAp73 protein). Data are represented as mean ± S.D. of at least three independent experiments, where *P < 0.05 and **P < 0.01.

Fig 2

Hsp72 is required for TAp73α anti-apoptotic effect. (A) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the presence of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (B) H82 cells were co-transfected with EGFP and asHsp72 vector (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (C) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the expression of HSF1, Hsp90 and Hsc70. G3PDH was used as protein loading control and numbers indicate the ratio between protein (HSF1, Hsp90 or Hsc70) and G3PDH band intensity. H82 cells were co-transfected with EGFP and asHsp72 vector (green) and the effect of asHsp72 expression on Hsp27 protein levels were detected using immunofluorescent staining for Hsp27 (red) and counterstaining of nuclei with Hoechst (blue). (D) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α). Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. (E) H82 cells were transfected with siRNAs pool designed to interfere specifically with the expression of human Hsp72 mRNA and total protein cell extracts were analysed by immunoblotting for the expression of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (F) H82 cells were co-transfected with EGFP, Hsp72 siRNAs pool and empty or TAp73α expression vectors. Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 3

Hsp72 represses TAp73β pro-apoptotic effect. (A) H82 cells were transfected with Hsp72 expression vector and extracts were analysed as described in Figure 2A. (B) H82 cells were co-transfected with EGFP and Hsp72 vector (green), and samples assayed as described in Figure 2B. (C) H82 cells were co-transfected with EGFP, Hsp72 vector and empty vector or TAp73β. Samples were treated, stained and assayed as described in Figure 2D. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 4

Hsp72 depletion favours Bax activation and loss of ΔΨm upon VP16 treatment in the presence of TAp73α. (A, B) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or TAp73α. Cells were treated with VP16 for 24 hrs, stained with TMRE and nuclei counterstained with Hoechst. Loss of ΔΨm was assayed by scoring of EGFP transfected cells without TMRE staining using microscopy (A) and FACS analysis (B). (C) H82 cells were transfected and treated as described (A). Samples were stained with anti-active Bax antibody (red) and nuclei counterstained with Hoechst (blue). (D) Cells were transfected and treated as in (A), stained with anti-active Bax antibody and analysed by FACS.

Fig 5

Hsp72 expression prevents TAp73β-induced mitochondrial dysfunction and Bax activation. (A, B) H82 cells were co-transfected with EGFP, Hsp72 vector and empty vector or TAp73β. Treatment and staining were performed as described in Figure 4A and B, and analysis was carried out using microscopy (A) and FACS analysis (B). (C, D) H82 cells were transfected, treated and stained as described in Figure 4C and D. Images are representatives of three independent experiments. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 6

TAp73α repress Etoposide-induced LMP in a Hsp72-dependent manner. H82 cells were co-transfected with EGFP, asHsp72, TAp73α, Hsp72 and/or TAp73β, treated with VP16 for 24 hrs and stained with Lysotracker Red. Cells were quantified live using fluorescence microscopy (A, B), or fixed and stained with Hoechst for confocal analysis (C). (A) and (B) are mean ± S.D. of at least three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001. (C) is a representative image.

Fig 7

The Hsp72-mediated anti-apoptotic effect of TAp73α can be counteracted by TAp73β. (A) H82 cells were transfected and assayed as described in Figure 1A. (B) H82 cells were transfected with TAp73α, TAp73β, ΔNp73α, TAp73α and TAp73β, or empty vector. Samples were immunoprecipitated using p73 antibody, and binding to the HSP72 promoter was detected with PCR. (C) H82 cells were transfected, treated and assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei (as described in Fig. 2D). Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.