Coupling endoplasmic reticulum stress to the cell-death program: a novel HSP90-independent role for the small chaperone protein p23

Abstract

The endoplasmic reticulum (ER) is the principal organelle for the biosynthesis of proteins, steroids and many lipids, and is highly sensitive to alterations in its environment. Perturbation of Ca(2+) homeostasis, elevated secretory protein synthesis, deprivation of glucose or other sugars, altered glycosylation and/or the accumulation of misfolded proteins may all result in ER stress, and prolonged ER stress triggers cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell-death modulators and effectors through the use of biochemical, pharmacological and genetic tools. In the present work, we describe the role of p23, a small chaperone protein, in preventing ER stress-induced cell death. p23 is a highly conserved chaperone protein that modulates HSP90 activity and is also a component of the steroid receptors. p23 is cleaved during ER stress-induced cell death; this cleavage, which occurs close to the carboxy-terminus, requires caspase-3 and/or caspase-7, but not caspase-8. Blockage of the caspase cleavage site of p23 was associated with decreased cell death induced by ER stress. Immunodepletion of p23 or inhibition of p23 expression by siRNA resulted in enhancement of ER stress-induced cell death. While p23 co-immunoprecipitated with the BH3-only protein PUMA (p53-upregulated modulator of apoptosis) in untreated cells, prolonged ER stress disrupted this interaction. The results define a protective role for p23, and provide further support for a model in which ER stress is coupled to the mitochondrial intrinsic apoptotic pathway through the activities of BH3 family proteins.

Figure 1

HSP90 protein expression during ER stress. HSP90 protein expression in MCF-7 cells and Apaf-1^−/− fibroblasts treated with 2.0 and 0.5 μM thapsigargin, respectively, for different time periods. Cell extracts (150 μg protein) were then analyzed by Western blot analysis for HSP90 expression. Increased levels of GRP78 protein expression in similar extracts indicate ER stress response. Surviving versus apoptotic cells were quantified as described in the Materials and Methods. Data (mean±S.E.) are from more than three independent experiments. Each figure is representative of at least three independent experiments

Figure 2

p23 protein expression during ER stress. p23 protein expression in Apaf-1^−/− fibroblasts and MCF-7 cells treated with various ER stress inducers for 24 h. (a) Apaf-1^−/− fibroblasts were treated with 1.0 μM brefeldin-A, 2.0 μg/ml tunicamycin, and 0.5 μM thapsigargin. MCF-7 cells were treated with 2.0 μM brefeldin-A, 2.0 μg/ml tunicamycin, and 2.0 μM thapsigargin. Cell extracts (150 μg protein) were then analyzed by Western blot analysis for p23 expression. MCF-7 cells do not elicit an ER stress response when cells are exposed to tunicamycin treatment. Cell extracts were also probed with GAPDH as a loading control. (b) p23 cleavage is accompanied by caspase processing. Cell extracts (150 μg of protein) were prepared from cells treated with 0.5 μM thapsigargin for various time periods. p23 is cleaved to a low Mw species (approximately 19 kD) and thapsigargin-induced p23 cleavage is also accompanied by caspase-9 and -7 processing. Surviving versus apoptotic cells were quantified as described in Materials and Methods. Data are from three independent experiments

Figure 3

ER stress-induced cleavage of p23 is a caspase-dependent phenomenon. (a and b) p23 protein expression in Apaf-1^−/− fibroblasts treated with 0.5 μM thapsigargin or 1.0 μM brefeldin-A in the presence or absence of ZVAD-fmk. Purified active caspase-3 (50 ng/ml), caspase-7 (100 ng/ml) or caspase-8 (1.0 μg/ml) was added to cell-free cytosolic extracts (150 μg protein) made from Apaf-1^−/− fibroblasts (lanes 5–7). Extracts were incubated at 37°C for 1 h, separated on SDS-PAGE gels, transferred to PVDF membrane and incubated with p23 antibody. p23 is cleaved to a low Mw (19 kD) species (a) that is blocked by ZVAD-fmk (b). (c) Potential caspase cleavage sites in p23. A total of eight ‘D’ (aspartate) residues exist in the carboxy-terminus that are potential sites for caspase activity. (d) p23 has several potential caspase sites at the carboxy-terminus. Site-directed mutagenesis of several of these sites were carried out as described in Materials and Methods. Cell-free extracts were made from 293T cells transfected with wild-type Flagp23, D142N, D145N, D150N, D153N, D150/153N and D145/150/153N constructs. Samples (150 μg protein) were incubated with 50 ng/ml caspase-3 or 100 ng/ml caspase-7 at 37°C for 1 h. Samples were analyzed by immunoblotting with anti-FLAG antibody to detect p23 and its cleavage product (19 kD). (e) p23 caspase mutant inhibits ER stress-induced cell death. MCF-7 cells were transfected with 6 μg of pcDNA3, pcDNAp23, p23D142N or p23D150/153N mutant constructs. After 24 h, cells were treated with 2.0 μM thapsigargin (Thaps) or 2.0 μM brefeldin-A (Bfl-A) for 24 h. Cells were gently lifted and washed once with PBS at room temperature. Surviving versus apoptotic cells were quantified as described in the Materials and Methods. *P< 0.05

Figure 4

Subcellular localization of p23. (a) Immunofluorescent staining for p23 before and after subjecting cortical neurons to hypoxia. For the detection of p23, cells were stained with anti-p23 monoclonal antibody as the primary antibody and 488-conjugated donkey anti-mouse IgG as the secondary antibody (green). Cells were also stained with anti-calreticulin polyclonal antibody and 555-conjugated donkey anti-rabbit IgG (red) as the secondary antibody. (b) Microsomes were isolated from cell-free extracts prepared from untreated or thapsigargin-treated Apaf-1^−/− fibroblasts (24 h) as described in Materials and Methods. A total of 50 μg of protein from the 400 000 × g supernatant (S) and microsomal (P) fractions was subjected to SDS-PAGE and Western blotting. Membranes were probed with anti-p23 monoclonal antibody. Cellular fractions were also probed with anti-PDI (protein disulfide isomerase) antibody and anti-β-tubulin as standard marker proteins for ER and cytosol, respectively. (c) p23 is required for the processing of caspase-9 in response to thapsigargin-induced ER stress. Untreated cell extracts and microsomes isolated from 24 h thapsigargin-treated cell extract were immunodepleted of p23 using the p23 monoclonal antibody. Samples were spun briefly to pellet the protein A/G-Sepharose conjugate. The supernatant was collected, subjected to SDS-PAGE, and probed for (1) p23 that would indicate the extent of immunodepletion and (2) PDI as ER marker protein to serve as loading controls. Additionally, a caspase-9 processing assay was carried out by incubating 100 μg of the supernatant protein at 37°C for 1 h with untreated cell extracts or extracts that had been immunodepleted of p23. Following the reactions, samples were analyzed by SDS-PAGE and Western blotting. Membranes were probed with anticaspase-9 antibody. Note that the reduction of p23 was approximately 75%

Figure 5

Transfection of siRNA-targeting p23. (a) Small interfering RNAs (siRNAs) were designed to target two regions each for p23. Apaf-1^−/− cells were first transfected with 80 nM siRNA as described in Materials and Methods. To estimate the efficiency of transfection, fluorescently labeled siRNA targeting the luciferase gene was used. Cells were gently lifted 36 h after siRNA transfection and washed once with PBS at room temperature. Cell lysates were immunoblotted with anti-p23 and anti-HSP90 antibodies. None of the siRNAs crossinhibited the expression of HSP90. Cell extracts were also probed with anti-GAPDH as a loading control. (comb) indicates cotransfection of the two siRNAs designed to target the p23 gene transcript (nucleotides 419–429 and 446–466). (b) Effect of RNA interference on ER stress-induced caspase activation and cell death. Apaf-1^−/− cells were first transfected with 80 nM siRNA as described in Materials and Methods. A combination of both siRNAs (nucleotides 419–429 and 446–466) was transfected to optimize the decrease of expression of p23 (Figure 5a, comb). At 24 h after transfection, cells were exposed to 50 nM thapsigargin (Thaps) for 12 and 24 h. Cells were gently lifted and washed once with PBS at room temperature. Cell extracts were subjected to SDS-PAGE and Western blotting. Membranes were probed with anti-caspase-9 or -7 antibody. (c) Thapsigargin-induced cell death was quantified by the Trypan blue procedure as described in Materials and Methods. *P< 0.05

Figure 6

Co-immunoprecipitation of p23, and PUMA (a and b) Co-immunoprecipitation was carried out in untransfected MCF-7 cells and cells transfected with 6 μg of WTp23FLAG cDNA (a) or FLAGp23D142N (b). Transfection was allowed to proceed for 24 h before the addition of ER stress inducers. ER stress was induced by the addition of 2.0 μM brefeldin-A (Bfl) or 2.0 μM thapsigargin (Thaps) for different time periods. Cells were gently lifted and washed once with PBS at room temperature. Immunoprecipitations were performed with anti-p23 antibody or anti-Flag monoclonal antibody (for Flag-tagged p23), and the resulting immunoprecipitates were analyzed by immunoblotting using antisera specific for PUMA or p23. (c) Cellular extracts were prepared from MCF-7 cells treated with 2.0 μM brefeldin-A (Bfl) or 2.0 μM thapsigargin (Thaps) for different time periods. Cells were gently lifted washed once with PBS at room temperature. Cell extracts were subjected to SDS-PAGE and Western blotting. Membranes were probed with anti-PUMA antibody, anti-P53 antibody and anti-GAPDH antibody

Figure 7

Co-immunoprecipitation of p23, HSP90, PUMA and Bax. (a) Co-immunoprecipitation was carried out in untransfected MCF-7 cells. ER stress was induced by the addition of 2.0 μM brefeldin-A (Bfl) or 2.0 μM thapsigargin (Thaps) for 24 h. Cells were gently lifted and washed once with PBS at room temperature. Immunoprecipitations were performed with anti-p23 antibody or anti-HSP90 antibody and the resulting immunoprecipitates were analyzed by immunoblotting using antisera specific for HSP90 or PUMA. (b) Co-immunoprecipitation was carried out in untransfected MCF-7 cells. ER stress was induced by the addition of 2.0 μM brefeldin-A (Bfl) or 2.0 μM thapsigargin (Thaps) for 24 h. Cells were gently lifted washed once with PBS at room temperature. Immunoprecipitations were performed with anti-Bax6A7 antibody that recognizes conformationally changed Bax, and the resulting immunoprecipitates were analyzed by immunoblotting using antisera specific for Bax (anti-Bax N20), PUMA and p23 antibody