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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.028654

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Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca²⁺ homeostasis after ER stress

Wonchae Choe^1,*

Sung Soo Kim^1,*

¹ Department of Biochemistry and Molecular Biology, Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species, BK-21, School of Medicine, Kyung Hee University, Seoul 130-701, Korea
² Department of Molecular and Cellular Biochemistry, Kangwon National University College of Medicine, Chunchon, Kangwon-do 200-701, Korea
³ Department of Biological Sciences, Bio/Molecular Informatics Center and Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Korea
⁴ Howard Hughes Medical Institute and Departments of Biological Chemistry and Internal Medicine, University of Michigan, 1150 W. Medical Center Dr, Ann Arbor, Michigan 48109, USA
⁵ Department of Biomedical Laboratory Science, Dongseo University, Busan 617-716, Korea

View larger version (48K): [in this window] [in a new window]   Fig. 1. Cyclophilin B is transcriptionally upregulated by ER stress. H9C2 cells were treated with 1 µM Tg and 10 µg/ml Tm under proliferation conditions (PM) for 48 hours or under differentiation conditions (DM) for 24 hours. Controls (con) are untreated cells. (A) Western blot analysis with ER stress marker antibodies to Bip, Grp94 and PDI. CypB protein level was quantified by densitometry. Actin was utilized as a loading control. Numbers under each band of CypB are representative of at least three different experiments and are expressed as the means ± s.d. *P<0.05 versus untreated cell in PM; #P<0.05, versus untreated cell in DM. (B) Micrographs of ER stress-induced cell death. (C) Western blot with antibodies to apoptosis markers: caspase 3 (cleaved form), Bax (mitochondrial), procaspase 12 and cytochrome c (cytosolic). When exposed to Tg or Tm, cleavage of caspase 3, reduction of procaspase 12, cytochrome c release and Bax translocation were observed. β-tubulin and HSP60 were used as a loading control for the cytosolic and mitochondrial fractions, respectively. Numbers under each band of procaspase 12 are representative of at least three different experiments and are expressed as the mean ± s.d. *P<0.05 versus untreated cell in PM; #P<0.05, versus untreated cell in DM. (D) Measurement of apoptosis-induced chromatin condensation by Hoechst 33342 staining. Yellow arrows indicate apoptosis-induced chromatin condensation and fragmentation. In the bar graph the data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus untreated cells in PM condition; #P<0.05 versus untreated cell in DM condition. (E,F) Semi-quantitative RT-PCR analysis of CypB transcripts. GAPDH mRNA amplification was used as a control for RT-PCR. Transcript levels of CypB in untreated cells were used as a reference. Numbers under the each band of CypB are representative of at least three different experiments and are expressed as the means ± s.d. *P<0.05 versus untreated cell in PM; #P<0.05, versus untreated cell in DM (E). Actinomycin D (AD) was employed to inhibit mRNA synthesis. *P<0.05 actinomycin D-treated cell versus untreated cell (F).

View larger version (56K): [in this window] [in a new window]   Fig. 2. Promoter analysis of cyclophilin B. (A) Sequence analysis of the CypB promoter with the Genomatix MatInspector program. Two putative ERSE elements of the CypB promoter are underlined. (B) Luciferase reporter assay of CypB promoter. Cells were incubated with 1 µM Tg or 10 µg/ml Tm for 24 hours in proliferation condition. Control were the cells without Tg or Tm treatment. The data are expressed as means ± s.d. from five independent experiments. P<0.05 versus untreated pGL3 basic; *P<0.05 versus untreated pCypB/400; #P<0.05 versus untreated pCypB/400. (C) Mutation analysis of the ERSE consensus-like sequence. Luciferase assay was conducted using pCypB/400-based luciferase reporter constructs containing an ERSE mutated promoter. 1, pGL3 basic vector; 2, pCypB/400; 3, CCAAg mutant construct; 4, aaccT mutant construct; 5, CtTGG mutant construct; 6, aGTGG mutant construct; 7, pCypB/400 containing ERSE consensus sequence. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pCypB/400; #P<0.05 versus Tm-treated pCypB/400. (D) Activation of the CypB promoter by ATF6 or XBP1. Luciferase assay was conducted with pCypB/400 and one of the following constructs: pcDNA, inactive ATF6, active ATF6, unspliced XBP1, spliced XBP1, or ATF6-siRNA. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus inactive ATF6 transfection; #P<0.05 versus unspliced XBP1 transfection; **P<0.05 versus active ATF6 transfection. pcDNA was employed as a negative control. All luciferase data are shown with the means ± s.d. relative to the basal activity of pCypB/400 (B-D). (E) EMSA and supershift EMSA of the CypB ERSE motif. Nuclear extracts prepared from H9C2 cells exposed to Tm for 24 hours or left unexposed, were incubated with the CypB-ERSE probe or a mutated probe (CGTtt) after preincubation with no antibody or with antiserum specific to ATF6. (F) ChIP assays with antibodies against ATF6, XBP1 or NF-Y along with extracts of H9C2 cells exposed to Tg or Tm for 24 hours or left unexposed. Input, sample representing amplification from a 1:100 dilution of total input chromatin. IgG: pre-immune serum. (G) Western blot with antibodies against ATF6, NF-Y or Lamin B using nuclear extracts. When exposed Tg and Tm, active ATF6 was increased whereas NF-Y remained unchanged. Lamin B was used as a loading control for nuclear extracts.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Overexpression of cyclophilin B attenuates ER stress-induced cell death. (A) Expression level of CypB/wt or CypB/R62A in the indicated transfectants was analyzed by western blotting. Three different clones in each case were analyzed to exclude the possibility of clonal specificity. CypB was tagged with myc. ER chaperon proteins such as Bip and Grp94 were detected as a reference to monitor the effect of overexpressed CypB on other ER chaperon proteins. Actin was used as a loading control. (B) Localization of overexpressed CypB. After 24 hours of treatment with Tg or Tm under proliferation condition, localization of GFP-CypB/wt or GFP-CypB/R62A in the cells was examined by microscopy. ER tracker was used to identify of the ER. Upper panels shows the localization of GFP alone. Middle panels shows the localization of GFP-CypB/wt, and the lower panels shows the location of GFP-CypB/R62A. ER tracker, blue; GFP, green. Bars, 20 µm. (C) Attenuation of ER stress-induced cell death by overexpressed CypB/wt in proliferation (PM) or differentiation condition (DM) was determined by MTT assay. Note that CypB/R62A overexpression further reduces cell viability. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pcDNA transfected cells; #P<0.05 versus Tm-treated pcDNA transfected cells in PM and DM, respectively. (D) Western blot with apoptosis marker antibodies: caspase 3 (cleaved form), cytochrome c (cytosolic), procaspase 12 and Bax (mitochondrial). Numbers under each band are representative of at least five different experiments and are expressed as the means ± s.d. *P<0.05 versus Tg-treated pcDNA. (E) Changes in induction of ER resident proteins in CypB/wt and CypB/R62A transfectants. Total cellular extracts from the cells were analyzed for Bip, Grp94 and PDI by western blot. Numbers under each band are representative of at least five different experiments and are expressed as the means ± s.d. *P<0.05 versus Tg-treated pcDNA. (F) The defensive role of CypB is not limited to cell type. Chang and DU145 cells were infected with no adenovirus, Ad-GFP, Ad-CypB/wt, or Ad-CypB/R62A. The expression levels of CypB were detected by western blotting. Their transduction frequency (60%) was monitored by GFP positivity. Numbers under each band are representative of at least five different experiments and are expressed as the means ± s.d. *P<0.05 versus non-infected Chang and DU145 cells, respectively. The survival rates of each infectant after ER stress were quantified by MTT assay. Ad-GFP, green fluorescence protein adenovirus; Ad-CypB/wt, CypB adenovirus; Ad-CypB/R62A, CypB/R62A adenovirus. The data represent the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated non-infected cells; #P<0.05 versus Tm-treated non-infected Chang and DU145 cells.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Overexpressed cyclophilin B suppresses Ca2+ release from the ER in response to ER stress. Relative changes in cytosolic Ca2+ level in the indicated transfectants (n=20) are shown in the left panels and are plotted in the right panels. (A) Green color represents intracellular Ca2+. Red arrow (left panel) and dotted line (right panel). (B) Each transfectant was treated with 4 mM EGTA to reduce extracellular background Ca2+ prior to treatment with 1 µM Tg. In A and B the red arrowheads (left panel) and dotted line (right panel) indicate the Tg treatment point. In B the blue arrow (left panel) and dotted line (right panel) indicate the EGTA treatment point.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Cyclophilin B overexpression suppresses ROS generation, mitochondrial membrane damage and chromosomal DNA fragmentation in response to ER stress. (A) ROS measurement and its quantitative analysis. The dotted line represents the basal level of ROS in cells. Values are means ± s.d. obtained from five independent experiments. (B) MMP measurement and its quantitative analysis. Arrowhead indicates cells with mitochondrial damage. M1 and M2 are cells of damaged and undamaged mitochondria, respectively. (C) Cell viability by MTT assay. Con, untreated; Tg, 1 µM Tg treatment; Tg+catalase, pretreated with 2000 U/ml catalase prior to Tg treatment. The data are means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pcDNA; #P<0.05 versus Tg-treated CypB/R62A. (D) TUNEL assay and its quantification. The green arrowheads in the top panels indicate TUNEL-positive cells. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pcDNA; #P<0.05 versus Tg-treated of CypB/R62A.

View larger version (30K): [in this window] [in a new window]   Fig. 6. siRNA knockdown of cyclophilin B enhances ER stress-induced cell death. (A) Knockdown efficiency of CypB-siRNA was determined by western blotting. ER chaperon proteins such as Grp94, Bip and PDI were detected as a reference to monitor the effect of CypB-siRNA on other ER chaperone proteins. (B) MTT assay. Relative cell viability of each cell line is shown based on that of the con-siRNA transfection. The data are means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated con-siRNA; #P<0.05 versus Tm-treated con-siRNA. (C) Western blotting with apoptosis marker antibodies: caspase 3 (cleaved form) and Bax. TUNEL assay performed after Tg treatment for 24 hours in con-siRNA and CypB-siRNA transfection. Green arrows indicate TUNEL-positive cells. Numbers under each band are representative of at least five different experiments and are expressed as the means ± s.d. *P<0.05 versus Tg-treated con-siRNA transfection. (D) The defensive role of CypB is partly caspase dependent. PcDNA-, CypB/wt- and CypB-siRNA-transfected cells were cultured with 50 µM z-VAD-fmk prior to Tg treatment. The percentage of viable cells was determined using the MTT assay. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tm-treated pcDNA transfectant; #P<0.05 versus Tm-treated CypB-siRNA transfectant.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Cyclophilin B interacts with Bip and Grp94. (A) GST pull-down assay. A GST pull-down assay was conducted using bacterially expressed recombinant GST-CypB/wt and GST-CypB/R62A fusion proteins and H2C2 cell extracts treated with or without Tg and Tm. Western blotting was conducted using Bip, Grp94, CypB or GST antibodies. GST alone was used as a negative control. (B) Co-immunoprecipitation of CypB with Bip and Grp94. Immunoprecipitation was conducted with non-immune serum (Non-IS) or anti-CypB antibody (CypB-Ab). (C) Yeast two-hybrid binding assays. pGBKT7, pGADT7, pGBKT7/CypB/wt, pGBKT7/CypB /R62A, pGADT7/Grp78 and pGADT7/Grp94 were used in the yeast two-hybrid assay with AH109. In the left panel, CypB/wt interacts with Bip or Grp94. In the right panel, CypB/R62A also interacts with Bip or Grp94.

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