Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells

Abstract

Neural cells undergo glutamate-induced apoptosis in ischaemic brain tissue, in which prostate apoptosis response-4 gene (Par-4) is involved. Human-bone mesenchymal stem cells can be utilized as an effective therapy for ischemic brain injury. In this study, we found that glutamate could induce apoptosis in human-bone mesenchymal stem cells, accompanied by increased expression of Par-4 gene and Smac release from mitochondria. Repressing Par-4 expression attenuated the glutamate-induced apoptosis. Both Par-4 protein and E2F1 protein could bind to E2F1-binding BS3 site on Smac promoter and participated in the formation of a proteins-DNA complex. Moreover, in the complex, E2F1, not Par-4, was found to be directly bound to the Smac promoter, suggesting that Par-4 exerted indirectly its transcriptional control on the Smac gene though interacting with E2F1. Expression of full-length Par-4 in human-bone mesenchymal cells resulted in increased activity of the Smac promoter. In addition, the indirect transcripional regulation of Par-4 on Smac depended on its COOH terminus-mediated interaction between Par-4 and E2F1. We conclude that the formation of proteins-DNA complex, containing Par-4 protein, E2F1 protein and the Smac promoter, contributes to the pro-apoptotic effect on glutamate-treated human-bone mesenchymal stem cells.

Figure 1.

Par-4 plays an important role in glutamate-induced apoptosis of hBMSCs. (A) Glutamate induced apoptosis in hBMSCs. hBMSCs were harvested after treatment with or without 1–100 μM glutamate for 6, 12 or 24 h. Cells were incubated with Annexin V-FITC and propidium iodide and analysed by flow cytometry. The percentages of apoptotic cells are presented in the bar graph. Results are means of triplicates and representative for results of three independent experiments. *, #, Δ, P < 0.05. (B) DNA Fragmentation assay in apoptotic hBMSCs. hBMSCs were harvested after treatment with (lane 1) or without (lane 2) 100 μM glutamate for 24 h. DNA were precipitated and electrophoresed in 1.5% agarose gel. (C) Glutamate induced an increase of Par-4 expression in hBMSCs in a dose-dependent manner. Western blotting analysis was used to determine Par-4 expression in nuclear extracts in hBMSCs exposed to various doses of glutamate. Intensity of Par-4 band from Western blotting were quantified and normalized with Lamin A/C. *P < 0.05, compared to the hBMSCs without treatment of glutamate. (D) Real time Quantitative PCR assays for inhibitory effect of siRNA targeting Par-4 gene. hBMSCs were transfected with either Par-4 siRNA-1,2,3,4, scrambled siRNA or mock. Empty vector was transfected as a control. After 48 h of transfection, relative amounts of Par-4 mRNA were measured by real-time quantitative RT–PCR and normalized against GAPDH. Bars depict the percentage of Par-4 mRNA versus control. *P < 0.05, versus control. (E) Reduced Par-4 expression by siRNA inhibited glutamate-induced apoptosis of hBMSCs. hBMSCs were transfected with either Par-4-siRNA-1, scrambled siRNA or mock. After 48 h of transfection, hBMSCs were harvested in the presence or absence of 100 μM glutamate for 24 h. The percentages of apoptosis in the cells were analysed by flow cytometry. The result is presented in the bar graph. Values are mean ± SE of five independent experiments. *P < 0.05, compared to the cells with transfection of empty vector but without exposure of glutamate. ^#P < 0.05, compared to the cells with transfection of empty vector and with exposure of glutamate. (F) Overexpression of Par-4 predisposed hBMSCs to undergo apoptosis induced by glutamate. hBMSCs were transfected with either pcDNA3.1-Par-4 or empty vector pcDNA3.1. After 48 h of transfection, hBMSCs were exposed to 100 μM glutamate for 24 h. The percentages of apoptosis in the cells were analysed by flow cytometry. *P < 0.05, compared to the cells with transfection of empty pcDNA3.1 but without exposure of glutamate. ^#P < 0.05, compared to the cells with transfection of empty pcDNA3.1 and with exposure of glutamate.

Figure 2.

Ectopic expression of Par-4 enhanced Smac protein and transcript. Blocking endogenous E2F1 abrogated the enhancement of Par-4-induced Smac expression. (A) Glutamate induced a release of Smac from mitochondria. hBMSCs were treated with glutamate at the dose from 1 to 100 µM. Subcellular localization of Smac was determined by western blotting after 24 h, and protein levels were quantified by computer-assisted densitometry. Tubulin levels were analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Intensities of bands from western blotting analysis were quantified. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are mean ± S.E. of three independent experiments performed in duplicate. *P < 0.05 versus the cells without treatment of glutamate. (B) Overexpression of Par-4 enhanced Smac protein in hBMSCs. hBMSCs were transfected with either pcDNA3.1-Par-4 or empty pcDNA3.1 vector. Subcellular localization of Smac was determined by Western blotting after 48 h, and protein levels were quantified by computer-assisted densitometry. Tubulin levels were analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are the mean ± SE of three independent experiments performed in duplicate. *P < 0.05 versus the cells transfected with empty vector. (C) Overexpression of Par-4 enhanced Smac transcript in hBMSCs. Northern blotting was used to determine Smac mRNA in hBMSCs. hBMSCs were transfected with either pcDNA3.1-Par-4 or empty pcDNA3.1 vector. After 24 h of transfection, total cellular RNAs were separated and hybridized to 32P-labelled DNA probes. GAPDH probe was used to normalize for differences in RNA loading. Intensities of bands from northern blotting analysis were quantitated and normalized with GAPDH. Arbitrary unit = (ASmac/AGAPDH)×100%. Values are mean ± SE of three independent experiments performed in duplicate. *P < 0.05 versus the cells transfected with empty vector. (D) Blocking endogenous E2F1 by siRNA abrogated the enhancement of Par-4-induced Smac protein in hBMSCs. hBMSCs were co-transfected with pcDNA3.1-Par-4 and psiSTRIKE-E2F1-siRNA-1 vector. As controls, these plasmids or empty vectors were respectively transfected into hBMSCs. Subcellular localization of Smac/DIABLO was determined by western blotting after 48 h, and protein levels were quantified by computer-assisted densitometry. Tubulin protein was analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Intensities of bands from western blotting analysis were quantitated. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are the mean ± S.E. of three independent experiments performed in duplicate. *P < 0.05 versus untransfected cells (the first bar at left). *P < 0.05 versus hBMSCs transfected only with pcDNA3.1-Par-4. (E) Blocking endogenous E2F1 by siRNA abrogated enhancement of Par-4-induced Smac transcript in hBMSCs. hBMSCs were co-transfected with pcDNA3.1-Par-4 and psiSTRIKE-E2F1-siRNA-1 vector. As a control, empty vector was respectively transfected into hBMSCs. After 24 h of transfection, relative amount of Par-4 mRNA was measured by real-time quantitative RT–PCR and normalized against GAPDH. Bars depict the percentage of Par-4 mRNA versus that of untransfected cells. Values are mean ± SE of three independent experiments. *P < 0.05 versus untransfected cells (control, the first bar at left). ^#P < 0.05 versus transfected cells only with pcDNA3.1-Par-4.

Figure 2.

Ectopic expression of Par-4 enhanced Smac protein and transcript. Blocking endogenous E2F1 abrogated the enhancement of Par-4-induced Smac expression. (A) Glutamate induced a release of Smac from mitochondria. hBMSCs were treated with glutamate at the dose from 1 to 100 µM. Subcellular localization of Smac was determined by western blotting after 24 h, and protein levels were quantified by computer-assisted densitometry. Tubulin levels were analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Intensities of bands from western blotting analysis were quantified. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are mean ± S.E. of three independent experiments performed in duplicate. *P < 0.05 versus the cells without treatment of glutamate. (B) Overexpression of Par-4 enhanced Smac protein in hBMSCs. hBMSCs were transfected with either pcDNA3.1-Par-4 or empty pcDNA3.1 vector. Subcellular localization of Smac was determined by Western blotting after 48 h, and protein levels were quantified by computer-assisted densitometry. Tubulin levels were analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are the mean ± SE of three independent experiments performed in duplicate. *P < 0.05 versus the cells transfected with empty vector. (C) Overexpression of Par-4 enhanced Smac transcript in hBMSCs. Northern blotting was used to determine Smac mRNA in hBMSCs. hBMSCs were transfected with either pcDNA3.1-Par-4 or empty pcDNA3.1 vector. After 24 h of transfection, total cellular RNAs were separated and hybridized to 32P-labelled DNA probes. GAPDH probe was used to normalize for differences in RNA loading. Intensities of bands from northern blotting analysis were quantitated and normalized with GAPDH. Arbitrary unit = (ASmac/AGAPDH)×100%. Values are mean ± SE of three independent experiments performed in duplicate. *P < 0.05 versus the cells transfected with empty vector. (D) Blocking endogenous E2F1 by siRNA abrogated the enhancement of Par-4-induced Smac protein in hBMSCs. hBMSCs were co-transfected with pcDNA3.1-Par-4 and psiSTRIKE-E2F1-siRNA-1 vector. As controls, these plasmids or empty vectors were respectively transfected into hBMSCs. Subcellular localization of Smac/DIABLO was determined by western blotting after 48 h, and protein levels were quantified by computer-assisted densitometry. Tubulin protein was analysed to confirm equal protein loading in cytosol. Hsp60 was used to monitor the absence of mitochondrial contamination in the cytosolic fraction and to verify equal protein loading in mitochondrial fraction. Intensities of bands from western blotting analysis were quantitated. Results are indicated as the ratio between Smac levels in mitochondria versus Smac levels in cytosol and are the mean ± S.E. of three independent experiments performed in duplicate. *P < 0.05 versus untransfected cells (the first bar at left). *P < 0.05 versus hBMSCs transfected only with pcDNA3.1-Par-4. (E) Blocking endogenous E2F1 by siRNA abrogated enhancement of Par-4-induced Smac transcript in hBMSCs. hBMSCs were co-transfected with pcDNA3.1-Par-4 and psiSTRIKE-E2F1-siRNA-1 vector. As a control, empty vector was respectively transfected into hBMSCs. After 24 h of transfection, relative amount of Par-4 mRNA was measured by real-time quantitative RT–PCR and normalized against GAPDH. Bars depict the percentage of Par-4 mRNA versus that of untransfected cells. Values are mean ± SE of three independent experiments. *P < 0.05 versus untransfected cells (control, the first bar at left). ^#P < 0.05 versus transfected cells only with pcDNA3.1-Par-4.

Figure 3.

Glutamate induced the formation of protein complex containing Par-4 and E2F1. Immunoprecipitations and western blotting analysis were performed with whole-cell extracts in hBMSCs with or without treatment of 100 µM glutamate for 24 h. The whole-cell extracts (0.15 mg protein/0.5 ml), were mixed with 10 μl of anti-Par-4 antibody or preimmune serum. The mixtures were incubated at 4°C for 1 h, and then 10 μl protein A Sepharose beads were added. The mixtures were incubated for 1 h and, after being washed, the beads were submitted for western blotting analysis with anti-E2F1 antibody. Immunoprecipitations with E2F1 antibody and Western blotting with Par-4 antibody were similarly performed. The other E2F1 family members were used as control.

Figure 4.

Par-4 and E2F1 bound to Smac promoter in hBMSCs with exposure of glutamate. (A and B) hBMSCs were treated with or without 100 µM glutamate for 24 h. And nuclear proteins were isolated. Before the binding reaction, 50 µg of nuclear extract was preincubated with either anti-Par-4 antibody (R334) or anti-E2F1 antibody. Subsequently, these nuclear extracts were co-incubated with the magnetic beads containing biotinylated oligonucleotide sequence of SB3 sites within Smac promoter for binding reaction. The beads were captured by a magnet, washed three times with a high salt buffer, and resuspended in Laemmli buffer. The samples were heated at 95°C for 5 min to elute all the proteins, loaded onto a 12% SDS–PAGE, electrophoresed, and transferred to a nitrocellulose membrane. Par-4 and E2F1 were detected using the Par-4 antibody (R334) or E2F1 antibody by immunoblotting. (C) DNA-binding activity of E2F1-binding sites BS3 (−200 to −193 bp) within Smac promoter was assessed by electrophoretic mobility shift assay. After treatment with or without 100 µM glutamate for 24 h, nuclear extracts were obtained from the hBMSCs. Nuclear extracts were incubated with the probes in the presence or absence of antibodies directed against Par-4 or E2F1. In competition studies, a 100-fold molar excess of unlabelled oligonucleotide was added to binding reaction mixture before the addition of the labelled probes. Results shown are representative of three independent experiments.

Figure 5.

Demonstration of in vivo binding of Par-4 and E2F1 to BS3 site (−200 to −193 bp relative to ATG) on the Smac/DIABLO promoter by chromatin immunoprecipitation. hBMSCs were treated with or without 100 µM glutamate for 24 h. Chromatin lysates were immunoprecipitated (IP) with either antibody against Par-4 or antibody against E2F1. The samples were processed as described under ‘Materials and Methods’ section. Immunoprecipitated DNA was amplified using primers representing the BS3 site (−200 to−193 bp relative to ATG) on Smac promoter. PCR analysis of the total input DNA was also preformed. Products of chromatin immunoprecipitation and PCR amplification were analysed by 2% agarose gel electrophoresis and beta-actin was performed as a negative control. Results are representative of three independent experiments.

Figure 6.

The indirectly regulation of Par-4 on Smac/DIABLO promoter was dependant on its COOH terminus-mediated interaction between Par-4 and E2F1 in the hBMSCs. (A) Luciferase activity assays. hBMSCs were co-transfected with pGL3-Smac-P-Luc reporter and pRL-CMV vector as an internal control for transfection efficiency. At the same time the cells were also transfected with either increasing amounts of pcDNA3.1-myc-Par-4 or pcDNA3.1-myc-ΔPar-4, or an empty pcDNA3.1 vector. Luciferase activity was determined at 24 h after transfection. Non-transfected cells were used as the background. All values were normalized for expression of Renilla luciferase and expressed as X-fold induction relative to the activity of pGL3-Smac-P-Luc reporter co-transfected with empty pcDNA3.1. Shown is a representative experiment of three independent experiments. Values are mean ± SE of three independent experiments. *P < 0.05 versus cells co-transfected with pGL3-Smac-P-Luc reporter and empty pcDNA3.1. (B) Immunoprecipitations and Western blotting analysis of nuclear extracts in hBMSCs. Cells was transfected with pcDNA3.1-myc-Par-4 or pcDNA3.1-myc-ΔPar-4. After 24 h of transfection, nuclear proteins were extracted. Then the nuclear extracts, (0.15 mg protein/0.5 ml), were mixed with 10 μl of anti-myc antibody or pre-immune serum. The mixtures were incubated at 4°C for 1 h, and then 10 μl protein A Sepharose beads were added. The mixtures were incubated for 1 h and, after being washed, the beads were submitted for western blotting analysis with anti-E2F1 antibody. Lanes 1 and 4, protein A gel only; lanes 2 and 5, cell lysate immunoprecipitated with anti-myc antibody; lanes 3 and 6, cell lysate incubated with pre-immune serum.