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Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis.

Seko Y, Fujimura T, Yao T, Taka H, Mineki R, Okumura K, Murayama K - Sci Rep (2015)

Bottom Line: It causes cell damage that leads to apoptosis via uncertain mechanisms.Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury.These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Nihonbashi-Bakurocho, Chuo-ku, Tokyo 103-0002, Japan.

ABSTRACT
Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

No MeSH data available.


Related in: MedlinePlus

Induction of apoptosis in cardiac myocytes by eIF5A.(a–f) Effects of re-eIF5A protein (10 μg/ml) on cultured cardiac myocytes. (a) Induction of apoptosis in cardiac myocytes as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue). Representative images at 72 h after the addition of re-eIF5A protein. (b) A time course of the percentage of apoptotic cardiac myocytes, as determined by TUNEL staining, induced by re-eIF5A (cytosolic), re-eIF5A (secreted), or mutant re-eIF5A (K50A) (secreted). The data are expressed as the mean ± s.e.m. (n = 6 for each). (c) Western blot analysis of the effects of secreted re-eIF5A on cytochrome c release from the mitochondria (upper panel) and on the activation of caspase-3 (middle panel). A Western blot for actin was used as a loading control. *P = 0.0001 vs. control; **P = 0.0001 vs. re-eIF5A (secreted); †P  = 0.0054 vs. control; ††P = 0.0107 vs. re-eIF5A (secreted) (mean ± s.e.m., n = 4 for each) (Dunnett's multiple comparison test). (d) Representative confocal images of the effect of secreted re-eIF5A on the subcellular localization of AIF. Nuclei (arrows) were stained with 1 μg/ml Hoechst 33342. (e) Western blot analysis of the effects of secreted re-eIF5A on subcellular translocation of AIF from the mitochondria to the nucleus. The ratio of the expression levels of AIF in the nuleus to the mitochondria were shown in the right panel. *P = 0.0001 vs. control (mean ± s.e.m., n = 4, unpaired t-test). (f) The induction of apoptosis in cardiac myocytes as determined by double-immunostaining for Annexin-V (upper panels, labeled with FITC) and cardiac myosin (lower panels, labeled with TRITC). (g) Electron microscopic determination of the hypercondensation of nuclear chromatin (an arrow) induced by secreted re-eIF5A. (d–g) Representative images at 48 h after the addition of secreted re-eIF5A.
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f3: Induction of apoptosis in cardiac myocytes by eIF5A.(a–f) Effects of re-eIF5A protein (10 μg/ml) on cultured cardiac myocytes. (a) Induction of apoptosis in cardiac myocytes as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue). Representative images at 72 h after the addition of re-eIF5A protein. (b) A time course of the percentage of apoptotic cardiac myocytes, as determined by TUNEL staining, induced by re-eIF5A (cytosolic), re-eIF5A (secreted), or mutant re-eIF5A (K50A) (secreted). The data are expressed as the mean ± s.e.m. (n = 6 for each). (c) Western blot analysis of the effects of secreted re-eIF5A on cytochrome c release from the mitochondria (upper panel) and on the activation of caspase-3 (middle panel). A Western blot for actin was used as a loading control. *P = 0.0001 vs. control; **P = 0.0001 vs. re-eIF5A (secreted); †P  = 0.0054 vs. control; ††P = 0.0107 vs. re-eIF5A (secreted) (mean ± s.e.m., n = 4 for each) (Dunnett's multiple comparison test). (d) Representative confocal images of the effect of secreted re-eIF5A on the subcellular localization of AIF. Nuclei (arrows) were stained with 1 μg/ml Hoechst 33342. (e) Western blot analysis of the effects of secreted re-eIF5A on subcellular translocation of AIF from the mitochondria to the nucleus. The ratio of the expression levels of AIF in the nuleus to the mitochondria were shown in the right panel. *P = 0.0001 vs. control (mean ± s.e.m., n = 4, unpaired t-test). (f) The induction of apoptosis in cardiac myocytes as determined by double-immunostaining for Annexin-V (upper panels, labeled with FITC) and cardiac myosin (lower panels, labeled with TRITC). (g) Electron microscopic determination of the hypercondensation of nuclear chromatin (an arrow) induced by secreted re-eIF5A. (d–g) Representative images at 48 h after the addition of secreted re-eIF5A.

Mentions: Re-eIF5A from RCP (that is, secreted re-eIF5A, 10 μg/ml) potently induced the apoptosis of cultured cardiac myocytes, as shown by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining (brown color) and cardiac myosin immunostaining (blue color) (Fig. 3a). However, hardly any cardiac myocytes underwent apoptosis in the untreated control and cytosolic re-eIF5A (10 μg/ml)-treated groups (Fig. 3a). Secreted re-eIF5A mutated at a single amino acid (K50→A50) [eIF5A (K50A)] (10 μg/ml), which renders it unable to be hypusinated12, only partially induced apoptosis (Fig. 3a). This suggested that hypusination is important but not essential for the apoptosis-inducing function of secreted-eIF5A. We also found that hypusination appears not to affect significantly the secretion of eIF5A (data not shown). A time course depicting the percentage of apoptotic cardiac myocytes is shown in Fig. 3b. Secreted re-eIF5A significantly increased the cytosolic expression of cytochrome c and the cleaved form of caspase-3, both of which peaked at 48 h (Fig. 3c), and significantly induced the translocation of apoptosis-inducing factor (AIF) from the cytosol (mitochondria) to the nucleus in cardiac myocytes at 48 h as determined by Hoechst 33342 (1 μg/ml) staining and AIF immunostaining as well as Western blot for AIF (Fig. 3d,e). The induction of the apoptosis of cardiac myocytes by secreted re-eIF5A was further confirmed by Annexin-V staining (Fig. 3f) and by the hypercondensation of nuclear chromatin, as assessed by electron microscopy (Fig. 3g). Secreted re-eIF5A induced the phosphorylation of the mitogen-activated protein kinase (MAPK) family members, IκB and ATF2 (Supplementary Fig. 6), markedly activating ERK1/2 and moderately activating other MAPK members, Akt, and signal transducers and activators of transcription (STATs) (Supplementary Fig. 7a,b). Whereas cytosolic-re-eIF5A did not activate these signaling pathways (data not shown).


Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis.

Seko Y, Fujimura T, Yao T, Taka H, Mineki R, Okumura K, Murayama K - Sci Rep (2015)

Induction of apoptosis in cardiac myocytes by eIF5A.(a–f) Effects of re-eIF5A protein (10 μg/ml) on cultured cardiac myocytes. (a) Induction of apoptosis in cardiac myocytes as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue). Representative images at 72 h after the addition of re-eIF5A protein. (b) A time course of the percentage of apoptotic cardiac myocytes, as determined by TUNEL staining, induced by re-eIF5A (cytosolic), re-eIF5A (secreted), or mutant re-eIF5A (K50A) (secreted). The data are expressed as the mean ± s.e.m. (n = 6 for each). (c) Western blot analysis of the effects of secreted re-eIF5A on cytochrome c release from the mitochondria (upper panel) and on the activation of caspase-3 (middle panel). A Western blot for actin was used as a loading control. *P = 0.0001 vs. control; **P = 0.0001 vs. re-eIF5A (secreted); †P  = 0.0054 vs. control; ††P = 0.0107 vs. re-eIF5A (secreted) (mean ± s.e.m., n = 4 for each) (Dunnett's multiple comparison test). (d) Representative confocal images of the effect of secreted re-eIF5A on the subcellular localization of AIF. Nuclei (arrows) were stained with 1 μg/ml Hoechst 33342. (e) Western blot analysis of the effects of secreted re-eIF5A on subcellular translocation of AIF from the mitochondria to the nucleus. The ratio of the expression levels of AIF in the nuleus to the mitochondria were shown in the right panel. *P = 0.0001 vs. control (mean ± s.e.m., n = 4, unpaired t-test). (f) The induction of apoptosis in cardiac myocytes as determined by double-immunostaining for Annexin-V (upper panels, labeled with FITC) and cardiac myosin (lower panels, labeled with TRITC). (g) Electron microscopic determination of the hypercondensation of nuclear chromatin (an arrow) induced by secreted re-eIF5A. (d–g) Representative images at 48 h after the addition of secreted re-eIF5A.
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f3: Induction of apoptosis in cardiac myocytes by eIF5A.(a–f) Effects of re-eIF5A protein (10 μg/ml) on cultured cardiac myocytes. (a) Induction of apoptosis in cardiac myocytes as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue). Representative images at 72 h after the addition of re-eIF5A protein. (b) A time course of the percentage of apoptotic cardiac myocytes, as determined by TUNEL staining, induced by re-eIF5A (cytosolic), re-eIF5A (secreted), or mutant re-eIF5A (K50A) (secreted). The data are expressed as the mean ± s.e.m. (n = 6 for each). (c) Western blot analysis of the effects of secreted re-eIF5A on cytochrome c release from the mitochondria (upper panel) and on the activation of caspase-3 (middle panel). A Western blot for actin was used as a loading control. *P = 0.0001 vs. control; **P = 0.0001 vs. re-eIF5A (secreted); †P  = 0.0054 vs. control; ††P = 0.0107 vs. re-eIF5A (secreted) (mean ± s.e.m., n = 4 for each) (Dunnett's multiple comparison test). (d) Representative confocal images of the effect of secreted re-eIF5A on the subcellular localization of AIF. Nuclei (arrows) were stained with 1 μg/ml Hoechst 33342. (e) Western blot analysis of the effects of secreted re-eIF5A on subcellular translocation of AIF from the mitochondria to the nucleus. The ratio of the expression levels of AIF in the nuleus to the mitochondria were shown in the right panel. *P = 0.0001 vs. control (mean ± s.e.m., n = 4, unpaired t-test). (f) The induction of apoptosis in cardiac myocytes as determined by double-immunostaining for Annexin-V (upper panels, labeled with FITC) and cardiac myosin (lower panels, labeled with TRITC). (g) Electron microscopic determination of the hypercondensation of nuclear chromatin (an arrow) induced by secreted re-eIF5A. (d–g) Representative images at 48 h after the addition of secreted re-eIF5A.
Mentions: Re-eIF5A from RCP (that is, secreted re-eIF5A, 10 μg/ml) potently induced the apoptosis of cultured cardiac myocytes, as shown by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining (brown color) and cardiac myosin immunostaining (blue color) (Fig. 3a). However, hardly any cardiac myocytes underwent apoptosis in the untreated control and cytosolic re-eIF5A (10 μg/ml)-treated groups (Fig. 3a). Secreted re-eIF5A mutated at a single amino acid (K50→A50) [eIF5A (K50A)] (10 μg/ml), which renders it unable to be hypusinated12, only partially induced apoptosis (Fig. 3a). This suggested that hypusination is important but not essential for the apoptosis-inducing function of secreted-eIF5A. We also found that hypusination appears not to affect significantly the secretion of eIF5A (data not shown). A time course depicting the percentage of apoptotic cardiac myocytes is shown in Fig. 3b. Secreted re-eIF5A significantly increased the cytosolic expression of cytochrome c and the cleaved form of caspase-3, both of which peaked at 48 h (Fig. 3c), and significantly induced the translocation of apoptosis-inducing factor (AIF) from the cytosol (mitochondria) to the nucleus in cardiac myocytes at 48 h as determined by Hoechst 33342 (1 μg/ml) staining and AIF immunostaining as well as Western blot for AIF (Fig. 3d,e). The induction of the apoptosis of cardiac myocytes by secreted re-eIF5A was further confirmed by Annexin-V staining (Fig. 3f) and by the hypercondensation of nuclear chromatin, as assessed by electron microscopy (Fig. 3g). Secreted re-eIF5A induced the phosphorylation of the mitogen-activated protein kinase (MAPK) family members, IκB and ATF2 (Supplementary Fig. 6), markedly activating ERK1/2 and moderately activating other MAPK members, Akt, and signal transducers and activators of transcription (STATs) (Supplementary Fig. 7a,b). Whereas cytosolic-re-eIF5A did not activate these signaling pathways (data not shown).

Bottom Line: It causes cell damage that leads to apoptosis via uncertain mechanisms.Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury.These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Nihonbashi-Bakurocho, Chuo-ku, Tokyo 103-0002, Japan.

ABSTRACT
Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

No MeSH data available.


Related in: MedlinePlus