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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

Upstream signaling mechanisms induced by secreted eIF5A in cardiac myocytes.(a) Secreted re-eIF5A (10 μg/ml) activated initiator caspases, including caspase-8 (IMG-5703; IMGENEX), caspase-10 (ab25045; Abcam), caspase-2 (ab2251; Abcam), and caspase-9 (#9508; Cell Signaling), in cultured cardiac myocytes. The anti-actin antibody was sc-1616 from Santa Cruz. (b) Secreted re-eIF5A (10 μg/ml) rapidly increased the phosphorylation of Shc at Tyr 239/240 (#2434; Cell Signaling) (lower panels), but not FADD at Ser 191 (sc-33399; Santa Cruz) (upper panels), in cultured cardiac myocytes. The control antibodies were anti-Shc (sc-967; Santa Cruz) and anti-FADD (ab24533; Abcam). (c) Secreted re-eIF5A (10 μg/ml) increased the phosphorylation of Jak 1 at Tyr 1022/1023 (sc-16773; Santa Cruz) and Tyk 2 at Tyr 1054/1055 (sc-11763), but not Jak 2 at Tyr 1007/1008 (sc-21870). The control antibodies were anti-Jak 1 (sc-295), anti-Tyk 2 (sc-169), and anti-Jak 2 (sc-34479). (d,e) The effects of Jak inhibitor I (Santa Cruz; 1 μM, pretreated for 24 h) on the induction of apoptosis in cardiac myocytes by secreted re-eIF5A (10 μg/ml, 72 h) as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue), as shown in Fig. 3a. *P < 0.0001 vs. secreted re-eIF5A alone (mean ± s.e.m., n = 4 each) (Dunnett's multiple comparison test) (e). (f) SPR analysis of the interaction between secreted re-eIF5A and cardiac myocyte membrane proteins. The surface of the sensor chip was coupled with 10μg/ml of secreted re-eIF5A in 10 mM acetate buffer (pH 5.5) at a flow rate of 5 μl/min. Then, cardiac myocyte membrane proteins (100 μg/ml in HBS-EP buffer) were analyzed for their interaction with the surface at a flow rate of 10 μl/min for 4 min. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18, 1 μg/ml for each) and BSA (100 μg/ml) were used as positive and negative controls, respectively. All experiments were performed at least in triplicate. The results shown are from one representative experiment.
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f4: Upstream signaling mechanisms induced by secreted eIF5A in cardiac myocytes.(a) Secreted re-eIF5A (10 μg/ml) activated initiator caspases, including caspase-8 (IMG-5703; IMGENEX), caspase-10 (ab25045; Abcam), caspase-2 (ab2251; Abcam), and caspase-9 (#9508; Cell Signaling), in cultured cardiac myocytes. The anti-actin antibody was sc-1616 from Santa Cruz. (b) Secreted re-eIF5A (10 μg/ml) rapidly increased the phosphorylation of Shc at Tyr 239/240 (#2434; Cell Signaling) (lower panels), but not FADD at Ser 191 (sc-33399; Santa Cruz) (upper panels), in cultured cardiac myocytes. The control antibodies were anti-Shc (sc-967; Santa Cruz) and anti-FADD (ab24533; Abcam). (c) Secreted re-eIF5A (10 μg/ml) increased the phosphorylation of Jak 1 at Tyr 1022/1023 (sc-16773; Santa Cruz) and Tyk 2 at Tyr 1054/1055 (sc-11763), but not Jak 2 at Tyr 1007/1008 (sc-21870). The control antibodies were anti-Jak 1 (sc-295), anti-Tyk 2 (sc-169), and anti-Jak 2 (sc-34479). (d,e) The effects of Jak inhibitor I (Santa Cruz; 1 μM, pretreated for 24 h) on the induction of apoptosis in cardiac myocytes by secreted re-eIF5A (10 μg/ml, 72 h) as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue), as shown in Fig. 3a. *P < 0.0001 vs. secreted re-eIF5A alone (mean ± s.e.m., n = 4 each) (Dunnett's multiple comparison test) (e). (f) SPR analysis of the interaction between secreted re-eIF5A and cardiac myocyte membrane proteins. The surface of the sensor chip was coupled with 10μg/ml of secreted re-eIF5A in 10 mM acetate buffer (pH 5.5) at a flow rate of 5 μl/min. Then, cardiac myocyte membrane proteins (100 μg/ml in HBS-EP buffer) were analyzed for their interaction with the surface at a flow rate of 10 μl/min for 4 min. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18, 1 μg/ml for each) and BSA (100 μg/ml) were used as positive and negative controls, respectively. All experiments were performed at least in triplicate. The results shown are from one representative experiment.

Mentions: Next, we analyzed upstream apoptotic signaling pathways, including initiator caspases such as caspase-8, which is recruited and activated by a death domain-containing adaptor protein such as Fas-associated death domain (FADD) upon DR binding. We confirmed that secreted re-eIF5A significantly activated caspases −8 (2.59 ± 0.21 [mean ± s.e.m.] fold at 2 h, n = 3 for each, P = 0.0174 vs. control [0 h] [paired t-test]), −10 (2.26 ± 0.11 fold at 8 h, P = 0.0073), −2 (2.43 ± 0.18 fold at 8 h, P = 0.0160), and −9 (3.52 ± 0.45 fold at 16 h, P = 0.0306) in cultured cardiac myocytes within 2–4 h after stimulation (Fig. 4a). We further examined whether secreted re-eIF5A activates FADD, which mediates DR signaling, or Shc, which mediates receptor tyrosine kinase signaling and activates the MAPK pathway. We found that secreted re-eIF5A rapidly activated Shc (2.77 ± 0.07 [mean ± s.e.m.] fold at 10 min, n = 3 for each, P = 0.0014 vs. control [0 min] [paired t-test]) but not FADD (0.93 ± 0.06 fold at 10 min, P = 0.3576) (Fig. 4b). This result suggested that secreted re-eIF5A induces apoptotic signaling via one of the various types of receptors, including growth factor receptors, cytokine receptors, and G protein-coupled receptors, rather than via a known DR, such as TNF-R1, Fas, DR3, DR4, or DR5.


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)

Upstream signaling mechanisms induced by secreted eIF5A in cardiac myocytes.(a) Secreted re-eIF5A (10 μg/ml) activated initiator caspases, including caspase-8 (IMG-5703; IMGENEX), caspase-10 (ab25045; Abcam), caspase-2 (ab2251; Abcam), and caspase-9 (#9508; Cell Signaling), in cultured cardiac myocytes. The anti-actin antibody was sc-1616 from Santa Cruz. (b) Secreted re-eIF5A (10 μg/ml) rapidly increased the phosphorylation of Shc at Tyr 239/240 (#2434; Cell Signaling) (lower panels), but not FADD at Ser 191 (sc-33399; Santa Cruz) (upper panels), in cultured cardiac myocytes. The control antibodies were anti-Shc (sc-967; Santa Cruz) and anti-FADD (ab24533; Abcam). (c) Secreted re-eIF5A (10 μg/ml) increased the phosphorylation of Jak 1 at Tyr 1022/1023 (sc-16773; Santa Cruz) and Tyk 2 at Tyr 1054/1055 (sc-11763), but not Jak 2 at Tyr 1007/1008 (sc-21870). The control antibodies were anti-Jak 1 (sc-295), anti-Tyk 2 (sc-169), and anti-Jak 2 (sc-34479). (d,e) The effects of Jak inhibitor I (Santa Cruz; 1 μM, pretreated for 24 h) on the induction of apoptosis in cardiac myocytes by secreted re-eIF5A (10 μg/ml, 72 h) as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue), as shown in Fig. 3a. *P < 0.0001 vs. secreted re-eIF5A alone (mean ± s.e.m., n = 4 each) (Dunnett's multiple comparison test) (e). (f) SPR analysis of the interaction between secreted re-eIF5A and cardiac myocyte membrane proteins. The surface of the sensor chip was coupled with 10μg/ml of secreted re-eIF5A in 10 mM acetate buffer (pH 5.5) at a flow rate of 5 μl/min. Then, cardiac myocyte membrane proteins (100 μg/ml in HBS-EP buffer) were analyzed for their interaction with the surface at a flow rate of 10 μl/min for 4 min. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18, 1 μg/ml for each) and BSA (100 μg/ml) were used as positive and negative controls, respectively. All experiments were performed at least in triplicate. The results shown are from one representative experiment.
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Related In: Results  -  Collection

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f4: Upstream signaling mechanisms induced by secreted eIF5A in cardiac myocytes.(a) Secreted re-eIF5A (10 μg/ml) activated initiator caspases, including caspase-8 (IMG-5703; IMGENEX), caspase-10 (ab25045; Abcam), caspase-2 (ab2251; Abcam), and caspase-9 (#9508; Cell Signaling), in cultured cardiac myocytes. The anti-actin antibody was sc-1616 from Santa Cruz. (b) Secreted re-eIF5A (10 μg/ml) rapidly increased the phosphorylation of Shc at Tyr 239/240 (#2434; Cell Signaling) (lower panels), but not FADD at Ser 191 (sc-33399; Santa Cruz) (upper panels), in cultured cardiac myocytes. The control antibodies were anti-Shc (sc-967; Santa Cruz) and anti-FADD (ab24533; Abcam). (c) Secreted re-eIF5A (10 μg/ml) increased the phosphorylation of Jak 1 at Tyr 1022/1023 (sc-16773; Santa Cruz) and Tyk 2 at Tyr 1054/1055 (sc-11763), but not Jak 2 at Tyr 1007/1008 (sc-21870). The control antibodies were anti-Jak 1 (sc-295), anti-Tyk 2 (sc-169), and anti-Jak 2 (sc-34479). (d,e) The effects of Jak inhibitor I (Santa Cruz; 1 μM, pretreated for 24 h) on the induction of apoptosis in cardiac myocytes by secreted re-eIF5A (10 μg/ml, 72 h) as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue), as shown in Fig. 3a. *P < 0.0001 vs. secreted re-eIF5A alone (mean ± s.e.m., n = 4 each) (Dunnett's multiple comparison test) (e). (f) SPR analysis of the interaction between secreted re-eIF5A and cardiac myocyte membrane proteins. The surface of the sensor chip was coupled with 10μg/ml of secreted re-eIF5A in 10 mM acetate buffer (pH 5.5) at a flow rate of 5 μl/min. Then, cardiac myocyte membrane proteins (100 μg/ml in HBS-EP buffer) were analyzed for their interaction with the surface at a flow rate of 10 μl/min for 4 min. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18, 1 μg/ml for each) and BSA (100 μg/ml) were used as positive and negative controls, respectively. All experiments were performed at least in triplicate. The results shown are from one representative experiment.
Mentions: Next, we analyzed upstream apoptotic signaling pathways, including initiator caspases such as caspase-8, which is recruited and activated by a death domain-containing adaptor protein such as Fas-associated death domain (FADD) upon DR binding. We confirmed that secreted re-eIF5A significantly activated caspases −8 (2.59 ± 0.21 [mean ± s.e.m.] fold at 2 h, n = 3 for each, P = 0.0174 vs. control [0 h] [paired t-test]), −10 (2.26 ± 0.11 fold at 8 h, P = 0.0073), −2 (2.43 ± 0.18 fold at 8 h, P = 0.0160), and −9 (3.52 ± 0.45 fold at 16 h, P = 0.0306) in cultured cardiac myocytes within 2–4 h after stimulation (Fig. 4a). We further examined whether secreted re-eIF5A activates FADD, which mediates DR signaling, or Shc, which mediates receptor tyrosine kinase signaling and activates the MAPK pathway. We found that secreted re-eIF5A rapidly activated Shc (2.77 ± 0.07 [mean ± s.e.m.] fold at 10 min, n = 3 for each, P = 0.0014 vs. control [0 min] [paired t-test]) but not FADD (0.93 ± 0.06 fold at 10 min, P = 0.3576) (Fig. 4b). This result suggested that secreted re-eIF5A induces apoptotic signaling via one of the various types of receptors, including growth factor receptors, cytokine receptors, and G protein-coupled receptors, rather than via a known DR, such as TNF-R1, Fas, DR3, DR4, or DR5.

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