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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

Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation.(a) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. (b) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). (c) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. (d) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). (e) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, *P = 0.0209) (Mann-Whitney U-test).
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f1: Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation.(a) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. (b) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). (c) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. (d) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). (e) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, *P = 0.0209) (Mann-Whitney U-test).

Mentions: We collected and concentrated a fraction with a relative molecular mass (Mr) > 10 kD from the phosphate-buffered saline (PBS) supernatants of cardiac myocytes subjected to hypoxia (60 min)/reoxygenation (10 min); this fraction was defined as reoxygenation-conditioned PBS (RCP) because it displayed extracellular signal-regulated kinase (ERK)-activating and apoptosis-inducing activities (data not shown). We also collected and concentrated PBS supernatants of non-stimulated cardiac myocytes under normoxia (10 min), designated as control-conditioned PBS (CCP). The proteins in the RCP and CCP fractions were separated via chromatofocusing (Fig. 1a). In each fraction, we monitored the induction of ERK activity, which appeared to be one of the most sensitive markers of the target factor examined, and found that fractions 49–52 (high-salt [1 M NaCl] fractions) in both the RCP and CCP groups displayed strong activity (activity, RCP > CCP; Fig. 1a) and that RCP fractions 5–8 (fractions in Solution A passed through from the column without binding) displayed much weaker activity than fractions 49–52. Then, aliquots of the fractions (49–52) from each group (corresponding to the same number of cells) were subjected to 2-D gel electrophoresis (Fig. 1b; left panel, CCP; right panel, RCP). Because the active components were not eluted by Solution B, they appeared to be acidic.


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)

Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation.(a) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. (b) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). (c) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. (d) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). (e) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, *P = 0.0209) (Mann-Whitney U-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562266&req=5

f1: Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation.(a) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. (b) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). (c) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. (d) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). (e) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, *P = 0.0209) (Mann-Whitney U-test).
Mentions: We collected and concentrated a fraction with a relative molecular mass (Mr) > 10 kD from the phosphate-buffered saline (PBS) supernatants of cardiac myocytes subjected to hypoxia (60 min)/reoxygenation (10 min); this fraction was defined as reoxygenation-conditioned PBS (RCP) because it displayed extracellular signal-regulated kinase (ERK)-activating and apoptosis-inducing activities (data not shown). We also collected and concentrated PBS supernatants of non-stimulated cardiac myocytes under normoxia (10 min), designated as control-conditioned PBS (CCP). The proteins in the RCP and CCP fractions were separated via chromatofocusing (Fig. 1a). In each fraction, we monitored the induction of ERK activity, which appeared to be one of the most sensitive markers of the target factor examined, and found that fractions 49–52 (high-salt [1 M NaCl] fractions) in both the RCP and CCP groups displayed strong activity (activity, RCP > CCP; Fig. 1a) and that RCP fractions 5–8 (fractions in Solution A passed through from the column without binding) displayed much weaker activity than fractions 49–52. Then, aliquots of the fractions (49–52) from each group (corresponding to the same number of cells) were subjected to 2-D gel electrophoresis (Fig. 1b; left panel, CCP; right panel, RCP). Because the active components were not eluted by Solution B, they appeared to be acidic.

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