Effect of hypoxia on the expression and activity of mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and MKP-3 in neuronal nuclei of newborn piglets: the role of nitric oxide.
Mitogen-activated protein kinase-1 (MAPK-1) and MAPK-3 regulate survival and programmed cell death of neurons under stress conditions. The activity of MAPK-1 and MAPK-3 is regulated by dual specificity phosphatases: MKP-1 and MKP-3. In previous studies, we have shown that cerebral hypoxia results in increased activation of MAPK-1 and MAPK-3. Furthermore, we have shown that the hypoxia-induced activation of MAPK is nitric oxide (NO)-mediated. The present study tested the hypothesis that hypoxia results in altered expression and activity of MKP-1 and MKP-3 in neuronal nuclei and the administration of 7-nitro-indazole (7-NINA; 1 mg/kg, 60 min prior to hypoxia), a selective nNOS inhibitor, will prevent the hypoxia-induced alteration in the expression and activity of MKP-1 and MKP-3. To test this hypothesis expression and activity of MKP-1 and MKP-3 were determined in neuronal nuclei of normoxic (Nx; n=5), hypoxic (Hx; n=5) and 7-NINA-pretreated-hypoxic (7-NINA-Hx; n=5). Hypoxia was achieved by exposing the animals to an FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were analyzed by Western blot using specific antibodies for MKP-1 and MKP-3 (Santa Cruz, CA, USA). The protein band density was determined by imaging densitometry and expressed as OD x mm2. The density of MKP-1 was 61.57+/-5.68, 155.86+/-44.02 and 69.88+/-25.54 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly, the density of MKP-3 was 66.46+/-5.88, 172.04+/-33.10 and 116.88+/-14.66 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). The data show an increased expression of MKP-1 and MKP-3 during hypoxia in neuronal nuclei of newborn piglets and the administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increased expression of MKP-1 and MKP-3. The activity of MKP-1 (pmol/min) was 176.17+/-16.95 in Nx, 97.56+/-10.64 in Hx and 130+/-14.42 in the 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly the activity of MKP-3 was 104.11+/-12.17 in Nx, 36.29+/-16.88 in Hx and 77.89+/-20.18 in the 7-NINA groups, respectively (P<0.05, ANOVA). The results demonstrate that cerebral hypoxia results in increased expression of MKP-1 and MKP-3 expression that was prevented by the administration of 7-NINA. In contrast, hypoxia resulted in decreased activity of MKP-1 and MKP-3 that was prevented by the administration of a nNOS inhibitor. We conclude that hypoxia-induced decrease in MKP-1 and MKP-3 activity is not due to altered expression but due to NO-mediated modification of the cysteine residue at the active site of these dual specificity phosphatases, a mechanism of their inactivation that leads to activation of MAP kinases.
Mishra
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Department of Pediatrics, Room 701, 7th Floor Heritage Building, Neonatal Research Laboratory, MCP, Drexel University College of Medicine and St. Christopher's Hospital for Children, 3300 Henry Avenue, Philadelphia, PA 19129, USA. mishra@drexel.edu
Delivoria-Papadopoulos
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology*; D054638:Dual Specificity Phosphatase 1; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D000860:Hypoxia; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D007191:Indazoles / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009474:Neurons / Q000166:cytology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000502:physiology*; D010725:Phosphocreatine / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013552:Swine
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D007191:Indazoles; D010725:Phosphocreatine; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D048052:Mitogen-Activated Protein Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; C080122:7-nitroindazole