EFFECTS OF AGING ON P38 SIGNALING PATHWAY IN MOUSE LIVER

由于活性氧（ROS）的作用，老化细胞积累氧化损伤的线粒体DNA （mtDNA）。这会导致活性氧产生增加，导致更多的DNA损伤和线粒体功能障碍。我们认为ROS影响p38 MAPK应激反应通路的功能。为了支持这一假设，我们在C57BL/6小鼠肝脏中发现了p38蛋白与年龄相关的变化，例如，磷酸化蛋白水平升高；它们的去磷酸化响应于3- NPA（一种配合物II（琥珀酸脱氢）的抑制剂）产生的ROS；而老年肝脏缺乏这种反应。我们提出(a)衰老组织中功能失调线粒体产生的RQS导致p38信号通路的基础活性增加；(b)衰老肝脏中p38通路蛋白对3-NPA反应的失败表明信号功能受损。我们将在以下5个目标中检验这一假设：

Aging cells accumulate oxidant-damaged mitochondriat DNA (mtDNA) due to reactive oxygen species (ROS). This leads to increased ROS production, to more DNA damage and mitochondrial dysfunction. We propose that ROS affects the function of the p38 MAPK stress response pathway. To support this hypothesis we have shown age-associated modifications to p38 proteins in C57BL/6 mouse livers, e.g., increased levels of phosphorylated protein ; their dephosphorylation in response to ROS generated by 3- NPA, an inhibitor of Complex II (succinic dehydrogense); and a lack of this response in aged liver. We propose that (a) RQS generated by dysfunctional mitochondria in aged tissue cause an increased basal activity of the p38 signaling pathway; (b) failure of the p38 pathway proteins to respond to 3-NPA in aged livers suggests signaling functions are compromised. We will test this hypothesis in the following 5 Aims: Aim 1 will identify the site and mechanisms of ROS generation by 3-NPA's inhibition of complex II (SDH) and the role of ROS generated by 3-NPA, rotenone and Antimycin A in p38 activation. Aim 2 will characterize the increased basal levels of p38 activity in aged mice (24 mo); the mechanism of p38 pathway activation by 3- NPA in young (3 mo) and middle-aged (12 mo) mice; and the failure of this response in aged livers. Aim 3 will characterize the age-specific 3-NPA-stimulated dephosphorylation of p38 MAPK activators. Aim 4 will study the mechanism of age-associated increase in activity of the transcription factors ATF-2 and CEBPbeta, and the failure of 3-NPA to activate these proteins, and characterize the role of p38 in activation of mitochondrial chaperones. Aim 5 will examine the effect of aging and oxidative stress on mitochondrial maintenance, using a microarray of 96 mitochondrial protein genes to determine the effects of aging on their expression in aged livers and in response to 3-NPA. We will determine the levels of carbonylated and nitrated mitochondrial 4-hydroxynonena-modified p38 MAPK proteins and correlate this oxidative damage to mitochondrial dysfunction. In this project we initiate an in-depth analysis of the molecular signaling interactions critical to cellular survival; we use gene array and proteomics technology to assist in understanding global effects of age-associated alteration of critical biological functions. Our Project wilt thus identify important causal factors in the age-associated decline in tissue function.