Characterization of age-associated effects on the transcriptional response to oxidative stress in the mouse heart and skeletal muscle.

摘要

The purpose of my graduate work was a global analysis of the effect of aging on the transcriptional response to stress. To accomplish this goal, high-density oligonucleotide arrays were used to quantify genome-scale differences in the transcriptional response to paraquat-induced oxidative stress in the heart and muscle of young and old mice. Results of this investigation revealed numerous examples of altered expression between young and old mice following oxidative insult in both tissue types. Among these was a reduction in the number of immediate early genes (IEGs) and growth arrest and DNA-damage inducible 45 (GADD45) gene families induced in older heart and muscle following paraquat treatment. Because these classes of genes are mediators or transcriptional targets of mitogen-activated protein kinase (MAPK) pathways, levels of active (phosphorylated) and total protein content of components of MAPK stress signaling were assayed in young and old skeletal muscle over the paraquat time course. It was found that constitutive levels of phospho-JNK, a critical signaling molecule for the MAPK c-Jun N-terminal protein kinase [JNK/stress-activated protein kinase (SAPK)] pathway, were elevated in old versus young skeletal muscle. Phospho-JNK levels increased almost immediately (1 hour post-paraquat treatment) in the young muscle following oxidative stress, whereas a delayed increase was observed in the older muscle over the same time period (5 hours post-paraquat treatment). In addition to the JNK-related findings, an age-related p53-mediated transcriptional program was identified in mouse skeletal muscle. The constitutive mRNA levels of genes induced by p53 was elevated in older muscle, while the levels for genes whose products have been shown to inhibit p53 activity were decreased with age. The age-related increase in p53 activity and expression of p53-related genes in mouse muscle as determined by DNA microarray analysis was also observed at the protein level as determined by Western blot (p53, p21 and GADD45a) and at the transcriptional level by RT-PCR (p21, GADD45a, p16, p53, bbc3, tnfrsf10b, pmaip1 and bok). These findings suggest that both p53 and JNK signaling are altered with age, and that this contributes to the transcriptional differences observed in the stress response of young and old mice.