Age-related changes in the regulation of stress reponse genes are due to altered structure and function of their trans-acting regulators. Our long-range goal is to identify the mechanisms for the age-relted effects on responses to stress factors. We propose that cytokines and reactive oxygen species (ROS), shown to increase in aging, affect the activity of pathways tranducing these mediators' singals, so that aging tissues exhibit characteristics of chronic stress. Our data suggest that the synthesis of C/EBPalpha and C/EBPbeta isoforms is regulated by lipopolysaccharide (LPS); that this involves alternative translational initiation(ATI) at specific in-frame AUG codons withinthe same mRNA; and that ATI characteristic of an inflammatory response in young livers occurs constitutively in aged animals. We propose that ATI occurs via a leaky ribosomal scanning (LRS) mechanism that is linked to specific signal transduction pathways.
Specific aim 1 will determine whether the regulation of C/EBP isoform synthesis involves ATI of their mRNAs in young livers and whether ATI is altered in the aged liver.
Specific Aim 2 will test whether ATI is constitutive level of activity.
Specific Aim 3 demonstrate a linkage of C/EBP isoform synthesis and activation to oxidative stress. We propose that generators of ROS, such as DNA-damaging agents, may trigger the signal pathways that regulate ATI. The role of C/EBP in AP- endonuclease (APE) induction by HOC1, in cellular adaptation to DNA-damaging agents, and APE's inducibility by LPS in aging will be examined. Collaborative experiments to study the effects of mitochondrial DNA-damaged agents, on the regulation of C/EBP isoform synthesis are based on the hypothesis that age-associated increases in ROS may, in part, be due to radicals produced by mitochondrial DNA damage;. We will determine whether p21 (Cip1) is a stress response gene whose expression and regulation involved interaction of C/EBPs with its promoter binding sites.
Specific Aim 4 will test the hypothesis that caloric restriction affects regulation of the biological process involving responses to ROS. The data gained by this project lay the foundation for future studies on tissue specific responses to stress, in vivo, and their susceptibility to age-related diseases.
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