Current theories suggest that progressive age-associated declines in tissue function are caused by changes in intrinsic processes, and that these can occur in the absence of disease. We hypothesize that components of eukaryotic gene regulatory processes may be intrinsically altered by aging to cause increased or decreased gene expression, in turn producing the observed declines in tissue functions. Following such systemic injuries as bacterial lipopolysaccharide (LPS) mediated acute inflammation, the liver responds with a striking increase in the synthesis of a subset of serum proteins, the acute phase reactants (APR). Our preliminary studies indicate that aging affects the regulation of one of the APR genes, the alpha1-acid glycoprotein (AGP) gene. Changes include (a) an increase in the constitutive level of the AGP MRNA pool, and (b) slowed induction of AGP by LPS. In this project we will use the AGP gene as a model to determine whether the structure and function of regulatory factors are affected by aging. AGP MRNA levels will be determined to establish complete LPS-stimulated induction curves in 2, 12, and 24 month Balb/cNNia mice, and nuclear run-on analyses will be done to ask if aging alters both constitutive and LPS-inducible levels of AGP gene transcription in aged mice. Preliminary studies have shown that the mouse AGP promoter has two trans-acting factor binding sites, namely region B (-104 to -91) and region C (-125 to -104), and that the binding activity of these factors may be altered in aged animals. In addition the proteins that binding activity of these factors may be altered in aged animals. In addition the proteins that bind to region C have been identified as C/EBPalpha (constitutive) and C/EBPbeta (LPS-inducible). To determine if trans-acting factors of the AGP promoter are altered in aged animals, activity of these factors will be determined by Dnase I (in vitro) and in vivo footprinting, and gel- shift/Scatchard plot analysis. Experiments will be done to ask whether age-associated structural changes in these trans-acting factors is the basis for their altered binding activity. These experiments include protein modification by phosphorylation and homodimer/heterodimer function. Trans-acting factor MRNA and protein levels will be analyzed to determine if aging affects the expression of their genes. An in vitro transcription assay system will also be developed in order to conduct functional assays of the trans-acting factors. Our long range goal is to determine how gene regulation at the transcriptional or post transcriptional level is intrinsically affected by aging.
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