The overall objective of this project is to determine the molecular basis for the lack of interleukin-6 (IL-6) mRNA in senescent cells. The determination of how the expression of a single gene is altered as cells become senescent may help to elucidate the mechanisms by which cells change as they age. The IL-6 gene is induced in young cells both by mitogenic factors and by factors involved in the acute phase response. However, serum and double-stranded RNA can not induce IL-6 expression in senescent cells.
The specific aims of this proposal are: (i) to determine whether IL-6 gene expression can be induced in senescent human fibroblasts (HDF) by inducers other than serum and double-stranded RNA; (ii) to determine whether the decreased expression of the IL-6 gene is unique to the senescent end state or whether there is a progressive age-related decrease in the responsiveness of the IL-6 gene to its inducers; (iii) to determine whether the lack of IL-6 mRNA in serum-stimulated senescent (HDF) reflects transcription repression or message instability; and (iv) to determine whether there is a difference in the proteins bound to the IL-6 promoter in senescent HDF versus quiescent HDF. Part (i) will help to pinpoint the pathways(s) no longer responsive in senescent cells. The induction of the IL-6 gene in quiescent and senescent cells by various agents will be tested by RNase protection assays to detect IL-6 transcripts. Part (ii) is relevant to the question of whether a decreased response in the IL-6 gene occurs in cells as they age in vivo. Using methods similar to part one, experiments will measure IL-6 response in cells at various points in the aging process. The results for part (iii) will be determined by nuclear run-on analysis and will provide clues into the methods used to control the expression of genes in senescent cells. In part (iv) the changes in proteins bound to the promoter will be determined by gel-shift assays and DNase I footprint analysis. This will give direct information on the changes in protein-DNA interactions in cells as they age, e.g. the loss of a transcription factor or the presence of a repressor, and will provide a model for the methods by which other genes are down-regulated in senescent cells.
