RESUMO
Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.
Assuntos
Senescência Celular , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes fos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Western Blotting , RNA Polimerases Dirigidas por DNA/metabolismo , Diploide , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Sondas de Oligonucleotídeos , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/biossíntese , Fator de Resposta Sérica , TATA Box , Fatores de Transcrição/isolamento & purificação , Transcrição GênicaRESUMO
The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process.
Assuntos
Senescência Celular , Metaloendopeptidases/análise , Inibidores de Proteases/análise , Fator de Crescimento Transformador beta/farmacologia , Células Cultivadas , Colagenases/análise , Colagenases/genética , Fibroblastos , Glicoproteínas/análise , Glicoproteínas/genética , Humanos , Metaloproteinase 3 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/genética , Proteínas/análise , Proteínas/genética , Acetato de Tetradecanoilforbol/farmacologia , Inibidor Tecidual de Metaloproteinase-2 , Inibidor Tecidual de Metaloproteinase-3 , Inibidores Teciduais de MetaloproteinasesRESUMO
Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in vitro and are widely used as a model of cellular aging. Despite growing evidence that cellular aging occurs as a result of altered gene expression, little is known about the activity of transcription factors that regulate gene expression in aging cells. Here we survey the relevant literature regarding altered gene expression and the role of transcription factors during cellular aging, focusing upon the serum response factor (SRF). SRF is hyperphosphorylated in senescent HDFs and fails to bind to the serum-response element in the c-fos promoter. Differential phosphorylation during replicative aging may contribute, at least in part, to the altered activity of SRF and possibly other transcription factors and to subsequent changes in the expression of serum-regulated genes in senescent HDFs.