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Decreased expression and activity of the immediate-early growth response (Egr-1) gene product during cellular
M Meyyappan1, K Wheaton, K T Riabowol
1Department of Medical Science, Southern Alberta Cancer Research Center, University of Calgary Health Sciences Center, Canada.
Abstract:
Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.
Insights
Cellular senescence in human diploid fibroblasts (HDFs) involves downregulation of the egr-1 gene. This decrease in egr-1 expression and activity is linked to reduced serum response factor (SRF) binding, impacting cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Human diploid fibroblasts (HDFs) exhibit limited proliferation in vitro, reaching cellular senescence.
- Altered gene expression in senescence is hypothesized to impede cell proliferation.
Purpose of the Study:
- To investigate the expression and activity of the early growth response-1 (egr-1) gene in senescent HDFs.
- To elucidate the mechanism underlying egr-1 downregulation during cellular senescence.
Main Methods:
- Quantitative analysis of egr-1 mRNA, protein levels, and DNA binding activity in senescent HDFs.
- Electrophoretic mobility shift assays to assess serum response factor (SRF) binding to egr-1 promoter elements.
Main Results:
- Senescent HDFs showed significant downregulation of egr-1 at mRNA, protein, and DNA binding levels.
- Reduced Egr-1 DNA binding correlated with decreased transcriptional activation in vivo.
- A marked decrease in SRF binding activity to the egr-1 promoter was observed in senescent cells.
Conclusions:
- Downregulation of egr-1 is a key feature of cellular senescence in HDFs.
- Reduced SRF binding to the egr-1 promoter contributes to egr-1 downregulation.
- A common mechanism involving SRF may regulate multiple mitogen-responsive genes during senescence.