Abstract
Background p16 INK4a tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16 INK4a gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts.Methodology/Principal FindingsMutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16 INK4a gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16 INK4a. Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16 INK4a gene expression. In addition, the enhanced binding of Sp1 to p16 INK4a promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.Conclusions/SignificanceAll these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16 INK4a expression during cell aging.
Highlights
Normal human cells undergo a finite number of cell divisions and enter a nondividing state called replicative senescence[1]
GC boxes are essential for p16INK4a promoter activity in senescent human fibroblast cells
To determine the crucial GC-rich region of the human p16INK4a promoter, plasmid pGL3-620 that contained the p16INK4a promoter with 620 bp upstream of the translation start site was used to generate GC boxes site mutation constructs pGL-Mut 1,5 (Tab. 1, and Fig. 1A) respectively. These plasmids were individually transfected into young (Fig. 1B) or senescent (Fig. 1C) human embryonic lung fibroblasts 2BS cells, and the promoter activities were measured by luciferase activities
Summary
Normal human cells undergo a finite number of cell divisions and enter a nondividing state called replicative senescence[1]. There are several independent pathways that control the process of replicative senescence in human cells [5,6,7,8,9]. Such pathways often involve the activation of the cell cycle inhibitors, p21CIP1/WAF1 [10,11,12] and p16INK4a [13,14,15,16,17,18], which are likely the genes that act to induce cellular senescence, and are direct targets of the genetic program that leads cells to senescence [19,20]. What’s more, the basal level of Sp1 is essential for this effect
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