Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.
Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.
Department of Life Science, College of Natural Science, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, South Korea.
Hyun-Seok
HS
Kim
Jee-In
JI
Heo
Seong-Hoon
SH
Park
Jong-Yeon
JY
Shin
Hong-Jun
HJ
Kang
Min-Ju
MJ
Kim
Sung Chan
SC
Kim
Jaebong
J
Kim
Jae-Bong
JB
Park
Jae-Yong
JY
Lee
D001665:Binding Sites; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016329:Sp1 Transcription Factor; D016159:Tumor Suppressor Protein p53