p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription.
Previous studies demonstrate that p16, a cyclin-dependent kinase inhibitor and a tumor suppressor, may inhibit matrix metalloproteinase-2 (MMP-2) expression in human cancer cells to suppress tumor invasion and metastasis. However, the detailed mechanism is still unclear. Our results show that p16 inhibits MMP-2 expression via transcriptional repression. Promoter deletion and mutation analysis indicates that p16 acts through the Sp1 transcription factor-binding site located between -72 and -64 bp region from the transcriptional start site of the human MMP-2 promoter to repress gene expression. DNA affinity precipitation assay (DAPA) and chromatin immuno-precipitation (CHIP) assay demonstrate that Sp1 proteins constitutively bind to this consensus sequence in vitro and in vivo. p16 attenuates Sp1 binding to the MMP-2 promoter to suppress gene transcription and overexpression of Sp1 may counteract p16-induced downregulation of MMP-2. CyclinA/CDK complex may directly phosphorylate Sp1 and enhance its DNA-binding activity. Thus, we investigated the effect of p16 on the interaction between cyclin A and Sp1. Our results indicate that p16 induces downregulation of cyclin A and CDK2, reduces the interaction between cyclin A and Sp1, and attenuates phosphorylation of Sp1. Ectoexpression of cyclin A counteracts p16-mediated inhibition of DNA binding of Sp1 and activates MMP-2 promoter activity and mRNA expression. Collectively, our results suggest that p16 suppresses MMP-2 by blocking Sp1-mediated gene transcription.
Wang
Chie-Hong
CH
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Chang
Hui-Chiu
HC
Hung
Wen-Chun
WC
D019925:Cyclin A / Q000032:analysis / Q000235:genetics / Q000502:physiology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000494:pharmacology*; D015536:Down-Regulation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D009361:Neoplasm Invasiveness / Q000503:physiopathology; D009362:Neoplasm Metastasis / Q000503:physiopathology; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000503:physiopathology; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000032:analysis / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology; D014407:Tumor Cells, Cultured
D019925:Cyclin A; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016329:Sp1 Transcription Factor; D020778:Matrix Metalloproteinase 2