p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription

J Cell Physiol. 2006 Jul;208(1):246-52. doi: 10.1002/jcp.20660.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cyclin A / analysis
  • Cyclin A / genetics
  • Cyclin A / physiology
  • Cyclin-Dependent Kinase Inhibitor p16 / pharmacology*
  • Down-Regulation / physiology
  • Gene Expression Regulation, Enzymologic / drug effects*
  • Gene Expression Regulation, Enzymologic / physiology
  • Gene Expression Regulation, Neoplastic / physiology
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Lung Neoplasms / chemistry
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Matrix Metalloproteinase 2 / analysis
  • Matrix Metalloproteinase 2 / genetics*
  • Matrix Metalloproteinase 2 / physiology
  • Neoplasm Invasiveness / physiopathology
  • Neoplasm Metastasis / physiopathology
  • Neovascularization, Pathologic / genetics
  • Neovascularization, Pathologic / physiopathology
  • Oligonucleotide Array Sequence Analysis
  • Promoter Regions, Genetic / drug effects
  • Promoter Regions, Genetic / physiology
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sp1 Transcription Factor / analysis
  • Sp1 Transcription Factor / physiology*
  • Transcription, Genetic / drug effects*
  • Transcription, Genetic / physiology
  • Tumor Cells, Cultured

Substances

  • Cyclin A
  • Cyclin-Dependent Kinase Inhibitor p16
  • Sp1 Transcription Factor
  • Matrix Metalloproteinase 2