Cyclin-dependent kinase 9 is required for tumor necrosis factor-alpha-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells

J Biol Chem. 2005 Jan 14;280(2):1103-11. doi: 10.1074/jbc.M406293200. Epub 2004 Nov 4.

Abstract

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma / enzymology*
  • Adenocarcinoma / genetics
  • Cell Line, Tumor
  • Culture Media, Conditioned / metabolism
  • Cyclin T
  • Cyclin-Dependent Kinase 9 / antagonists & inhibitors
  • Cyclin-Dependent Kinase 9 / metabolism*
  • Cyclins / metabolism
  • Dichlororibofuranosylbenzimidazole / pharmacology
  • Gene Expression Regulation, Enzymologic / drug effects*
  • Genes, Reporter / genetics
  • Humans
  • Lung Neoplasms / enzymology*
  • Lung Neoplasms / genetics
  • Matrix Metalloproteinase 2 / genetics
  • Matrix Metalloproteinase 9 / genetics*
  • Promoter Regions, Genetic / genetics
  • Protein Binding / drug effects
  • Transcription, Genetic / drug effects
  • Transcription, Genetic / genetics
  • Tumor Necrosis Factor-alpha / antagonists & inhibitors
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Up-Regulation / drug effects

Substances

  • CCNT1 protein, human
  • Culture Media, Conditioned
  • Cyclin T
  • Cyclins
  • Tumor Necrosis Factor-alpha
  • Dichlororibofuranosylbenzimidazole
  • Cyclin-Dependent Kinase 9
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 9