Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes

Biochim Biophys Acta. 2008 Sep;1783(9):1605-12. doi: 10.1016/j.bbamcr.2008.04.005. Epub 2008 Apr 18.

Abstract

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Chondrocytes / drug effects
  • Chondrocytes / metabolism*
  • Chromatin Immunoprecipitation
  • Humans
  • Isoquinolines / pharmacology
  • Promoter Regions, Genetic
  • Pyridines / pharmacology
  • Pyrroles / pharmacology
  • RNA Interference
  • Signal Transduction
  • Smad Proteins / metabolism*
  • Smad2 Protein / antagonists & inhibitors
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism
  • Smad3 Protein / antagonists & inhibitors
  • Smad3 Protein / genetics
  • Smad4 Protein / antagonists & inhibitors
  • Smad4 Protein / genetics
  • Tissue Inhibitor of Metalloproteinase-3 / biosynthesis
  • Tissue Inhibitor of Metalloproteinase-3 / genetics*
  • Transforming Growth Factor beta / antagonists & inhibitors
  • Transforming Growth Factor beta / pharmacology*

Substances

  • 6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride
  • Isoquinolines
  • Pyridines
  • Pyrroles
  • Smad Proteins
  • Smad2 Protein
  • Smad3 Protein
  • Smad4 Protein
  • Tissue Inhibitor of Metalloproteinase-3
  • Transforming Growth Factor beta