RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

Exp Cell Res. 2007 Jan 1;313(1):168-78. doi: 10.1016/j.yexcr.2006.10.001. Epub 2006 Oct 6.


Osteoclast differentiation is tightly regulated by receptor activator of NF-kappaB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+1 to -1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to +1 bp to -446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from -446 bp to -1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (-1123 bp to -1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Bone Remodeling / genetics
  • Bone Remodeling / physiology
  • Cell Differentiation
  • Cell Line
  • DNA Primers / genetics
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Gene Expression Regulation, Developmental
  • Gene Expression Regulation, Enzymologic
  • Matrix Metalloproteinase 9 / genetics*
  • Mice
  • NFATC Transcription Factors / antagonists & inhibitors
  • NFATC Transcription Factors / genetics
  • NFATC Transcription Factors / metabolism
  • Osteoclasts / cytology*
  • Osteoclasts / metabolism*
  • Promoter Regions, Genetic
  • RANK Ligand / metabolism*
  • RANK Ligand / pharmacology
  • RNA, Small Interfering / genetics
  • Recombinant Proteins / pharmacology
  • Signal Transduction
  • TNF Receptor-Associated Factor 6 / antagonists & inhibitors
  • TNF Receptor-Associated Factor 6 / genetics
  • TNF Receptor-Associated Factor 6 / metabolism


  • DNA Primers
  • NFATC Transcription Factors
  • Nfatc1 protein, mouse
  • RANK Ligand
  • RNA, Small Interfering
  • Recombinant Proteins
  • TNF Receptor-Associated Factor 6
  • Tnfsf11 protein, mouse
  • Extracellular Signal-Regulated MAP Kinases
  • Matrix Metalloproteinase 9
  • Mmp9 protein, mouse