title: RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation.
abstract: 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.
authors.affiliation: Charles P. Darby Children's Research Institute, 173 Ashley Avenue, Charleston, SC 29425, USA.
authors.forename: Kumaran
authors.initials: K
authors.lastname: Sundaram
authors.forename: Riko
authors.initials: R
authors.lastname: Nishimura
authors.forename: Joseph
authors.initials: J
authors.lastname: Senn
authors.forename: Rimon F
authors.initials: RF
authors.lastname: Youssef
authors.forename: Steven D
authors.initials: SD
authors.lastname: London
authors.forename: Sakamuri V
authors.initials: SV
authors.lastname: Reddy
mesh_terms: D000818:Animals; D001483:Base Sequence; D016723:Bone Remodeling / Q000235:genetics / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D051379:Mice; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000378:metabolism* / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D053245:RANK Ligand; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D048029:TNF Receptor-Associated Factor 6; C505689:Tnfsf11 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse