Sundaram Kumaran, Nishimura Riko, Senn Joseph, Youssef Rimon F, London Steven D, Reddy Sakamuri V
Charles P. Darby Children's Research Institute, 173 Ashley Avenue, Charleston, SC 29425, USA.
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.
破骨细胞分化受核因子κB受体激活剂配体（RANKL）信号通路的严格调控。基质金属蛋白酶-9（MMP-9），一种IV型胶原酶，在破骨细胞中高表达，在细胞外基质降解中起重要作用；然而，调控MMP-9基因表达的分子机制尚不清楚。在本研究中，我们证明RANKL信号通路可诱导破骨细胞前体细胞中MMP-9基因的表达。我们进一步表明，RANKL通过TRAF6而非TRAF2调控MMP-9基因的表达。有趣的是，用药物抑制剂SB203580阻断p38丝裂原活化蛋白激酶（MAPK）的活性可增加MMP-9的活性，而ERK1/2抑制剂PD98059则可降低RANKL在RAW264.7细胞中诱导的MMP-9活性。这些数据表明，在破骨细胞分化过程中，RANKL通过p3上下游调节MMP-9的表达。将MMP-9基因（相对于ATG起始密码子为+1至-1174 bp）启动子-荧光素酶报告质粒瞬时转染至RAW264.7细胞，并给予RANKL刺激，结果显示MMP-9基因启动子活性显著增加（20倍）；然而，相对于+1 bp至-446 bp启动子区域和空载体转染细胞，其活性并无显著变化。这些结果表明，相对于起始密码子，-446 bp至-1174 bp的MMP-9启动子序列对RANKL刺激有反应。对小鼠MMP-9基因启动子区域的序列分析进一步确定了活化T细胞核因子1（NFATc1）转录因子的结合基序（-1123 bp至-1153 bp）的存在。使用小干扰RNA（siRNA）和VIVIT肽抑制剂抑制NFATc1可显著降低RANKL对MMP-9活性的刺激。我们通过寡核苷酸下拉实验进一步证实，RANKL刺激增强了NFATc1与MMP-9基因启动子元件的结合。此外，在RAW264.7细胞中过表达组成型活性NFAT，在无RANKL的情况下，MMP-9基因启动子活性显著增加（5倍）。综上所述，我们的结果表明，RANKL通过TRAF6发出信号，并且NFATc1是RANKL信号通路的下游效应器，在破骨细胞分化过程中调节MMP-9基因的表达。
