Amcheslavsky Alla, Bar-Shavit Zvi
The Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University Faculty of Medicine, Jerusalem, Israel.
J Bone Miner Res. 2007 Aug;22(8):1301-10. doi: 10.1359/jbmr.070501.
CpG-ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c-fos, an essential molecule for osteoclast differentiation. Therefore, CpG-ODN blocks osteoclast differentiation.
Activation of either Toll-like receptor 9 (TLR9) or RANK induces similar responses in osteoclast precursors. Paradoxically, activation of TLR9 results in inhibition of RANKL-induced osteoclastogenesis.
We used bone marrow-derived osteoclast precursors. Analyses of signaling molecules phosphorylation were performed using Western blotting. Different levels of gene expression analyses were performed using RT-PCR, Northern, and run-on analyses (for RNA), and EMSA, Western, and pulse-chase experiments (for protein). Phosphatase activity was measured spectrophotometrically.
We found that RANKL and TLR9 ligand, oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN), induce sustained and transient extracellular signal-regulated kinase (ERK) phosphorylation, respectively. Furthermore, together they induce a transient phosphorylation of ERK. The duration of ERK phosphorylation is a key factor in determining induction of c-fos, a protein critical for osteoclastogenesis. Indeed, we found that CpG-ODN does not induce c-fos and inhibits its induction by RANKL by enhancing c-fos mRNA and protein degradation. Our observation that CpG-ODN, but not RANKL, induces the expression of the phosphatase PP2A suggests that CpG-ODN exerts its inhibitory activity by induction of ERK dephosphorylation. Moreover, together with the phosphatase inhibitor okadaic acid, CpG-ODN induces sustained ERK phosphorylation and c-fos expression.
Our findings suggest that the increased rate of c-fos degradation by the TLR9 ligand mediates the inhibition of RANKL-induced osteoclast differentiation. The TLR9 ligand, through induction of dephosphorylation, prevents the sustained ERK phosphorylation needed for maintaining high c-fos levels that are essential for osteoclast differentiation.
除了刺激早期破骨细胞前体中的破骨细胞生成信号外，CpG-ODN还诱导磷酸酶，使细胞外信号调节激酶（ERK）磷酸化模式从持续型转变为瞬时型。这种转变导致c-fos降解，而c-fos是破骨细胞分化所必需的分子。因此，CpG-ODN可阻断破骨细胞分化。
Toll样受体9（TLR9）或核因子κB受体活化因子（RANK）的激活在破骨细胞前体中诱导相似的反应。矛盾的是，TLR9的激活导致RANKL诱导的破骨细胞生成受到抑制。
我们使用了骨髓来源的破骨细胞前体。通过蛋白质印迹法进行信号分子磷酸化分析。使用逆转录聚合酶链反应（RT-PCR）、Northern杂交和连续分析（用于RNA），以及电泳迁移率变动分析（EMSA）、蛋白质印迹法和脉冲追踪实验（用于蛋白质）进行不同水平的基因表达分析。通过分光光度法测量磷酸酶活性。
我们发现RANKL和TLR9配体，即含有未甲基化CpG二核苷酸的寡脱氧核苷酸（CpG-ODN），分别诱导持续和瞬时的细胞外信号调节激酶（ERK）磷酸化。此外，它们共同诱导ERK的瞬时磷酸化。ERK磷酸化的持续时间是决定c-fos诱导的关键因素，而c-fos是破骨细胞生成所必需的蛋白质。实际上，我们发现CpG-ODN不会诱导c-fos，并通过增强c-fos mRNA和蛋白质降解来抑制RANKL对其的诱导。我们观察到CpG-ODN而非RANKL可诱导磷酸酶PP2A的表达，这表明CpG-ODN通过诱导ERK去磷酸化发挥其抑制活性。此外，与磷酸酶抑制剂冈田酸一起，CpG-ODN可诱导持续的ERK磷酸化和c-fos表达。
我们的研究结果表明，TLR9配体导致的c-fos降解速率增加介导了对RANKL诱导的破骨细胞分化的抑制。TLR9配体通过诱导去磷酸化，阻止了维持破骨细胞分化所必需的高c-fos水平所需的持续ERK磷酸化。
