Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.
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.


BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Amcheslavsky
Alla
A
The Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University Faculty of Medicine, Jerusalem, Israel.
Bar-Shavit
Zvi
Z
D000818:Animals; D002454:Cell Differentiation; D004790:Enzyme Induction; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008024:Ligands; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D051217:Toll-Like Receptor 9 / Q000378:metabolism*
C408982:CPG-oligonucleotide; D008024:Ligands; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; D051217:Toll-Like Receptor 9; D048049:Extracellular Signal-Regulated MAP Kinases; D010744:Phosphoric Monoester Hydrolases