Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.
We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways.


BACKGROUND
Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity.


METHODS
Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein.


RESULTS
In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs.


CONCLUSIONS
sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.
Bone, Joint, and Cancer Unit, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
Karl D
KD
Häusler
Nicole J
NJ
Horwood
Yoshiro
Y
Chuman
Jane L
JL
Fisher
Jennifer
J
Ellis
T John
TJ
Martin
Jeffrey S
JS
Rubin
Matthew T
MT
Gillespie
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism; D002117:Calcitriol / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D017370:Interleukin-11 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism; D016133:Polymerase Chain Reaction; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D051153:Wnt Proteins; D000094742:Secreted Frizzled-Related Proteins
D002117:Calcitriol; D002352:Carrier Proteins; D005982:Glutathione Transferase; D036341:Intercellular Signaling Peptides and Proteins; D017370:Interleukin-11; D015850:Interleukin-6; D016173:Macrophage Colony-Stimulating Factor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010281:Parathyroid Hormone; D053245:RANK Ligand; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D051153:Wnt Proteins; C485539:Sfrp1 protein, mouse; D000094742:Secreted Frizzled-Related Proteins; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse