Abstract
Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.
Highlights
Osteoclastogenesis, the formation of specialized multinucleated giant cells that resorb the inorganic and organic components of bone, is a complex multistep process
We have shown that antibody-mediated blockade of TSP-1 led to inhibition of human osteoclast (OCL) formation when cultures of dendritic cells or monocytes were induced by myeloma cells or RANKL and CSF-1 [9]
It is thought that cells of the osteoblast lineage induce osteoclastogenesis from marrow precursors through a paracrine signaling cascade in which RANKL produced by osteoblasts engages RANK on osteoclast precursors
Summary
We have shown that antibody-mediated blockade of TSP-1 led to inhibition of human osteoclast (OCL) formation when cultures of dendritic cells or monocytes were induced by myeloma cells or RANKL and CSF-1 [9]. As was the case when osteoclasts were generated from CD47Ϫ/Ϫ precursors, treating CD36Ϫ/Ϫ or DKO cultures with a neutralizing antibody to TSP-1 completely abrogated osteoclastogenesis (Fig. 2A, panels b and c, right-hand columns). Despite suppression of CD47 expression by nearly 70%, osteoclast formation still occurred, albeit at a reduced level These data suggest that deficiency of CD47 alone does not fully recapitulate the effects of antibody-mediated blockade of TSP-1 in both murine and human cells. P907 was still able to rescue the inhibitory effect of the anti-TSP-1 antibody on osteoclastogenesis when expression of CD47 was suppressed with an siRNA (Fig. 5D). The fourth set of data (filled and open squares) show the effect of adding p907 to osteoclast precursors in which CD47 has been suppressed and treated with anti-TSP-1. When PTH was infused into either CD47Ϫ/Ϫ or CD36Ϫ/Ϫ mice, there were no significant differences in either the hypercalcemic response or the increase in CTx in these animals compared with WT controls (Fig. 9)
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