Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling

Abstract

Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [125I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.

Sclerostin inhibits osteoblast differentiation without affecting BMP2/SMAD1/5 or Wnt3a/β-catenin signaling but through activation of platelet-derived growth factor receptor signaling in vitro

“…It has been proposed that sclerostin antagonizes canonical Wnt/LRP6 signaling by promoting clathrindependent internalization of LRP6. 35 It is likely that sclerostin Sclerostin activates PDGFR signaling C Thouverey and J Caverzasio mediates LRP6 endocytosis through binding to a co-receptor, as it has been shown for DKK1. As PDGFR has been shown to be internalized following activation by PDGF and that PDGFR can form complex with LRP6 in other cell type, 36 PDGFR could be the co-receptor involved in sclerostin-elicited endocytosis of LRP6.…”

Sostdc1 deficiency accelerates fracture healing by promoting the expansion of periosteal mesenchymal stem cells

“…Sostdc1 has been described as both a Bmp and a Wnt antagonist in a context-dependent manner [13], and it interacts with Lpr4, 5, and 6 Wnt co-receptors and with Bmp ligands [6, 13–16], in vivo . While Sostdc1 shares 55% protein sequence homology to its paralog Sost, it reportedly displays higher specificity for the Lrp4 co-receptor [14], in contrast to the preferred binding to the Lrp5 and 6 co-receptors by Sost [17, 18]. Mesenchyme-derived Sostdc1 inhibits Wnt signaling in the epithelium through the activation of Hedgehog signaling, which in turn suppresses Wnt signaling via Sostdc1 up-regulation in the mesenchyme [12, 19].…”

Determining the Role of Sost and Sostdc1 During Fracture Healing

“…The Sost protein is a potent WNT antagonist secreted by osteocytes that normally functions to inhibit bone formation in osteoblasts by inhibiting WNT signaling through binding to the LRP5/6 co-receptors [72] [84] [74] with a preference in binding to LRP6 over LRP5 [85]. However, recently it has been discovered that Sost binds to the LRP4 co-receptor as well [86].…”

“…However, recently it has been discovered that Sost binds to the LRP4 co-receptor as well [86]. Normally, when Sost binds to the Wnt co-receptors (LRP 5, 6, 4), Sost gets internalized via the Clathrin dependent pathway and degraded in a proteasome manner [85].…”

“…Sostdc1 has been described as both a Bmp and a Wnt antagonist in a contextdependent manner [182], and it interacts with Lpr4, 5, and 6 Wnt co-receptors and with Bmp ligands [3,103,[182][183][184], in vivo. While Sostdc1 shares 55% protein sequence homology to its paralog Sost, it reportedly displays higher specificity for the Lrp4 coreceptor [183], in contrast to the preferred binding to the Lrp5 and 6 co-receptors by Sost [85,137]. Mesenchyme-derived Sostdc1 inhibits Wnt signaling in the epithelium through the activation of Hedgehog signaling, which in turn suppresses Wnt signaling via Sostdc1 up-regulation in the mesenchyme [185,186].…”