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

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 whi...

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Main Authors:	van Dinther, Maarten, Zhang, Juan, Weidauer, Stella E., Boschert, Verena, Muth, Eva-Maria, Knappik, Achim, de Gorter, David J. J., van Kasteren, Puck B., Frisch, Christian, Mueller, Thomas D., ten Dijke, Peter
Format:	Online
Language:	English
Published:	Public Library of Science 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639248/

id	pubmed-3639248
recordtype	oai_dc
spelling	pubmed-36392482013-05-01 Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling van Dinther, Maarten Zhang, Juan Weidauer, Stella E. Boschert, Verena Muth, Eva-Maria Knappik, Achim de Gorter, David J. J. van Kasteren, Puck B. Frisch, Christian Mueller, Thomas D. ten Dijke, Peter Research Article 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. Public Library of Science 2013-04-29 /pmc/articles/PMC3639248/ /pubmed/23638027 http://dx.doi.org/10.1371/journal.pone.0062295 Text en © 2013 van Dinther et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	van Dinther, Maarten Zhang, Juan Weidauer, Stella E. Boschert, Verena Muth, Eva-Maria Knappik, Achim de Gorter, David J. J. van Kasteren, Puck B. Frisch, Christian Mueller, Thomas D. ten Dijke, Peter
spellingShingle	van Dinther, Maarten Zhang, Juan Weidauer, Stella E. Boschert, Verena Muth, Eva-Maria Knappik, Achim de Gorter, David J. J. van Kasteren, Puck B. Frisch, Christian Mueller, Thomas D. ten Dijke, Peter Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
author_facet	van Dinther, Maarten Zhang, Juan Weidauer, Stella E. Boschert, Verena Muth, Eva-Maria Knappik, Achim de Gorter, David J. J. van Kasteren, Puck B. Frisch, Christian Mueller, Thomas D. ten Dijke, Peter
author_sort	van Dinther, Maarten
title	Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
title_short	Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
title_full	Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
title_fullStr	Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
title_full_unstemmed	Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling
title_sort	anti-sclerostin antibody inhibits internalization of sclerostin and sclerostin-mediated antagonism of wnt/lrp6 signaling
description	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.
publisher	Public Library of Science
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639248/
_version_	1611973610646274048

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

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