title: 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 [(125)I]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.
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Maarten
authors: M
authors: van Dinther
authors: Juan
authors: J
authors: Zhang
authors: Stella E
authors: SE
authors: Weidauer
authors: Verena
authors: V
authors: Boschert
authors: Eva-Maria
authors: EM
authors: Muth
authors: Achim
authors: A
authors: Knappik
authors: David J J
authors: DJ
authors: de Gorter
authors: Puck B
authors: PB
authors: van Kasteren
authors: Christian
authors: C
authors: Frisch
authors: Thomas D
authors: TD
authors: Mueller
authors: Peter
authors: P
authors: ten Dijke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000918:Antibody Specificity; D002454:Cell Differentiation; D002460:Cell Line; D006023:Glycoproteins / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D060492:Low Density Lipoprotein Receptor-Related Protein-5 / Q000378:metabolism; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D057134:Antibodies, Neutralizing; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555483:Lrp5 protein, mouse; C555476:Lrp6 protein, mouse; C486030:Sost protein, mouse; D051153:Wnt Proteins
references: J Clin Endocrinol Metab. 2012 Feb;97(2):311-25
references: 22238383
references: Cell Mol Life Sci. 2010 Aug;67(15):2551-62
references: 20229235
references: Proc Natl Acad Sci U S A. 1982 Apr;79(7):2291-5
references: 6179094
references: J Biol Chem. 2005 Jul 22;280(29):26770-5
references: 15908424
references: Nature. 2000 Sep 28;407(6803):530-5
references: 11029007
references: J Cell Sci. 2010 Feb 1;123(Pt 3):360-8
references: 20053636
references: J Bone Joint Surg Am. 2012 Sep 19;94(18):1670-80
references: 22992878
references: Nature. 2000 Sep 28;407(6803):535-8
references: 11029008
references: J Bone Miner Res. 2007 Jan;22(1):19-28
references: 17032150
references: Hum Mol Genet. 2002 Oct 1;11(20):2385-93
references: 12351574
references: Nat Struct Mol Biol. 2011 Oct 09;18(11):1204-10
references: 21984209
references: J Bone Miner Res. 2011 May;26(5):1012-21
references: 21542004
references: J Bone Miner Res. 2012 Oct;27(10):2065-74
references: 22589174
references: J Biol Chem. 2010 Dec 31;285(53):41614-26
references: 20952383
references: Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3299-304
references: 12626757
references: J Mol Biol. 2008 Feb 29;376(4):1182-200
references: 18191144
references: J Biol Chem. 2011 Jun 3;286(22):19489-500
references: 21471202
references: Am J Hum Genet. 2001 Mar;68(3):577-89
references: 11179006
references: J Bone Miner Res. 2008 Jun;23(6):860-9
references: 18269310
references: J Biol Chem. 2010 Mar 19;285(12):9172-9
references: 20093360
references: Mol Cell Biol. 2002 Sep;22(17):6100-10
references: 12167704
references: Dev Cell. 2011 Nov 15;21(5):862-73
references: 22000856
references: EMBO J. 2003 Dec 1;22(23):6267-76
references: 14633986
references: Nature. 2001 May 17;411(6835):321-5
references: 11357136
references: Nat Rev Rheumatol. 2011 Sep 20;7(11):631-8
references: 21931340
references: Hum Mol Genet. 2001 Mar 1;10(5):537-43
references: 11181578
references: J Biol Chem. 2005 May 20;280(20):19883-7
references: 15778503
references: Cytokine Growth Factor Rev. 2009 Oct-Dec;20(5-6):399-407
references: 19896889
references: Curr Biol. 2001 Jun 26;11(12):951-61
references: 11448771
references: J Bone Miner Res. 2006 Nov;21(11):1738-49
references: 17002572
references: Am J Hum Genet. 2002 Jan;70(1):11-9
references: 11741193
references: Nat Rev Drug Discov. 2011 Feb;10(2):141-56
references: 21283108
references: Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15473-8
references: 20713706
references: J Biol Chem. 1984 Sep 10;259(17):10995-1000
references: 6088525
references: Bone. 1996 Nov;19(5):429-36
references: 8922640
references: Nature. 2002 Jun 6;417(6889):664-7
references: 12050670
references: Dev Cell. 2008 Jul;15(1):37-48
references: 18606139
references: J Orthop Res. 2012 Oct;30(10):1541-8
references: 22457198
references: PLoS One. 2010 Sep 13;5(9):e12682
references: 20856934
references: J Bone Miner Res. 2011 Jan;26(1):19-26
references: 20593411
references: J Bone Miner Res. 2010 May;25(5):948-59
references: 20200929
references: N Engl J Med. 2002 May 16;346(20):1513-21
references: 12015390
references: Cell Differ Dev. 1990 May;30(2):109-16
references: 2201423
references: Lancet. 2011 Apr 9;377(9773):1276-87
references: 21450337
references: Cytokine Growth Factor Rev. 2005 Jun;16(3):319-27
references: 15869900
references: Nat Cell Biol. 2001 Jul;3(7):683-6
references: 11433302
references: J Bone Miner Res. 2009 Apr;24(4):578-88
references: 19049336
references: J Biol Chem. 2009 Apr 17;284(16):10890-900
references: 19208630
references: Cell. 1992 Feb 21;68(4):673-82
references: 1739974
references: J Musculoskelet Neuronal Interact. 2004 Jun;4(2):139-42
references: 15615113
references: Curr Osteoporos Rep. 2012 Sep;10(3):190-8
references: 22711369
references: J Cell Biol. 1995 Jul;130(1):217-26
references: 7790373
references: Mol Cell Biol. 2002 Feb;22(4):1172-83
references: 11809808
references: Structure. 2011 Oct 12;19(10):1433-42
references: 21944579
references: Biochem Biophys Res Commun. 2009 Feb 27;380(1):160-5
references: 19166819