Bone density ligand, Sclerostin, directly interacts with LRP5 but not LRP5G171V to modulate Wnt activity.
We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway.


BACKGROUND
The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6.


METHODS
For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections.


RESULTS
In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5(G171V)) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6(G158V)) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of alpha-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6.


CONCLUSIONS
Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.
Ellies
Debra L
DL
Stowers Institute for Medical Research, kansa City, Missouri 64110, USA.
Viviano
Beth
B
McCarthy
John
J
Rey
Jean-Philippe
JP
Itasaki
Nobue
N
Saunders
Scott
S
Krumlauf
Robb
R
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D001842:Bone and Bones; D003545:Cysteine / Q000737:chemistry; D005819:Genetic Markers / Q000502:physiology*; D006023:Glycoproteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D051153:Wnt Proteins / Q000378:metabolism; D014981:Xenopus
D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins; C555482:LRP5 protein, human; C555475:LRP6 protein, human; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555483:Lrp5 protein, mouse; C555476:Lrp6 protein, mouse; C422903:SOST protein, human; C486030:Sost protein, mouse; D051153:Wnt Proteins; D003545:Cysteine