Bone Density Ligand, Sclerostin, Directly Interacts With LRP5 but Not LRP5G171V to Modulate Wnt Activity

Abstract

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.Introduction: 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. Materials and 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

Sclerostin: A gem from the genome leads to bone-building antibodies

“…(42,43) Above and beyond the reported associations with LRP5 and BMPs, hints that sclerostin's mechanism of action at the molecular level might contain additional complexity came from the observation that sclerostin's inhibitory activity was notably variable across different osteoblast-lineage cell-based assays, much more so than the Wnt signaling inhibitor DKK1 and the BMP signaling inhibitor Gremlin. (44) With regard to other possible molecular interactions for sclerostin, data from in vitro experiments had shown that sclerostin could bind LRP6 and inhibit Wnt signaling, (32)(33)(34) similar to the findings for LRP5. This, coupled with mouse genetic data showing that LRP6 appeared to play a role in achieving and/or maintaining normal bone mass, (45)(46)(47) suggested that sclerostin also might interact with LRP6 in vivo.…”

“…(32)(33)(34) Additionally, it was shown that both sclerostin and another Wnt signaling antagonist, Dickkopf1 (DKK1), had decreased binding affinity to HBM LRP5 and reduced ability to inhibit HBM LRP5-mediated Wnt signaling. (34)(35)(36)(37)(38) As such, the gain of function for HBM LRP5 appeared to be primarily from enhanced resistance to inhibition by sclerostin and/or DKK1. Thus the totality of genetic and in vitro data indicated that, in vivo, sclerostin very likely did bind to the extracellular domain of LRP5 on cells of the osteoblast lineage and inhibit Wnt signaling.…”

Wnt signaling and skeletal development

“…However, recent studies demonstrate that sclerostin can inhibit the canonical Wnt signaling pathway by interacting with Lrp5 and 6 [66,67]. Importantly, sclerostin is almost exclusively expressed in osteocytes [68].…”

“…Importantly, sclerostin is almost exclusively expressed in osteocytes [68]. The HBM Lrp5 variant (Lrp5 G171V ) exhibits reduced [69] or abolished [66] SOST binding. It is proposed that Lrp5 HBM mutations render Lrp5 more resistant to SOST inhibition [69].…”

High serum sclerostin predicts the occurrence of osteoporotic fractures in postmenopausal women: The center of excellence for osteoporosis research study

“…(9) Sclerostin antagonizes the Wnt signaling via binding to low-density lipoprotein receptor-related proteins (LRP)-5 and LRP-6. (10,11) This stabilizes cytoplasmic b-catenin and causes its nuclear translocation, which activates gene transcription and promotes osteoblast proliferation, differentiation, and activity, resulting in new bone formation. Loss of LRP-5 function gave rise to osteoporosis-pseudoglioma, characterized by low BMD.…”