Osteoblastic sclerostin loop3-LRP4 interaction could be required by sclerostin to inhibit bone formation

Sclerostin has become a novel bone anabolic target. Humanized therapeutic sclerostin antibody romosozumab which mainly targeted sclerostin loop2 was demonstrated to promote bone formation for postmenopausal osteoporosis. However, severe cardiac ischemic events found in clinical trials of romosozumab significantly limited its clinical use.

In our published work, it was notably found that sclerostin loop3 contributed to the antagonistic effect of sclerostin on bone formation, while the cardiovascular protective action of sclerostin was independent of sclerostin loop3. Targeting sclerostin loop3 by our developed sclerostin loop3-specific aptamer could promote bone formation, increase bone mass, and improve bone microarchitecture integrity in both ovariectomized (OVX) rats with established osteoporosis and Col1a2+/G610C mice (osteogenesis imperfecta, OI), while had no influence in cardiovascular events
progression. The sclerostin loop3 aptamer for OI with cardiovascular safety was granted Orphan Drug Designation (DRU-2019-6966) and Rare Pediatric Disease Designation (RPD-2022-667) by US-FDA. However, how sclerostin loop3 participates in the antagonistic effect of sclerostin on bone formation remains unclear.

Sclerostin was reported to antagonize bone anabolic Wnt/β-catenin signal pathway by binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) of osteoblasts via sclerostin loop2. In
this project, sclerostin loop3 was identified to bind to LRP4, rather than LRP6, in osteoblasts by our pull-down assay and BLI analysis. Lrp4 deficiency in osteocytes/osteoblasts was reported to promote progressive cancellous and cortical bone gain in mice, indicating the critical role of LRP4 in inhibiting bone formation. To determine whether sclerostin loop3-LRP4 interaction participated in inhibiting
bone formation, Lrp4m mutation and LRP4-Pep peptide tool were designed to genetically and pharmacologically block the interaction based on the identified interaction residues within LRP4 to sclerostin loop3 in osteoblasts.

Genetically, Lrp4m mutation-induced blockade of sclerostin loop3-LRP4 interaction attenuated the antagonistic effects of sclerostin on Wnt signaling and osteogenic potential in osteoblasts in vitro. Moreover, the bone formation of osteoblastic (OB)-Lrp4m mice was significantly higher than that of the wild-type littermates. Further, to determine whether the promotive effect of osteoblastic Lrp4m
mutation on bone formation was induced by genetic blockade of sclerostin loop3-LRP4 interaction in vivo, the sostko.OB-Lrp4m mouse model was constructed to shield the effect of endogenous sclerostin. No significant difference was found in bone formation between sostko.OB-Lrp4m mice and sostko mice.

Pharmacologically, LRP4-Pep peptide tool-induced blockade of sclerostin loop3-LRP4 interaction attenuated the antagonistic effects of sclerostin on Wnt signaling and osteogenic potential in osteoblasts in vitro. Moreover, after the treatment of the exogenous LRP4-Pep, the serum levels of bone formation biomarkers (ALP and OCN) in SOSTki mice were significantly elevated.

Together, it was hypothesized that osteoblastic sclerostin loop3-LRP4 interaction could be required by sclerostin to inhibit bone formation. To test the hypothesis using the above established genetic mouse models and pharmacologic peptide tool, we have the following two specific Aims:
Aim 1. To genetically determine the role of osteoblastic sclerostin loop3-LRP4 interaction in the antagonistic effect of sclerostin on bone formation by comparing the bone formation between sostko.OB-Lrp4m mice and sostko mice, with and without treatment of recombinant sclerostin.
Aim 2. To pharmacologically determine the role of sclerostin loop3-LRP4 interaction in the antagonistic effect of sclerostin on bone formation by comparing the bone formation of SOSTki mice with and without treatment of the exogenous peptide tool LRP4-Pep.

If the hypothesis could be supported, it would facilitate understanding how sclerostin loop3 participates in the antagonistic effect of sclerostin on bone formation. Translationally, specific blockade of osteoblastic sclerostin loop3-LRP4 interaction would provide a precise sclerostin inhibition strategy to promote bone formation with no safety concern in cardiovascular system.