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

Abstract

Sclerostin negatively regulates bone formation. The marketed antibody against sclerostin loop2 promoted bone formation but may have caused severe cardiovascular events in clinical use. In our published studies, sclerostin loop3 was found to be involved in inhibitory effects of sclerostin on bone formation, whereas cardiovascular protective effects of sclerostin in mice were independent of loop3. It is necessary to investigate how sclerostin loop3 participates in the inhibitory effects of sclerostin on bone formation to facilitate developing precise strategies that promote bone formation without increasing cardiovascular risk. In this study, sclerostin loop3 was identified to bind to LRP4, thereby facilitating binding of sclerostin to LRP6 in osteoblasts. Blockade of sclerostin loop3-LRP4 interaction by both Lrp4 mutation (Lrp4m) and blocking peptide (LRP4-Pep) diminished the antagonistic effect of sclerostin on Wnt/β-catenin signaling in osteoblasts in vitro. Consistently, Lrp4m promoted bone formation in Lrp4m mice in vivo. Mechanistically, osteoblast-conditional correction of Lrp4m to wild-type Lrp4 resulted in significantly lower bone formation than Lrp4m mice, indicating that the promotive effects of Lrp4m on bone formation acted in osteoblasts in vivo. Moreover, re-expression of sclerostin dramatically inhibited bone formation in sost^−/− mice, whilst the inhibitory effects of sclerostin were significantly weaker in sost^−/−.Lrp4m mice. Pharmacologically, LRP4-Pep diminished the inhibitory effects of sclerostin on bone formation in SOST^ki mice. Taken together, osteoblastic sclerostin loop3-LRP4 interaction, as an anchor, was required by sclerostin to bind to LRP6, thereby inhibiting bone formation. Translationally, blockade of sclerostin loop3-LRP4 interaction in osteoblasts would provide precise therapeutic strategies to promote bone formation without increasing cardiovascular risk.

References

[1] Holdsworth G, et al. . Characterization of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors. J. Biol. Chem., 2012, 287: 26464-26477

[2] McClung MR, et al. . Romosozumab in postmenopausal women with low bone mineral density. N. Engl. J. Med., 2014, 370: 412-420

[3] Cosman F, et al. . Romosozumab treatment in postmenopausal women with osteoporosis. N. Engl. J. Med., 2016, 375: 1532-1543

[4] Saag KG, et al. . Romosozumab or alendronate for fracture prevention in women with osteoporosis. N. Engl. J. Med., 2017, 377: 1417-1427

[5] Lewiecki EM, et al. . A phase III Randomized Placebo-Controlled Trial to Evaluate Efficacy and Safety of Romosozumab in Men With Osteoporosis. J. Clin. Endocrinol. Metab., 2018, 103: 3183-3193

[6] Kawaguchi H. Serious adverse events with Romosozumab Use in Japanese patients: need for clear formulation of contraindications Worldwide. J. Bone Min. Res., 2020, 35: 994-995

[7] Vestergaard Kvist, A. et al. Cardiovascular safety profile of romosozumab: a pharmacovigilance analysis of the US Food and Drug Administration Adverse Event Reporting System (FAERS). J. Clin. Med.10, 1660 (2021).

[8] Turk JR, et al. . Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Regul. Toxicol. Pharmacol., 2020, 115 104697

[9] Yu Y, et al. . Targeting loop3 of sclerostin preserves its cardiovascular protective action and promotes bone formation. Nat. Commun., 2022, 13 4241

[10] Wang L, et al. . Therapeutic aptamer targeting sclerostin loop3 for promoting bone formation without increasing cardiovascular risk in osteogenesis imperfecta mice. Theranostics, 2022, 12: 5645-5674

[11] Niu Y, et al. . Aptamer-immobilized bone-targeting nanoparticles in situ reduce sclerostin for osteoporosis treatment. Nano Today, 2022, 45: 101529

[12] Bourhis E, et al. . Wnt antagonists bind through a short peptide to the first beta-propeller domain of LRP5/6. Structure, 2011, 19: 1433-1442

[13] Pekkinen M, et al. . Novel mutations in the LRP5 gene in patients with Osteoporosis-pseudoglioma syndrome. Am. J. Med. Genet. A, 2017, 173: 3132-3135

[14] Stürznickel J, Rolvien T, Delsmann A, Butscheidt S. Clinical phenotype and relevance of LRP5 and LRP6 variants in patients with Early-Onset Osteoporosis (EOOP). J. Bone Min. Res., 2021, 36: 271-282

[15] Xiong L, et al. . Lrp4 in osteoblasts suppresses bone formation and promotes osteoclastogenesis and bone resorption. Proc. Natl. Acad. Sci. USA, 2015, 112: 3487-3492

[16] Kim SP, et al. . Lrp4 expression by adipocytes and osteoblasts differentially impacts sclerostin’s endocrine effects on body composition and glucose metabolism. J. Biol. Chem., 2019, 294: 6899-6911

[17] Svetlana K, Biplab C, Chen VAD, Niv P, Noam L. Competitive blocking of LRP4–sclerostin binding interface strongly promotes bone anabolic functions. Cell. Mol. Life Sci., 2022, 79: 113-125

[18] Maeda, K. et al. The regulation of bone metabolism and disorders by wnt signaling. Int. J. Mol. Sci.20, 5525 (2019).

[19] Shen, W. et al. Discovery of a novel dual-targeting D-peptide to block CD24/Siglec-10 and PD-1/PD-L1 interaction and synergize with radiotherapy for cancer immunotherapy. J. Immunother. Cancer11, e007068 (2023).

[20] Sadowski M, et al. . A synthetic peptide blocking the apolipoprotein E/beta-amyloid binding mitigates beta-amyloid toxicity and fibril formation in vitro and reduces beta-amyloid plaques in transgenic mice. Am. J. Pathol., 2004, 165: 937-948

[21] Bullock WA, et al. . Lrp4 mediates bone homeostasis and mechanotransduction through interaction with sclerostin in vivo. iScience, 2019, 20: 205-215

[22] Lara-Castillo N, Johnson ML. LRP receptor family member associated bone disease. Rev. Endocr. Metab. Disord., 2015, 16: 141-148

[23] Leupin O, et al. . Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function. J. Biol. Chem., 2011, 286: 19489-19500

[24] Kotake K, Mitsuboshi S, Omori Y, Kawakami Y, Kawakami Y. Evaluation of risk of cardiac or cerebrovascular events in romosozumab users focusing on comorbidities: analysis of the japanese adverse drug event report database. J. Pharm. Technol., 2023, 39: 23-28

[25] Bentzon JF, Otsuka F, Virmani R, Falk E. Mechanisms of plaque formation and rupture. Circ. Res., 2014, 114: 1852-1866

[26] Palasubramaniam J, Wang X, Peter K. Myocardial infarction—from atherosclerosis to thrombosis. Arterioscler. Thrombosis. Vasc. Biol., 2019, 39: e176-e185

[27] Volkamer A, Kuhn D, Rippmann F, Rarey M. DoGSiteScorer: a web server for automatic binding site prediction, analysis and druggability assessment. Bioinformatics, 2012, 28: 2074-2075

[28] Xue C, et al. . Aconine attenuates osteoclast-mediated bone resorption and ferroptosis to improve osteoporosis via inhibiting NF-κB signaling. Front. Endocrinol., 2023, 14: 1234563

[29] Qi JL, Zhang ZD, Dong Z, Shan T, Yin ZS. mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway. J. Orthop. Surg. Res., 2024, 19: 190

[30] Qiu S, Divine G, Warner E, Rao SD. Reference intervals for bone histomorphometric measurements based on data from healthy premenopausal women. Calcif. Tissue Int., 2020, 107: 543-550

[31] Surowiec RK, et al. . Ex vivo exposure to calcitonin or raloxifene improves mechanical properties of diseased bone through non-cell mediated mechanisms. Bone, 2023, 173: 116805

[32] Zhang ZK, et al. . A newly identified lncRNA MAR1 acts as a miR-487b sponge to promote skeletal muscle differentiation and regeneration. J. Cachexia, Sarcopenia Muscle, 2018, 9: 613-626

[33] Montilla-García Á, et al. . Grip strength in mice with joint inflammation: a rheumatology function test sensitive to pain and analgesia. Neuropharmacology, 2017, 125: 231-242

[34] Krishna SM, et al. . Wnt signaling pathway inhibitor sclerostin inhibits angiotensin II-Induced aortic aneurysm and atherosclerosis. Arterioscler Thromb. Vasc. Biol., 2017, 37: 553-566

[35] He, P. et al. Cholesterol chip for the study of cholesterol-protein interactions using SPR. Biosensors12, 788 (2022).

[36] Jiang R, et al. . One-step bioprocess of inulin to product inulo-oligosaccharides using bacillus subtilis secreting an extracellular endo-inulinase. Appl. Biochem. Biotechnol., 2019, 187: 116-128

[37] Chen H, et al. . MicroRNA-224-5p nanoparticles balance homeostasis via inhibiting cartilage degeneration and synovial inflammation for synergistic alleviation of osteoarthritis. Acta Biomater., 2023, 167: 401-415

[38] Lyu S, Zhang C, Hou X, Wang A. Tag-based pull-down assay. Methods Mol. Biol., 2022, 2400: 105-114

[39] Hirano S. Western blot analysis. Methods Mol. Biol., 2012, 926: 87-97

[40] Grentzmann G, Ingram JA, Kelly PJ, Gesteland RF, Atkins JF. A dual-luciferase reporter system for studying recoding signals. Rna, 1998, 4: 479-486

[41] McNabb DS, Reed R, Marciniak RA. Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae. Eukaryot. Cell, 2005, 4: 1539-1549

[42] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25: 402-408

[43] Zhao XY, et al. . iPS cells produce viable mice through tetraploid complementation. Nature, 2009, 461: 86-90

[44] Wang T, et al. . Engineering immunomodulatory and osteoinductive implant surfaces via mussel adhesion-mediated ion coordination and molecular clicking. Nat. Commun., 2022, 13 160

[45] Song R, Wang D, Zeng R, Wang J. Synergistic effects of fibroblast growth factor-2 and bone morphogenetic protein-2 on bone induction. Mol. Med. Rep., 2017, 16: 4483-4492

[46] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal. Biochem., 2004, 329: 77-84

[47] Song CY, Guo Y, Chen FY, Liu WG. Resveratrol promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells through miR-193a/SIRT7 Axis. Calcif. Tissue Int., 2022, 110: 117-130

[48] Jonkman J, Brown CM. Tutorial: guidance for quantitative confocal microscopy. Nat. Protoc., 2020, 15: 1585-1611

[49] Consortium, T. U. et al. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res.51, 523–531 (2023).

[50] Abramson J, et al. . Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 630: 493-500

[51] Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci.30, 70–82 (2021).

[52] Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX1-2, 19–25 (2015).

[53] Building Water Models: A Different Approach. J. Phys. Chem. Lett.5, 3863–3871 (2014).

[54] Best RB, Hummer G. Optimized Molecular Dynamics Force Fields Applied to the Helix−Coil Transition of Polypeptides. J. Phys. Chem. B113, 9004–15 (2009).

[55] Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem.32, 2319–2327 (2011).

[56] Hunter, J. D. Matplotlib: a 2D Graphics Environment. Comput. Sci. Eng.9, 90–95 (2007).

[57] Dreyer T, et al. . Recombinant sclerostin inhibits bone formation in vitro and in a mouse model of sclerosteosis. J. Orthop. Transl., 2021, 29: 134-142

[58] Kogawa M, et al. . Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size. Am. J. Physiol. Cell Physiol., 2018, 314: C53-c61

[59] Yan L, et al. . New insight into enzymatic hydrolysis of peptides with site-specific amino acid d-isomerization. Bioorg. Chem., 2020, 105: 104389

[60] Meng Y, Zhang H, Li Y, Li Q, Zuo L. Effects of unfractionated heparin on renal osteodystrophy and vascular calcification in chronic kidney disease rats. Bone, 2014, 58: 168-176

Funding

Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)(2018YFA0800804)

Research Grants Council, University Grants Committee (RGC, UGC)(12102524)

Innovation and Technology Commission (ITF)(UIM/298)

Guangdong Science and Technology Department (Science and Technology Department, Guangdong Province)(2019B1515120089)

National Natural Science Foundation of China (National Science Foundation of China)(82300988)

Hunan Provincial Science and Technology Department (Department of Science and Technology of Hunan Province)(2022WK2010)

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