Aging impairs the osteocytic regulation of collagen integrity and bone quality

Abstract

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFβ signaling (TβRII^ocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFβ signaling and PLR, but aging did not worsen the existing PLR suppression in male TβRII^ocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFβ. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFβ-dependent maintenance of collagen integrity.

References

1. Bliuc D., Alarkawi D., Nguyen T. V., Eisman J. A.& Center, J. R. Risk of subsequent fractures and mortality in elderly women and men with fragility fractures with and without osteoporotic bone density: the Dubbo osteoporosis epidemiology study. J. Bone Miner. Res. 30, 637-646 (2015).
2. Wainwright, S. A.et al.Hip fracture in women without osteoporosis. J. Clin. Endocrinol. Metab. 90, 2787-2793 (2005).
3. Alliston T.Biological regulation of bone quality. Curr. Osteoporos. Rep. 12, 366-375 (2014).
4. Seeman E.& Delmas, P. D. Bone quality—the material and structural basis of bone strength and fragility. N. Engl. J. Med. 354, 2250-2261 (2006).
5. Milovanovic P.& Busse, B. Inter-site variability of the human osteocyte lacunar network: implications for bone quality. Curr. Osteoporos. Rep. 17, 105-115 (2019).
6. Blank M.& Sims, N. A. Cellular processes by which osteoblasts and osteocytes control bone mineral deposition and maturation revealed by stage-specific EphrinB2 knockdown. Curr. Osteoporos. Rep. 17, 270-280 (2019).
7. Boskey A. L.Bone composition: relationship to bone fragility and antiosteoporotic drug effects. Bonekey Rep. 2, 447(2013).
8. Saito M.& Marumo, K. Effects of collagen crosslinking on bone material properties in health and disease. Calcif. Tissue Int. 97, 242-261 (2015).
9. Garnero P.The contribution of collagen crosslinks to bone strength. BoneKEy Rep. 1, 182(2012).
10. Boskey A. L.& Imbert, L. Bone quality changes associated with aging and disease: a review. Ann. N.Y. Acad. Sci. 1410, 93-106 (2017).
11. Zimmermann, E. A.et al.Age-related changes in the plasticity and toughness of human cortical bone at multiple length scales. Proc. Natl. Acad. Sci. 108, 14416-14421 (2011).
12. Farr J. N., Kaur J., Doolittle M. L.& Khosla, S. Osteocyte cellular senescence. Curr. Osteoporos. Rep. 18, 559-567 (2020).
13. Wang, T., Huang, S.& He, C. Senescent cells: a therapeutic target for osteoporosis. Cell Prolif. 55, e13323(2022).
14. Balooch, G.et al. TGF-beta regulates the mechanical properties and composition of bone matrix. Proc. Natl. Acad. Sci. USA 102, 18813-18818 (2005).
15. Chang, J. L.et al.Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-beta and Runx2 in bone is required for hearing. EMBO Rep. 11, 765-771 (2010).
16. Filvaroff, E.et al. Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass. Development 126, 4267-4279 (1999).
17. Mohammad, K. S.et al.Pharmacologic inhibition of the TGF-beta type I receptor kinase has anabolic and anti-catabolic effects on bone. PLoS One 4, e5275 (2009).
18. Kinoshita, A.et al.Domain-specific mutations in TGFB1 result in Camurati- Engelmann disease. Nat. Genet. 26, 19-20 (2000).
19. Grafe, I.et al.Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta. Nat. Med. 20, 670-675 (2014).
20. Wu, M., Chen, G.& Li, Y.-P. TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res. 4, 16009(2016).
21. Tang S. Y.& Alliston, T. Regulation of postnatal bone homeostasis by TGFβ. BoneKEy Rep. 2, 255(2013).
22. Dallas, S., Alliston, T. & Bonewald, L. F. Transforming growth factor-β. Principles of Bone Biology1145-1166 (Elsevier, 2008).
23. Weivoda, M. M.et al.Osteoclast TGF‐β receptor signaling induces Wnt1 secretion and couples bone resorption to bone formation. J. Bone Miner. Res. 31, 76-85 (2016).
24. Bonewald, L. F. & Mundy, G. R. Role of transforming growth factor-beta in bone remodeling. Clin. Orthop. Relat. R 250, 261-276 (1990).
25. Heldin C.-H.& Moustakas, A. Signaling receptors for TGF-β family members. Csh Perspect. Biol. 8, a022053(2016).
26. Dole, N. S.et al.Osteocyte-intrinsic TGF-β signaling regulates bone quality through perilacunar/canalicular remodeling. Cell Rep. 21, 2585-2596 (2017).
27. Dole N. S., Yee C. S., Mazur C. M., Acevedo C.& Alliston, T. TGFβ regulation of perilacunar/canalicular remodeling is sexually dimorphic. J. Bone Miner. Res. 35, 1549-1561 (2020).
28. Qing, H.et al.Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation. J. Bone Miner. Res. 27, 1018-1029 (2012).
29. Kaya, S.et al.Lactation‐induced changes in the volume of osteocyte lacunarcanalicular space alter mechanical properties in cortical bone tissue. J. Bone Miner. Res. 32, 688-697 (2017).
30. Kegelman, C. D.et al.YAP and TAZ mediate osteocyte perilacunar/canalicular remodeling. J. Bone Miner. Res. 35, 196-210 (2020).
31. Tang S., Herber R. P., Ho S.& Alliston, T. Matrix metalloproteinase-13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance. J. Bone Miner. Res. 27, 1936-1950 (2012).
32. Fowler, T. W.et al.Glucocorticoid suppression of osteocyte perilacunar remodeling is associated with subchondral bone degeneration in osteonecrosis. Sci. Rep. 7, 44618(2017).
33. Alemi, A. S.et al.Glucocorticoids cause mandibular bone fragility and suppress osteocyte perilacunar-canalicular remodeling. Bone Rep. 9, 145-153 (2018).
34. Schurman, C. A., Verbruggen, S. W.& Alliston, T. Disrupted osteocyte connectivity and pericellular fluid flow in bone with aging and defective TGF-$\beta $ signaling. Proc. Natl. Acad. Sci. USA 118, e2023999118 (2021).
35. Kerschnitzki, M.et al.Architecture of the osteocyte network correlates with bone material quality. J. Bone Miner. Res. 28, 1837-1845 (2013).
36. Poundarik A. A., Boskey A., Gundberg C.& Vashishth, D. Biomolecular regulation, composition and nanoarchitecture of bone mineral. Sci. Rep. 8, 1191(2018).
37. Boskey, A. L. Variations in bone mineral properties with age and disease. J. Musculoskel. Neuron 2, 532-534 (2002).
38. Thomas C. J., Cleland T. P., Sroga, G. E. & Vashishth, D. Accumulation of carboxymethyl-lysine (CML) in human cortical bone. Bone 110, 128-133 (2018).
39. Hein G., Wiegand R., Lehmann G., Stein, G. & Franke, S. Advanced glycation end-products pentosidine and Nϵ-carboxymethyllysine are elevated in serum of patients with osteoporosis. Rheumatology 42,1242-1246 (2003).
40. Nyman, J. S.et al. Age-related effect on the concentration of collagen crosslinks in human osteonal and interstitial bone tissue. Bone 39, 1210-1217 (2006).
41. Tomasek J. J., Meyers S. W., Basinger J. B., Green D. T.& Shew, R. L. Diabetic and age-related enhancement of collagen-linked fluorescence in cortical bones of rats. Life Sci. 55, 855-861 (1994).
42. Oxlund, H., Sekilde, L. & Ørtoft, G. Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis. Bone 19, 479-484 (1996).
43. Snedeker J. G.& Gautieri, A. The role of collagen crosslinks in ageing and diabetes—the good, the bad, and the ugly. Muscles Ligaments Tendons J. 4, 303-308 (2014).
44. Farr, J. N.et al.Identification of senescent cells in the bone microenvironment. J. Bone Miner. Res. 31, 1920-1929 (2016).
45. Porter A., Wang L., Han L.& Lu, X. L. Bio-orthogonal click chemistry methods to evaluate the metabolism of inflammatory challenged cartilage after traumatic overloading. ACS Biomater. Sci. Eng. 8, 2564-2573 (2022).
46. Chen H., Zhou X., Fujita H., Onozuka M.& Kubo, K.-Y. Age-related changes in trabecular and cortical bone microstructure. Int. J. Endocrinol. 2013, 213234(2013).
47. Ritchie, R. O., Buehler, M. J. & Hansma, P. Plasticity and toughness in bone. Phys. Today 62, 41-47 (2009).
48. Walker, E. C.et al.Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signaling in osteocytes. Elife 9, e56666 (2020).
49. Boskey A. L.& Coleman, R. Aging and bone. J. Dent. Res. 89, 1333-1348 (2010).
50. Patton, D. M.et al.The relationship between whole bone stiffness and strength is age and sex dependent. J. Biomech. 83, 125-133 (2019).
51. Cole J. H.& van der Meulen, M. C. H. Whole bone mechanics and bone quality. Clin. Orthop. Relat. Res. 469, 2139-2149 (2011).
52. Reznikov, N., Shahar, R.& Weiner, S. Bone hierarchical structure in three dimensions. Acta Biomater. 10, 3815-3826 (2014).
53. Gupta, H. S.et al.Cooperative deformation of mineral and collagen in bone at the nanoscale. Proc. Natl. Acad. Sci. 103, 17741-17746 (2006).
54. Unal, M., Creecy, A.& Nyman, J. S. The role of matrix composition in the mechanical behavior of bone. Curr. Osteoporos. Rep. 16, 205-215 (2018).
55. Hudson D. M.& Eyre, D. R. Collagen prolyl 3-hydroxylation: a major role for a minor post-translational modification? Connect. Tissue Res. 54, 245-251 (2013).
56. Trackman P. C.Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone. Matrix Biol. 52, 7-18 (2016).
57. Krane S. M.The importance of proline residues in the structure, stability and susceptibility to proteolytic degradation of collagens. Amino Acids 35, 703 (2008).
58. Yamauchi M.& Sricholpech, M. Lysine post-translational modifications of collagen. Essays Biochem. 52, 113-133 (2012).
59. Acevedo, C.et al.Contributions of material properties and structure to increased bone fragility for a given bone mass in the UCD-T2DM rat model of type 2 diabetes. J. Bone Miner. Res. 27, 219-1075 (2018).
60. Boskey A. L.Assessment of bone mineral and matrix using backscatter electron imaging and FTIR imaging. Curr. Osteoporos. Rep. 4, 71-75 (2006).
61. Imbert, L.et al.Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy. PLoS One 13, e0202833 (2018).
62. Paschalis, E. P., Mendelsohn, R.& Boskey, A. L. Infrared assessment of bone quality: a review. Clin. Orthop. Relat. Res. 469, 2170-2178 (2011).
63. Zhang, A.et al.O-Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations. J. Biol. Chem. 295, 15742-15753 (2020).
64. Wang, W.-M.et al.Transforming growth factor-β induces secretion of activated ADAMTS-2 A procollagen III N-proteinase. J. Biol. Chem. 278, 19549-19557 (2003).
65. Tominaga K.& Suzuki, H. I. TGF-β signaling in cellular senescence and agingrelated pathology. Int. J. Mol. Sci. 20, 5002(2019).
66. Basisty, N.et al.A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599(2020).
67. Zhang, Y.et al.A new role for oxidative stress in aging: the accelerated aging phenotype in Sod1-/- mice is correlated to increased cellular senescence. Redox Biol. 11, 30-37 (2017).
68. Pignolo, R. J., Law, S. F.& Chandra, A. Bone aging, cellular senescence, and osteoporosis. JBMR Plus 5, e10488 (2021).
69. Creecy, A., Damrath, J. G.& Wallace, J. M. Control of bone matrix properties by osteocytes. Front. Endocrinol. 11, 578477(2021).
70. Heveran C. M.& Boerckel, J. D. Osteocyte remodeling of the lacunar-canalicular system: what’s in a name? Curr. Osteoporos. Rep. 21, 11-20 (2022).
71. Buehler M. J.Nature designs tough collagen: explaining the nanostructure of collagen fibrils. Proc. Natl. Acad. Sci. 103, 12285-12290 (2006).
72. Staines, KA., MacRae, VE. & Farquharson, C. The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling. J. Endocrinol. 214, 214-55 (2012). Epub 2012 Jun 13. Erratum in: J Endocrinol. 2013 Nov;219(2):X1.
73. Qin, C., Baba, O.& Butler, W. T. Post-translational modifications of SIBLING proteins and their roles in osteogenesis and dentinogenesis. Crit. Rev. Oral Biol. Med. 15, 126-136 (2004).
74. Monteiro, D. A.et al.Fluid shear stress generates a unique signaling response by activating multiple TGFβ family type I receptors in osteocytes. FASEB J. 35, e21263(2021).
75. Lim J., Burclaff J., He G., Mills J. C.& Long, F. Unintended targeting of Dmp1- Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice. Bone Res. 5, 16049(2017).
76. Mazur, C. M.et al.Osteocyte dysfunction promotes osteoarthritis through MMP13- dependent suppression of subchondral bone homeostasis. Bone Res. 7, 34(2019).
77. Youlten, S. E.et al.Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease. Nat. Commun. 12, 2444(2021).
78. Shiflett, L. A.et al.Collagen dynamics during the process of osteocyte embedding and mineralization. Front. Cell Dev. Biol. 7, 178(2019).
79. Lu, Y.et al.DMP1-targeted Cre expression in odontoblasts and osteocytes. J. Dent. Res. 86, 320-325 (2007).
80. Levéen, P.et al. Induced disruption of the transforming growth factor beta type II receptor gene in mice causes a lethal inflammatory disorder that is transplantable. Blood 100, 560-568 (2002).
81. Yee C. S., Schurman C. A., White C. R.& Alliston, T. Investigating osteocytic perilacunar/canalicular remodeling. Curr. Osteoporos. Rep. 17, 157-168 (2019).
82. Dole N. S., Yee C. S., Schurman C. A., Dallas S. L.& Alliston, T. Assessment of osteocytes: techniques for studying morphological and molecular changes associated with perilacunar/canalicular remodeling of the bone matrix. Methods Mol. Biol. 2230, 303-323 (2021).
83. Kaya S., Schurman C. A., Dole N. S., Evans D. S.& Alliston, T. Prioritization of genes relevant to bone fragility through the unbiased integration of aging mouse bone transcriptomics and human GWAS analyses. J. Bone Miner. Res. 37, 804-817 (2022).
84. Dobin, A.et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2012).
85. Love, M. I., Huber, W.& Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 31(2014).
86. Nguyen, J., Massoumi, R.& Alliston, T. CYLD, a mechanosensitive deubiquitinase, regulates TGFβ signaling in load-induced bone formation. Bone 131, 115148 (2019).
87. Tiede-Lewis, L. M.et al. Degeneration of the osteocyte network in the C57BL/6 mouse model of aging. Aging 9, 2190-2208 (2017).
88. Bouxsein, M. L.et al.Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res. 25, 1468-1486 (2010).
89. Jepsen K. J., Silva M. J., Vashishth D., Guo X. E.& van der Meulen, M. C. Establishing biomechanical mechanisms in mouse models: practical guidelines for systematically evaluating phenotypic changes in the diaphyses of long bones. J. Bone Miner. Res. 30, 951-966 (2015).
90. Hexemer A., Bras W., Glossinger J., Schaible E., Gann E., Kirian R., MacDowell A., Church M., Rude B., & Padmore H. (2010). A SAXS/WAXS/GISAXS beamline with multilayer monochromator. J. Phys.: Conf. Ser. 247, Article 012007.
91. Barth, H. D.et al. Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone. Biomaterials 32, 8892-8904 (2011).
92. Ilavsky J.Nika: software for two‐dimensional data reduction. J. Appl. Crystallogr. 45, 324-328 (2012).
93. Paschalis, E. P.et al.Fourier transform infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links. Calcified Tissue Int. 96, 18-29 (2015).
94. Paschalis, E. P.et al.Spectroscopic characterization of collagen cross‐links in bone. J. Bone Miner. Res. 16, 1821-1828 (2001).
95. Schmidt, F. N.et al. Assessment of collagen quality associated with nonenzymatic cross-links in human bone using Fourier-transform infrared imaging. Bone 97, 243-251 (2017).
96. Creecy A., Brown K. L., Rose K. L., Voziyan P.& Nyman, J. S. Post-translational modifications in collagen type I of bone in a mouse model of aging. Bone 143, 115763 (2021).
97. Jiang, X.et al.Method development of efficient protein extraction in bone tissue for proteome analysis. J. Proteome Res. 6, 2287-2294 (2007).
98. Rose, J. P.et al.Robust and highly efficient extractions of proteins from bones enable deep, high-throughput proteomic quantification to gain insights into bone biology. https://doi.org/10.1101/2022.11.20.517228 (2022).
99. Escher, C.et al. Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 12, 1111-1121 (2012).
100. Gillet, L. C.et al.Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis*. Mol. Cell Proteom. 11, O111.016717(2012).
101. Collins, B. C.et al.Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry. Nat. Commun. 8, 291(2017).
102. Bruderer, R.et al.Optimization of experimental parameters in data-independent mass spectrometry significantly increases depth and reproducibility of results*. Mol. Cell Proteom. 16, 2296-2309 (2017).
103. Burger T.Gentle introduction to the statistical foundations of false discovery rate in quantitative proteomics. J. Proteome Res. 17, 12-22 (2018).
104. Wickham, H. ggplot2: Elegant Graphics for Data Analysis. (Springer-Verlag, 2016). https://doi.org/10.1007/978-0-387-98141-3.

Funding

Tamara Alliston ([e-mail])