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Matrix-embedded cells control osteoclast formation

Abstract

Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.

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Figure 1: Deletion of RANKL in Prx1-Cre expressing cells causes osteopetrosis.
Figure 2: Deletion of RANKL in Osx1-Cre- and Ocn-Cre-expressing cells causes osteopetrosis.
Figure 3: Deletion of RANKL from Dmp1-Cre expressing cells reduces bone remodeling.
Figure 4: Osx1-Cre–mediated RANKL deletion in adult mice does not alter osteoclast number in cancellous bone.
Figure 5: Tail suspension of mice lacking RANKL in osteocytes.

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References

  1. Kronenberg, H.M. Developmental regulation of the growth plate. Nature 423, 332–336 (2003).

    Article  CAS  Google Scholar 

  2. Parfitt, A.M. Targeted and nontargeted bone remodeling: relationship to basic multicellular unit origination and progression. Bone 30, 5–7 (2002).

    Article  CAS  Google Scholar 

  3. Teitelbaum, S.L. & Ross, F.P. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4, 638–649 (2003).

    Article  CAS  Google Scholar 

  4. Whyte, M.P. et al. Osteoprotegerin deficiency and juvenile Paget's disease. N. Engl. J. Med. 347, 175–184 (2002).

    Article  CAS  Google Scholar 

  5. Manolagas, S.C. Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21, 115–137 (2000).

    CAS  Google Scholar 

  6. Kong, Y.Y. et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397, 315–323 (1999).

    Article  CAS  Google Scholar 

  7. Sobacchi, C. et al. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat. Genet. 39, 960–962 (2007).

    Article  CAS  Google Scholar 

  8. Kearns, A.E., Khosla, S. & Kostenuik, P.J. Receptor activator of nuclear factor κB ligand and osteoprotegerin regulation of bone remodeling in health and disease. Endocr. Rev. 29, 155–192 (2008).

    Article  CAS  Google Scholar 

  9. O'Brien, C.A. Control of RANKL gene expression. Bone 46, 911–919 (2010).

    Article  CAS  Google Scholar 

  10. Kobayashi, Y., Udagawa, N. & Takahashi, N. Action of RANKL and OPG for osteoclastogenesis. Crit. Rev. Eukaryot. Gene Expr. 19, 61–72 (2009).

    Article  CAS  Google Scholar 

  11. Sims, N.A. & Gooi, J.H. Bone remodeling: multiple cellular interactions required for coupling of bone formation and resorption. Semin. Cell Dev. Biol. 19, 444–451 (2008).

    Article  CAS  Google Scholar 

  12. Takahashi, N. et al. Osteoblastic cells are involved in osteoclast formation. Endocrinology 123, 2600–2602 (1988).

    Article  CAS  Google Scholar 

  13. Rodan, G.A. & Martin, T.J. Role of osteoblasts in hormonal control of bone resorption–a hypothesis. Calcif. Tissue Int. 33, 349–351 (1981).

    Article  CAS  Google Scholar 

  14. Corral, D.A. et al. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. Proc. Natl. Acad. Sci. USA 95, 13835–13840 (1998).

    Article  CAS  Google Scholar 

  15. Galli, C. et al. Commitment to the osteoblast lineage is not required for RANKL gene expression. J. Biol. Chem. 284, 12654–12662 (2009).

    Article  CAS  Google Scholar 

  16. Li, X. et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J. Bone Miner. Res. 23, 860–869 (2008).

    Article  Google Scholar 

  17. Akune, T. et al. PPARγ insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J. Clin. Invest. 113, 846–855 (2004).

    Article  CAS  Google Scholar 

  18. Ogata, N., Kawaguchi, H., Chung, U.I., Roth, S.I. & Segre, G.V. Continuous activation of G alpha q in osteoblasts results in osteopenia through impaired osteoblast differentiation. J. Biol. Chem. 282, 35757–35764 (2007).

    Article  CAS  Google Scholar 

  19. Weinstein, R.S., Jilka, R.L., Parfitt, A.M. & Manolagas, S.C. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J. Clin. Invest. 102, 274–282 (1998).

    Article  CAS  Google Scholar 

  20. Weinstein, R.S. et al. Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J. Clin. Invest. 109, 1041–1048 (2002).

    Article  CAS  Google Scholar 

  21. Logan, M. et al. Expression of Cre recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 33, 77–80 (2002).

    Article  CAS  Google Scholar 

  22. Dougall, W.C. et al. RANK is essential for osteoclast and lymph node development. Genes Dev. 13, 2412–2424 (1999).

    Article  CAS  Google Scholar 

  23. Rodda, S.J. & McMahon, A.P. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133, 3231–3244 (2006).

    Article  CAS  Google Scholar 

  24. Kobayashi, T. et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc. Natl. Acad. Sci. USA 105, 1949–1954 (2008).

    Article  CAS  Google Scholar 

  25. Zhang, M. et al. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J. Biol. Chem. 277, 44005–44012 (2002).

    Article  CAS  Google Scholar 

  26. Lu, Y. et al. DMP1-targeted Cre expression in odontoblasts and osteocytes. J. Dent. Res. 86, 320–325 (2007).

    Article  CAS  Google Scholar 

  27. Masuyama, R. et al. Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. J. Clin. Invest. 116, 3150–3159 (2006).

    Article  CAS  Google Scholar 

  28. Usui, M. et al. Murine and chicken chondrocytes regulate osteoclastogenesis by producing RANKL in response to BMP2. J. Bone Miner. Res. 23, 314–325 (2008).

    Article  CAS  Google Scholar 

  29. Gebhard, S. et al. Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter. Matrix Biol. 27, 693–699 (2008).

    Article  CAS  Google Scholar 

  30. Zhao, S. et al. MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J. Bone Miner. Res. 17, 2068–2079 (2002).

    Article  CAS  Google Scholar 

  31. Collin-Osdoby, P. et al. Receptor activator of NF-κB and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in human osteoclastogenesis. J. Biol. Chem. 276, 20659–20672 (2001).

    Article  CAS  Google Scholar 

  32. Kong, Y.Y. et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402, 304–309 (1999).

    Article  CAS  Google Scholar 

  33. Yasuda, H. et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95, 3597–3602 (1998).

    Article  CAS  Google Scholar 

  34. O'Brien, C.A., Gubrij, I., Lin, S.C., Saylors, R.L. & Manolagas, S.C. STAT3 activation in stromal osteoblastic cells is required for induction of the receptor activator of NF-κB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D-3 or parathyroid hormone. J. Biol. Chem. 274, 19301–19308 (1999).

    Article  CAS  Google Scholar 

  35. Kostenuik, P.J. et al. Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL. J. Bone Miner. Res. 24, 182–195 (2009).

    Article  CAS  Google Scholar 

  36. Tatsumi, S. et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab. 5, 464–475 (2007).

    Article  CAS  Google Scholar 

  37. Lacey, D.L. et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176 (1998).

    Article  CAS  Google Scholar 

  38. Bonewald, L.F. The amazing osteocyte. J. Bone Miner. Res. 26, 229–238 (2011).

    Article  CAS  Google Scholar 

  39. Kamioka, H., Honjo, T. & Takano-Yamamoto, T. A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 28, 145–149 (2001).

    Article  CAS  Google Scholar 

  40. Knothe Tate, M.L. “Whither flows the fluid in bone?” An osteocyte's perspective. J. Biomech. 36, 1409–1424 (2003).

    Article  Google Scholar 

  41. Zelzer, E. et al. VEGFA is necessary for chondrocyte survival during bone development. Development 131, 2161–2171 (2004).

    Article  CAS  Google Scholar 

  42. Aguirre, J.I. et al. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J. Bone Miner. Res. 21, 605–615 (2006).

    Article  Google Scholar 

  43. Herman, B.C., Cardoso, L., Majeska, R.J., Jepsen, K.J. & Schaffler, M.B. Activation of bone remodeling after fatigue: differential response to linear microcracks and diffuse damage. Bone 47, 766–772 (2010).

    Article  CAS  Google Scholar 

  44. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).

    Article  CAS  Google Scholar 

  45. O'Brien, C.A. et al. IL-6 is not required for parathyroid hormone stimulation of RANKL expression, osteoclast formation, and bone loss in mice. Am. J. Physiol. Endocrinol. Metab. 289, E784–E793 (2005).

    Article  CAS  Google Scholar 

  46. Parfitt, A.M. et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 2, 595–610 (1987).

    Article  CAS  Google Scholar 

  47. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)). Methods. 25, 402–408 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P.E. Cazer, S.B. Berryhill, W. Webb, R. Shelton, A. Deloose, L.K. Climer, K. Vyas, L. Han, A.D. Warren, E.A. Hogan and J.J. Goellner for technical support and advice; and M. Almeida and H. Zhao for helpful discussions. We thank the following individuals for providing Cre-deleter strains: C.J. Tabin (Harvard Medical School), Prx1-Cre; H.M. Kronenberg (Harvard Medical School), Osx1-Cre; K. von der Mark (University of Erlangen-Nuremberg) and B. de Crombrugghe (M.D. Anderson Cancer Center), ColX-Cre; T.L. Clemens (Johns Hopkins University School of Medicine), Ocn-Cre; and J.Q. Feng (Baylor College of Dentistry) Dmp1-Cre. We thank P.D. Pajevic (Harvard Medical School) for the collagenase digestion protocol; D. Chen (University of Rochester School of Medicine) for the X-gal staining protocol; T. Bellido (Indiana University School of Medicine) and I. Aguirre (University of Florida) for advice on tail suspension; the staff of the UAMS Department of Laboratory Animal Medicine; and L. Suva and R. Skinner of the UAMS Skeletal Imaging Core. This work was supported by the following grants from the US National Institutes of Health: AR049794 (to C.A.O.) and AG13918 (to S.C.M.). Support was also provided by the Central Arkansas Veteran's Healthcare System (Merit Reviews to C.A.O., S.C.M., R.L.J. and R.S.W.), by the UAMS Translational Research Institute (1UL1RR029884) and by UAMS tobacco settlement funds.

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Contributions

J.X. carried out the conditional deletion breeding, analysis of gene expression, histomorphometry, immunochemistry and tail-suspension studies. M.O. carried out the R26R breeding and histological analysis of X-gal staining. C.A.O. designed experiments, created the RANKL-flox mice and prepared the first draft of the manuscript. R.L.J., R.S.W., S.C.M. and C.A.O. provided reagents, contributed methods, discussed results and revised the manuscript.

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Correspondence to Charles A O'Brien.

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Xiong, J., Onal, M., Jilka, R. et al. Matrix-embedded cells control osteoclast formation. Nat Med 17, 1235–1241 (2011). https://doi.org/10.1038/nm.2448

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