The Products of Bone Resorption: Comparison
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Please note this is a comparison between Version 2 by Vivi Li and Version 1 by Gordon L. Klein.

Surprisingly little is known about the factors released from bone during resorption and the metabolic roles they play. This entry describes what we have learned about factors released from bone, mainly through the study of burn injuries, and what roles they play in post-burn metabolism. From these studies, we know that calcium, phosphorus, and magnesium, along with transforming growth factor (TGF)-β, are released from bone following resorption. Additionally, studies in mice from Karsenty’s laboratory have indicated that undercarboxylated osteocalcin is also released from bone during resorption. Questions arising from these observations are discussed as well as a variety of potential conditions in which release of these factors could play a significant role in the pathophysiology of the conditions. Therapeutic implications of understanding the metabolic roles of these and as yet other unidentified factors are also raised. While much remains unknown, that which has been observed provides a glimpse of the potential importance of this area of study.

  • bone resorption
  • calcium
  • phosphorus
  • TGF-β
  • undercarboxylated osteocalcin

1. Introduction

In recent years we have learned much about the mechanisms of bone formation and resorption, the cells involved, and many of the factors that affect and link the two processes. What we have not learned is the breadth of factors released by bone on resorption, the relationship of these factors to each other, what controls their release, and how they are utilized by the body’s metabolism under a wide variety of conditions. The study of certain conditions, such as burn injury, provides clues to what is released by bone and how they may operate. However, the large number of conditions, such as hyperparathyroidism, Paget’s disease, renal osteodystrophy, inflammation, and immobilization potentially affected by bone factor release are variable. Therefore, it is important to understand how the release system works in one condition at a time so as not to become overwhelmed by the complexity of the task facing us. The importance of understanding the mechanisms involved in bone factor release and utilization lies in the potential to regulate these releases for therapeutic purposes depending on the underlying pathologic condition.

2. What Factors Are Released by Resorbing Bone?

Given that we are still discovering elements and factors that are released by resorbing bone, it is reasonable to assume that there are many factors still awaiting discovery. However, we can only work with those which we currently know and are beginning to understand. Those which we currently know of are the following: calcium, phosphorus, magnesium, transforming growth factor-β and osteocalcin. What we have learned from the study of burns is that resorbing bone releases calcium, phosphorus, magnesium and transforming growth factor (TGF)-β while we do not know how much undercarboxylated osteocalcin is released and under what circumstances. While each of the first four listed factors is released in burn-induced bone resorption, the serum concentration of these does not necessarily reflect their metabolic fate inasmuch as some may have paracrine effects that serve to reduce circulating concentrations of these factors. We will explore each within the context of burn injury.

2.1. Calcium

Inasmuch as 99% of the body’s calcium is stored in bone [4], the release of calcium is the most immediate factor to examine. Bone is resorbed due to the robust systemic inflammatory response that develops due to the destruction of the skin barrier to entry of microorganisms into the body as well as possibly the effects of systemic glucocorticoids produced by the stress response. Calcium handling by burned children and adults appears to be different and this has been dealt with in two earlier publications [5,6]. In children [7], as in sheep [8], the inflammatory response leads to an upregulation of the parathyroid calcium-sensing receptor (CaSR), the G-protein coupled calcium-sensing receptor located on the membrane of the parathyroid chief cells [8,9,10,11]. This action lowers the amount of circulating calcium needed to suppress PTH secretion, in essence a lowering of the calcium threshold for suppression of PTH secretion. The result is a hypocalcemic hypoparathyroidism. Studies in adults with burn injury report that ionized calcium is normal to be slightly elevated while PTH concentration in serum is also normal to be slightly elevated [12,13]. Is this a significant finding? A study by Rossol et al. [14] showed that extracellular calcium can upregulate the nod-like receptor (NLR) P3 inflammasome, which will stimulate the monocytes and macrophages of the innate immune system to produce IL-1. Another study by Klein et al. [15] reported that cultures of peripheral blood mononuclear cells of adult volunteers resulted in chemokine production or suppression in direct or inverse relationship to the calcium content of the culture medium. Taken together, these studies provide evidence supporting intensification and/or prolongation of the inflammatory response. It has been shown by Finnerty et al. [16] that adults suffering the same extent of burn injury as children have greater burn morbidity. Thus, the higher circulating calcium in burned adults may prolong or intensify the inflammatory response while the ability of the hypoparathyroid pediatric burn patients to excrete excess calcium by means of the upregulated CaSR may have the effect of diminishing the inflammatory response in those patients. The mechanism for initiating failure to upregulate the CaSR in response to burns is at this point not identified.

Inasmuch as 99% of the body’s calcium is stored in bone [1], the release of calcium is the most immediate factor to examine. Bone is resorbed due to the robust systemic inflammatory response that develops due to the destruction of the skin barrier to entry of microorganisms into the body as well as possibly the effects of systemic glucocorticoids produced by the stress response. Calcium handling by burned children and adults appears to be different and this has been dealt with in two earlier publications [2][3]. In children [4], as in sheep [5], the inflammatory response leads to an upregulation of the parathyroid calcium-sensing receptor (CaSR), the G-protein coupled calcium-sensing receptor located on the membrane of the parathyroid chief cells [5][6][7][8]. This action lowers the amount of circulating calcium needed to suppress PTH secretion, in essence a lowering of the calcium threshold for suppression of PTH secretion. The result is a hypocalcemic hypoparathyroidism. Studies in adults with burn injury report that ionized calcium is normal to be slightly elevated while PTH concentration in serum is also normal to be slightly elevated [9][10]. Is this a significant finding? A study by Rossol et al. [11] showed that extracellular calcium can upregulate the nod-like receptor (NLR) P3 inflammasome, which will stimulate the monocytes and macrophages of the innate immune system to produce IL-1. Another study by Klein et al. [12] reported that cultures of peripheral blood mononuclear cells of adult volunteers resulted in chemokine production or suppression in direct or inverse relationship to the calcium content of the culture medium. Taken together, these studies provide evidence supporting intensification and/or prolongation of the inflammatory response. It has been shown by Finnerty et al. [13] that adults suffering the same extent of burn injury as children have greater burn morbidity. Thus, the higher circulating calcium in burned adults may prolong or intensify the inflammatory response while the ability of the hypoparathyroid pediatric burn patients to excrete excess calcium by means of the upregulated CaSR may have the effect of diminishing the inflammatory response in those patients. The mechanism for initiating failure to upregulate the CaSR in response to burns is at this point not identified.

2.2. Phosphorus and Magnesium

The first evidence obtained that bone phosphorus and magnesium are important in burn metabolism came to light when Borsheim et al. [17] reported results of stable isotope studies of muscle protein balance in burned children who had participated in a double-blind randomized controlled trial of a bisphosphonate to prevent bone resorption following burn injury. While the outcome of the anti-resorptive study showed success in prevention of bone resorption [18,19] when examining the muscle protein kinetics it was found that while muscle protein breakdown was reduced in the bisphosphonate group, muscle protein synthesis was also reduced [17]. Inasmuch as the ATP requirements for muscle function go up significantly following burns, it is likely that bone resorption provided the phosphorus and magnesium necessary to promote synthesis of the ATP to be used in muscle protein synthesis.

The first evidence obtained that bone phosphorus and magnesium are important in burn metabolism came to light when Borsheim et al. [14] reported results of stable isotope studies of muscle protein balance in burned children who had participated in a double-blind randomized controlled trial of a bisphosphonate to prevent bone resorption following burn injury. While the outcome of the anti-resorptive study showed success in prevention of bone resorption [15][16] when examining the muscle protein kinetics it was found that while muscle protein breakdown was reduced in the bisphosphonate group, muscle protein synthesis was also reduced [14]. Inasmuch as the ATP requirements for muscle function go up significantly following burns, it is likely that bone resorption provided the phosphorus and magnesium necessary to promote synthesis of the ATP to be used in muscle protein synthesis.

Moreover, the upregulation of the CaSR by the inflammatory response lowered PTH and FGF23 [20] thus helping to preserve phosphate for use in generating more ATP. Paradoxically, CaSR upregulation also leads to more urinary magnesium excretion thus perhaps putting a greater strain on bone to release more magnesium in the face of CaSR upregulation [21]. This issue has not been addressed.

Moreover, the upregulation of the CaSR by the inflammatory response lowered PTH and FGF23 [17] thus helping to preserve phosphate for use in generating more ATP. Paradoxically, CaSR upregulation also leads to more urinary magnesium excretion thus perhaps putting a greater strain on bone to release more magnesium in the face of CaSR upregulation [18]. This issue has not been addressed.

2.3. Transforming Growth Factor (TGF)-β

Bone matrix is rich in TGF-β. The molecule is synthesized in osteoblasts, stored in the matrix, and released from the matrix by the action of osteoclastic proteolysis of the latent TGF-β binding protein-1 mediated by matrix metalloproteinases (MMP)-2 and -9 [22]. Waning et al. [23] identified TGF-β release from bone as the cause of the paracrine effect on muscle wasting in patients with bone metastases from either breast or lung cancer. The mechanism explaining this was the oxidation of the ryanodine receptor with consequent calcium wasting resulting in weakness and wasting of the muscle. In burn injury, the results reported by Borsheim et al. [17] were explained by Pin et al. [24]. They reported that in vitro studies of murine myoblasts cultured with serum from burn patients who received treatment either with a bisphosphonate to prevent bone resorption or a saline placebo demonstrated that burn injury resulted in a reduction in myotube size, an increase in the catabolic pathway as represented by ubiquitin concentration and a reduction in phosphorylation of the anabolic AKT/mTOR pathway. Myoblasts cultured with serum from patients treated with bisphosphonates, in contrast, demonstrated partial rescue of myotube size, a significant reduction in ubiquitin concentration in the medium and an increase in phosphorylation of the AKT/mTOR pathway. More importantly, when the myoblast culture experiments were repeated with the addition of anti-TGF-β antibody to the cultures, rescue of myotube size in the placebo group reached the magnitude initially seen in those cultures with serum from the bisphosphonate-treated patients while in the cultures of myoblasts with serum from bisphosphonate-treated patients, there were negligible changes in myotube size from the original experiments without the anti-TGF-β antibody. These data support TGF-β release from bone as a significant factor in muscle wasting in two discrete groups of patients, mature women with breast cancer metastases to bone and pediatric burn patients.

Bone matrix is rich in TGF-β. The molecule is synthesized in osteoblasts, stored in the matrix, and released from the matrix by the action of osteoclastic proteolysis of the latent TGF-β binding protein-1 mediated by matrix metalloproteinases (MMP)-2 and -9 [19]. Waning et al. [20] identified TGF-β release from bone as the cause of the paracrine effect on muscle wasting in patients with bone metastases from either breast or lung cancer. The mechanism explaining this was the oxidation of the ryanodine receptor with consequent calcium wasting resulting in weakness and wasting of the muscle. In burn injury, the results reported by Borsheim et al. [14] were explained by Pin et al. [21]. They reported that in vitro studies of murine myoblasts cultured with serum from burn patients who received treatment either with a bisphosphonate to prevent bone resorption or a saline placebo demonstrated that burn injury resulted in a reduction in myotube size, an increase in the catabolic pathway as represented by ubiquitin concentration and a reduction in phosphorylation of the anabolic AKT/mTOR pathway. Myoblasts cultured with serum from patients treated with bisphosphonates, in contrast, demonstrated partial rescue of myotube size, a significant reduction in ubiquitin concentration in the medium and an increase in phosphorylation of the AKT/mTOR pathway. More importantly, when the myoblast culture experiments were repeated with the addition of anti-TGF-β antibody to the cultures, rescue of myotube size in the placebo group reached the magnitude initially seen in those cultures with serum from the bisphosphonate-treated patients while in the cultures of myoblasts with serum from bisphosphonate-treated patients, there were negligible changes in myotube size from the original experiments without the anti-TGF-β antibody. These data support TGF-β release from bone as a significant factor in muscle wasting in two discrete groups of patients, mature women with breast cancer metastases to bone and pediatric burn patients.

2.4. Osteocalcin

Work from the laboratory of Karsenty et al. [25,26] and others has shown that the undercarboxylated form of osteocalcin, also synthesized by osteoblasts, is an important factor in muscle fiber uptake of glucose and nutrients, thus constituting an anabolic factor. It is released from the bone matrix by IL-6 generated by muscle in response to osteocalcin. IL-6 will then stimulate osteoblast synthesis of the ligand of the receptor activator of nuclear transcription factor κB (RANK ligand or RANKL), which will stimulate osteoclastogenesis, increase bone resorption and release more undercarboxylated osteocalcin for anabolic action on muscle. Additionally, undercarboxylated osteocalcin stimulates pancreatic β cells to secrete insulin and renders muscle more sensitive to insulin action. It should be pointed out that undercarboxylated osteocalcin has not been specifically studied in burn patients. Only total serum osteocalcin has been examined and those results have all been low [27], not surprisingly since bone is being lost due to resorption. However, undercarboxylated osteocalcin is mentioned here inasmuch as it exerts anabolic action as opposed to the catabolic action of TGF-β, therefore serving as a potential counterbalance to TGF-β and raising the question, to be posed in the next section, of how does the bone decide, if it indeed does decide, to release catabolic as opposed to anabolic factors for metabolic use.

Work from the laboratory of Karsenty et al. [22][23] and others has shown that the undercarboxylated form of osteocalcin, also synthesized by osteoblasts, is an important factor in muscle fiber uptake of glucose and nutrients, thus constituting an anabolic factor. It is released from the bone matrix by IL-6 generated by muscle in response to osteocalcin. IL-6 will then stimulate osteoblast synthesis of the ligand of the receptor activator of nuclear transcription factor κB (RANK ligand or RANKL), which will stimulate osteoclastogenesis, increase bone resorption and release more undercarboxylated osteocalcin for anabolic action on muscle. Additionally, undercarboxylated osteocalcin stimulates pancreatic β cells to secrete insulin and renders muscle more sensitive to insulin action. It should be pointed out that undercarboxylated osteocalcin has not been specifically studied in burn patients. Only total serum osteocalcin has been examined and those results have all been low [24], not surprisingly since bone is being lost due to resorption. However, undercarboxylated osteocalcin is mentioned here inasmuch as it exerts anabolic action as opposed to the catabolic action of TGF-β, therefore serving as a potential counterbalance to TGF-β and raising the question, to be posed in the next section, of how does the bone decide, if it indeed does decide, to release catabolic as opposed to anabolic factors for metabolic use.

References

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  2. Klein, G.L.; Benjamin, D.; Herndon, D.N. Calcemic response differs between adults and children: A review of the literature. Osteoporos. Sarcopenia 2017, 3, 170–173.
  3. Klein, G.L. The role of calcium in inflammation-associated bone resorption. Biomolecules 2018, 8, 69.
  4. Klein, G.L.; Nicolai, M.; Langman, C.B.; Cuneo, B.F.; Sailer, D.E.; Herndon, D.N. Dysregulation of calcium homeostasis after severe burn injury in children: Possible role of magnesium depletion. J. Pediatrics 1997, 131, 246–251.
  5. Murphey, E.D.; Chattopadhyay, N.; Bai, M.; Kifor, O.; Harper, D.; Traber, D.L.; Hawkins, H.K.; Brown, E.M.; Klein, G.L. Up-regulation of the parathyroid calcium-sensing receptor after burn injury in sheep: A potential contributory factor to post-burn hypocalcemia. Crit. Care Med. 2000, 28, 3885–3890.
  6. Nielsen, P.K.; Rasmussen, A.K.; Butters, R.; Feldt-Rasmussen, U.; Bendtzen, K.; Diaz, R.; Brown, E.M.; Olgaard, K. Inhibition of PTH secretion by interleukin-1 beta in bovine parathyroid glands in vitro is associated with an up-regulation of the calcium-sensing receptor mRNA. Biochem. Biophys. Res. Commun. 1997, 238, 880–885.
  7. Toribio, R.E.; Kohn, C.W.; Capen, C.C.; Rosol, T.J. Parathyroid hormone (PTH) secretion, PTH mRNA and calcium-sensing receptor mRNA expression in equine parathyroid cells, and effects of interleukin(IL)-1, IL-6, and tumor necrosis factor-alpha on equine parathyroid cell function. J. Mol. Endocrinol. 2003, 31, 609–620.
  8. Canaff, L.; Zhou, X.; Hendy, G.N. The pro-inflammatory cytokine, interleukin-6 up-regulates calcium-sensing receptor gene transcription via Stat1/3 and Sp 1/3. J. Biol. Chem. 2008, 283, 13586–13600.
  9. Klein, G.L.; Herndon, D.N.; Rutan, T.C.; Sherrard, D.J.; Coburn, J.W.; Langman, C.B.; Thomas, M.L.; Haddad, J.G., Jr.; Cooper, C.W.; Miller, N.L.; et al. Bone disease in burn patients. J. Bone Min. Res. 1993, 8, 337–345.
  10. Rousseau, A.F.; Damas, P.; Ledoux, D.; Lukas, P.; Carlisi, A.; LeGoff, C.; Gadisseur, R.; Cavalier, E. Vitamin D status after a high dose of cholecalciferol in healthy and burn subjects. Burns 2015, 41, 1028–1034.
  11. Rossol, M.; Pierer, M.; Raulien, N.; Quandt, D.; Meusch, U.; Rothe, K.; Schubert, K.; Schoneberg, T.; Schaefer, M.; Krugel, U.; et al. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium-sensing receptors. Nat. Commun. 2012, 3, 1329.
  12. Klein, G.L.; Castro, S.; Garofalo, R.P. The calcium-sensing receptor as a mediator of inflammation. Semin. Cell Dev. Biol. 2016, 49, 52–56.
  13. Finnerty, C.C.; Jeschke, M.G.; Qian, W.J.; Kaushal, A.; Xiao, W.; Liu, T.; Gritsenko, M.A.; Moore, R.J.; Camp, D.G., 2nd; Moldawer, L.L.; et al. Investigators of the Inflammation and the Host Response Glue Grant. Determination of the best patient outcome by large-scale quantitative discovery proteomics. Crit. Care Med. 2013, 41, 1421–1434.
  14. Borsheim, E.; Herndon, D.N.; Hawkins, H.K.; Suman, O.E.; Cotter, M.; Klein, G.L. Pamidronate attenuates muscle loss after pediatric burn injury. J. Bone Min. Res. 2014, 29, 1369–1372.
  15. Klein, G.L.; Wimalawansa, S.J.; Kulkarni, G.; Sherrard, D.J.; Sanford, A.P.; Herndon, D.N. The efficacy of acute administration of pamidronate on the conservation of bone mass following severe burn injury in children: A double-blind, randomized, controlled study. Osteoporos. Int. 2005, 16, 631–635.
  16. Przkora, R.; Herndon, D.N.; Sherrard, D.J.; Chinkes, D.L.; Klein, G.L. Pamidronate preserves bone mass for at least two years following acute administration for pediatric burn injury. Bone 2007, 41, 297–302.
  17. Klein, G.L.; Herndon, D.N.; Le, P.T.; Andersen, C.R.; Benjamin, D.; Rosen, C.J. The effect of burn on serum concentrations of sclerostin and FGF-23. Burns 2015, 41, 1532–1535.
  18. Miller, R.T. Control of renal calcium, phosphate, electrolyte and water excretion by the calcium sensing receptor. Best. Pract. Res. Clin. Endocrinol. Metab. 2013, 27, 345–358.
  19. Dallas, S.L.; Rosser, J.L.; Mundy, G.R.; Bonewald, L.F. Proteolysis of latent transforming growth factor beta (TGF beta) binding protein 1 by osteoclasts. A cellular mechanism for release of TGF beta from bone matrix. J. Biol. Chem. 2002, 277, 21352–21360.
  20. Waning, D.L.; Mohammad, K.S.; Reiken, S.; Xie, W.; Andersson, D.C.; John, S.; Chiechi, A.; Wright, L.E.; Umanskaya, A.; Niewolna, M.; et al. Excess TGF-β mediates muscle weakness associated with bone metastases in mice. Nat. Med. 2015, 21, 1262–1271.
  21. Pin, F.; Bonetto, A.; Bonewald, L.F.; Klein, G.L. Molecular mechanisms responsible for the rescue effect of pamidronate on muscle atrophy in pediatric burn patients. Front. Endocrinol. 2019, 10, 543.
  22. Mera, P.; Laue, K.; Ferron, M.; Confavreux, C.; Wei, J.; Galan-Diez, M.; Lacampagne, A.; Mitchell, S.J.; Mattison, J.A.; Chen, Y.; et al. Osteocalcin signaling in myofibers is necessary and sufficient for optimum adaptation to exercise. Cell Metab. 2016, 23, 1078–1092.
  23. Chowdhury, S.; Schulz, L.; Palmisano, B.; Singh, P.; Berger, J.M.; Yadav, V.K.; Mera, P.; Ellingsgaard, H.; Hidalgo, J.; Bruning, J.; et al. Muscle-derived interleukin-6 increases exercise capacity by signaling in osteoblasts. J. Clin. Investig. 2020, 130, 2888–2902.
  24. Klein, G.L.; Wolf, S.E.; Langman, C.B.; Rosen, C.J.; Mohan, S.; Keenan, B.S.; Matin, S.; Steffen, C.; Nicolai, M.; Sailer, D.E.; et al. Effects of therapy with recombinant human growth hormone on insulin-like growth factor system components and serum levels of biochemical markers of bone formation in children after severe burn injury. J. Clin. Endocrinol. Metab. 1998, 83, 21–24.
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