Back to summary |Download this issue

Pierre J. MARIE, PhD
Inserm U606 and
University of Paris Diderot
Lariboisière Hospital
Paris, FRANCE

by P. J . Marie, France

Bone remodeling, a process by which bone resorption by osteoclasts is followed by bone formation by osteoblasts, is an essential physiological process regulating bone mass and strength. During growth, bone formation exceeds bone resorption, resulting in bone expansion. In the young adult, bone resorption is balanced by bone formation, resulting in maintenance of bone mass. The cellular mechanisms underlying the age-related alterations in bone resorption and formation are now better known. With aging, bone formation decreases due to reduction in osteoblast number, activity, and life span, whereas bone resorption increases as a result of sex hormone deprivation. These two mechanisms contribute to the decreased bone mass and increased risk of fractures seen in the aging population. Current effective antiresorbing drugs reduce bone remodeling in osteoporotic subjects. An ideal way to prevent age-related bone loss would be not only to reduce bone resorption, but also to promote bone formation. There is therefore an important need to develop therapeutic strategies capable of promoting bone formation in osteoporotic subjects. Current research efforts are focusing on strategies to target signaling pathways that positively control bone formation and bone mass. This may lead to the development of novel therapeutic approaches that promote osteoblastogenesis to counteract the defective bone formation and bone loss related to aging.

The skeleton is a unique tissue providing support and mineral balance for the organism. It is formed during growth and is maintained during adult life by continual renewal of the matrix, a process called bone remodeling. Bone remodeling is ensured by two cell types: osteoclasts, which resorb the calcified bone matrix, and osteoblasts, which are responsible for new bone matrix synthesis. During growth, bone formation exceeds bone resorption, resulting in bone expansion. In the young adult, bone resorption is balanced by bone formation, resulting in maintenance of bone mass. With aging and after the menopause, an imbalance in bone resorption relative to formation results in negative bone balance at the tissue level. This may lead to osteoporosis, a common skeletal disease characterized by reduced bone mass, deterioration of bone microarchitecture, and increased susceptibility to fractures.¹ The causes of increased bone resorption relative to bone formation in women after the menopause are now better known (Figure 1). Estrogen deficiency in perimenopausal women (and to a lesser extent, the decline in testosterone levels in men) results in accelerated bone remodeling with bone resorption exceeding bone formation. This leads to an increased number of bone remodeling units, perforation of trabeculae, endocortical erosion (responsible for trabecular disconnection), alteration of trabecular microarchitecture, and reduced bone strength.^2,3 Several mechanisms are involved in the acceleration of the bone remodeling occurring in estrogen deficiency, including increased cytokine production by monocytes, lymphocytes, and osteoblast/stromal cells in the bone microenvironment, as well as an increased receptor activated nuclear factor-êB ligand (RANKL)/osteoprote-gerin ratio that determines osteoclast differentiation.^2,3 Although the increased bone resorption activity associated with the menopause is related to increased bone formation due to the coupling phenomenon, bone formation remains insufficient to compensate for the increased bone resorbing activity (Figure 1). This is a key issue when considering the prevention and treatment of age-related bone loss, since once trabeculae are perforated, it is almost impossible to replace the missing trabeculae within the bone marrow and to rebuild appropriate connections with other trabeculae.

Figure 1. Age-related alteration in bone mass.

Age-related defective bone formation

Bone formation is a complex process involving the commitment of osteoprogenitor cells, their differentiation into preosteoblasts, and mature osteoblasts, whose function it is to synthesize bone matrix that becomes progressively mineralized. Osteoblast commitment, differentiation, and function are all governed by several transcription factors, resulting in the expression of phenotypic genes and the acquisition of the osteoblast phenotype.⁴ The sequence of osteogenic differentiation is characterized by the expression of alkaline phosphatase and the synthesis and deposition of type I collagen and bone matrix proteins, followed by the onset of mineralization. At the end of bone formation, most osteoblasts become flattened lining cells, some become osteocytes, and others undergo apoptosis. A fraction of osteoblasts also die by apoptosis, a process that directly affects osteoblast life span and the duration of the bone formation phase.⁵ It has been established from animal models and human metabolic bone diseases that bone formation is more dependent on osteoblast number, which can be expanded, than on osteoblast activity, which is physiologically limited.⁶

Aging is associated with decreased bone formation relative to bone resorption (Figure 1). There are two major causes that underlie the relative, age-related alteration in bone formation. As mentioned above, bone resorption increases as a result of hormone deprivation in the perimenopausal years. The coupling mechanism during bone remodeling results in increased bone formation, as reflected, for example, by the increase in bone remodeling markers occurring at menopause.³ However, the increased bone formation cannot compensate for the increased resorption, and this imbalance results in bone loss after menopause. Several mechanisms can be involved in the defective bone formation relative to bone resorption in estrogen deficiency. First, the osteoblast capacity of forming bone is limited, as evaluated by the mineral apposition rate, and this limited capacity to form bone matrix by osteoblasts is not increased in estrogen deficiency. Second, although the proliferative capacity of osteoblastic cells is increased by estrogen deficiency,^7,8 most probably in response to the local release of growth factors from bone, this is not sufficient to compensate for the increase in bone resorption. A third mechanism underlying the relative lack of bone formation in estrogen deficiency is the alteration of osteoblast life span.⁵ Estrogens prevent osteoblast apoptosis, and estrogen deficiency results in increased osteoblast apoptosis that leads to a decrease in the duration of the bone formation phase. The decreased osteoblast life span does not allow bone to compensate for the increased bone resorbing activity of osteoclasts.

Figure 2. Main mechanisms involved in age-related defective bone formation.

Age-related bone loss is associated with a second phenomenon, characterized by a slow, continuous decrease in bone forming activity, independent of sex hormone deficiency (Figure 1). This decreased bone forming activity that occurs with aging was first documented in humans as the decline in the amount of bone formed by osteoblasts in each remodeling unit.⁹ Although this slow decrease in bone matrix formed does not lead to perforation of trabeculae, the effect results in thinning of the bone trabeculae, increased trabecular separation, and decreased cortical thickness with age.³ This is an important and underestimated mechanism that contributes to the deterioration of bone microarchitecture and strength associated with fractures in osteoporotic subjects.^10,11

_ Cellular causes of age-related decrease in bone formation
The development of appropriate therapeutic strategies in osteoporosis requires a better understanding of the mechanisms underlying defective bone formation occurring with aging and the menopause. Multiple mechanisms are believed to contribute to the age-related decline in bone formation (Figure 2). It is well known that mesenchymal stromal cells within the bone marrow are able to differentiate into osteoblasts or adipocytes under stimulation by hormonal or local factors, a process called cell plasticity. It has been found that the decreased osteoblastogenesis that occurs with aging may result from preferential differentiation of mesenchymal stromal cells into adipocytes, as a result of increased lipid oxidation causing oxidative stress and activation of the transcription factor peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) that governs adipocyte differentiation.¹² Pharmacological inactivation of PPAR-γ2 was consistently found to increase osteoblast differentiation and bone formation in mice.¹³

A second mechanism that might contribute to defective bone formation with aging is a decrease in the preosteoblastic cell proliferative capacity.¹⁴ This decrease in cell proliferative capacity is likely to contribute to the age-related decline in osteoblast number in humans.¹⁵ Another important possible mechanism is the age-related intrinsic decrease in osteoblast function, possibly related to local decreases in the production of anabolic factors such as insulin-like growth factor 1 (IGF-1) or transforming growth factor β (TGF-β).¹⁶ Another like- ly causative mechanism is the decreased maximal life span and accelerated senescence of bone marrow stromal cells with aging. This phenomenon may be linked to the age-related increase in oxidative stress in bone¹² or to the increased local cytokine production occurring in bone after the menopause.³ All these pathogenic mechanisms may concur to decrease osteoblast number and function and contribute to age-related decline in bone formation relative to bone resorption (Figure 2).

Besides these intrinsic causes, several exogenous factors may be involved in defective age-related osteoblastogenesis. One well-known extrinsic factor that may alter osteoblast differentiation is the progressive decline in physical activity in aged subjects. Decreased mechanical strain is known to reduce osteogenic differentiation and to increase adipogenic differentiation of mesenchymal stromal cells, presumably by changes in the local production of growth factors and Wnt signaling.^17,18 Thus, it is likely that the reduced physical activity that occurs with age reduces bone formation. Other important exogenous factors that may contribute to defective osteoblastogenesis in the aging population include insufficient protein intake,¹⁹ excess alcohol and tobacco consumption, as well as medications, such as long-term glucocorticoid treatment.²⁰ It is thus likely that the alterations in osteoblastogenesis and the resulting decline in bone formation that occurs with aging result from multiple intrinsic and extrinsic causes.

Promoting bone formation: an enduring therapeutic challenge in osteoporosis

Given the fact that estrogen deficiency results in excessive bone resorption relative to bone formation, pharmacological compounds that decrease bone resorption are efficient at treating osteoporosis.³ Bisphosphonates are known to act by reducing bone remodeling (both resorption and formation), which leads to the prevention of bone loss and to a reduction in fracture incidence in osteoporosis. Denosumab, a fully human monoclonal antibody to RANKL that blocks its binding to receptor activated nuclear factor-êB and hence osteoclast differentiation, was recently shown to strongly reduce the risk of fractures in women with osteoporosis.21 Although efficient at decreasing bone remodeling activity, the long-term effects of bisphosphonates or denosumab on bone properties remain unknown.

Since age-related bone loss is associated with insufficient bone formation relative to bone resorption, a major advance in the therapeutic field would be to promote bone formation while reducing bone resorption. In this context, strontium ranelate was found to act by dissociating bone resorption and bone formation in vitro. A number of studies have shown that strontium ranelate activates osteoblast replication, differentiation, activity, and survival and reduces osteoclast function and survival.²² Accordingly, this drug was found to increase the mineral apposition rate, to improve trabecular microarchitecture, and to reduce fracture risk in osteoporotic subjects.^23-25 This compound thus offers an ideal way of favoring bone formation without increasing bone resorption in age-related bone loss.

An enduring challenge in the prevention or treatment of agerelated bone loss is whether one should prevent or protect the age-related decrease in bone formation. Up to now, the number of anabolic agents that promote osteoblastogenesis has been very limited. Nature has provided us with some physiological tools to promote bone formation. For example, bone morphogenetic proteins (BMPs) are natural anabolic molecules that physiologically promote osteoblast differentiation in vitro and in vivo.²⁵ However, BMPs can only be used as therapeutic agents for local bone repair because of their short half-life and possible side effects on nonskeletal stem cell development. Other natural skeletal growth factors such as IGF-1 or TGF-β have been shown to promote bone formation and reduce bone loss in experimental models of osteoporosis.^26,27 However, these growth factors cannot be used easily in clinics because of their possible modulation of bone resorption as well as side effects. Two decades ago, fluoride, a mitogenic agent for osteoblastic cells, was tested in osteoporosis. Unfortunately, although fluoride is effective in increasing osteoblast replication in osteoporosis,²⁸ osteoblast function is altered with fluoride treatment, which failed to improve bone strength.²⁹ Finally, some statins have been shown to promote bone formation in experimental studies in animals.³⁰ However, there is still no evidence for a clear anabolic effect on bone for these agents in humans.

A major step forward was the finding that, in contrast to continuous treatment, intermittent parathyroid hormone (PTH) increases bone formation in osteoporotic patients.³¹ At the cellular level, PTH acts on osteoblasts by activating protein kinase A (PKA), which phosphorylates the osteoblast transcription factor Runx2, which in turn upregulates the expression of osteoblast genes. Additionally, intermittent PTH activates extracellular signal-regulated kinase 1/2 (ERK1/2), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase signaling, which upregulate osteoblast proliferation, differentiation, and survival (Figure 3, page 14). Furthermore, IGF-1, TGF-β, and fibroblast growth factor expression is upregulated by intermittent PTH, resulting in increased osteogenesis.³² All these mechanisms contribute to increase the recruitment of osteoblast progenitors and to decrease osteoblast apoptosis, resulting in increased bone formation relative to bone resorption.

At the tissue level, the increased bone formation induced by PTH (1-34) or (1-84) results in increased trabecular bone mass and cortical thickness, leading to a marked reduction in fracture risk in osteoporotic patients.^31,33 This finding emphasizes the point that anabolic treatments may be more effective than antiresorbing drugs in the maintenance of bone quality and quantity in osteoporosis. Although the development of intermittent PTH as an anabolic agent is a major advance in the treatment of osteoporosis, this treatment has some limitations linked to its short half-life and cost. Alternatively, the use of an oral calcilytic molecule that blocks the parathyroid cell calcium receptor, thus stimulating the endogenous release of PTH, may prove to be useful for promoting bone formation in osteopenic disorders.³⁴

Figure 3. Main signaling pathways involved in the anabolic effect of PTH on bone.

_ Therapeutic perspectives in promoting bone formation
As emphasized above, there is still a need to develop efficient and safe drugs that are able to promote bone formation in osteoporosis. One possibility is to target theWnt/β-catenin signaling pathway that was found to upregulate osteoblastogenesis, postnatal bone formation, and bone mass in animals and humans.^35-37 How does Wnt signaling control bone formation? It was found that activation of the canonical Wnt/β-catenin pathway promotes osteoblastic cell proliferation and differentiation and reduces adipogenic differentiation from mesenchymal stromal cells through modulation of Runx2 and PPAR-γ2. Additionally,Wnt signaling promotes osteoblast survival (Figure 4). These effects, in addition toexisting crosstalks betweenWnt, BMP-2, and PTH signaling, contribute to the positive effects ofWnt signaling on osteoblastogenesis and bone mass.^35-37 Interestingly, mechanical loading upregulates Wnt signaling and prevents adipogenic differentiation in mesenchymal stem cells.³⁸ Moreover, attenuation of Wnt/β-catenin signaling contributes to age-related bone loss in mice,³⁹ suggesting that the combination of reduced β-catenin signaling and mechanical stimulation may be involved in age-related decline of bone formation in humans.

Figure 4. The canonical Wnt signaling pathway and control of bone formation.

Recent data have challenged the role of low-density lipoprotein receptor–related protein 5 (LRP-5) in the control of bone formation. Yadav et al⁴⁰ showed that LRP-5 may not play a major role in osteoblast function, but rather that bone mass is regulated by a β-catenin- and Wnt-independent effect of LRP-5 deletion on serotonin secretion from the gut. If confirmed, this discovery may lead to novel therapeutic approaches aimed at antagonizing serotonin synthesis in the gut and/or serotonin action on osteoblasts. Nevertheless, the fact remains that Wnt signaling in the bone microenvironment is likely to play a role in the control of bone mass.⁴¹ The important role of Wnt signaling in the control of bone mass suggests that this pathway may be a potential therapeutic target in osteoporosis. According to this concept, activation of canonical Wnt signaling using glycogen synthase kinase 3 inhibitors were shown to promote bone formation and to prevent bone loss in aged or ovariectomized osteopenic mice.^42,43 However, the therapeutic use of Wnt signaling agonists in clinical settings is limited due to the potential activation of cancer cells. Future research is needed to determine whether pharmacological inhibition of natural antagonists of Wnt signaling, such as Frizzled or Dkk1, results in safe activation of Wnt signaling in bone. Alternatively, noncanonical Wnt signaling, which has been shown to promote bone formation,⁴⁴ may be another target for developing new anabolic therapeutic approaches in osteopenic disorders.

Figure 5. Mode of action of sclerostin on osteoblastogenesis.

Recent studies have opened a new area of translational research based on the clinical observation that the loss of function of sclerostin, the product of the SOST gene, results in increased bone mass.^45,46 Sclerostin is produced by osteocytes and is a potent inhibitor of bone formation. It does this by antagonizing LRP-5 receptor signaling,Wnt signaling, and bone formation (Figure 5). Interestingly, sclerostin expression is negatively regulated by loading⁴⁷ and PTH,⁴⁸ suggesting that it may be a physiological modulator of bone formation. These findings led to the exciting concept that targeting sclerostin may lead to increased bone formation and bone mass in vivo. Indeed, targeted deletion of the sclerostin gene results in increased bone formation and bone strength in mice.⁴⁹ More interestingly, a sclerostin antibody treatment was shown to increase bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis.⁵⁰ This raises the hope that this novel therapeutic strategy may result in increased bone formation and bone mass in age-related osteopenic disorders.

Conclusion and perspectives

The available data indicate that aging is associated with impaired bone formation relative to bone resorption, indicating that osteoporosis is (also) a disease of bone formation. This has important implications for developing novel, efficient, anabolic therapeutic strategies in age-related bone loss.

Up to now, a limited number of molecules, including teriparatide and, to a lesser extent, strontium ranelate, have been shown to activate bone formation in clinical studies. Ongoing investigations are currently focused on targeting the Wnt signaling pathway that governs osteoblastogenesis and bone formation. It is hoped that this approach will lead to the development of safe anabolic agents that are able to promote bone formation in age-related osteopenic disorders. _

References

1. Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest. 2005;115:3318-3325.
2. Martin TJ, Seeman E. Bone remodelling: its local regulation and the emergence of bone fragility. Best Pract Res Clin Endocrinol Metab. 2008;22:701-722.
3. Riggs BL, Parfitt AM. Drugs used to treat osteoporosis: the critical need for a uniform nomenclature based on their action on bone remodeling. J Bone Miner Res. 2005;20:177-184.
4. Marie PJ. Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys. 2008;473:98-105.
5. Weinstein RS, Manolagas SC. Apoptosis and osteoporosis. Am J Med. 2000; 108:153-164.
6. Marie PJ. Human osteoblastic cells: a potential tool to assess the etiology of pathologic bone formation. J Bone Miner Res. 1994;9:1847-1850.
7. Modrowski D, Miravet L, Feuga M, Marie PJ. Increased proliferation of osteoblast precursor cells in estrogen deficient rats. Am J Physiol. 1993;264 (2 pt 1): E190-E196.
8. Marie PJ, Sabbagh A, de Vernejoul MC, Lomri A. Osteocalcin and DNA synthesis in vitro and histomorphometric indices of bone formation in postmenopausal osteoporosis. J Clin End Metab. 1989;69:272-279.
9. Lips P, Courpron P, Meunier PJ. Mean wall thickness of trabecular bone packets in the human iliac crest: changes with age. Calcif Tissue Res. 1978;26:13-17.
10. Boutroy S, Bouxsein ML, Munoz F, Delmas PD. In vivo assessment of trabecular bone microarchitecture by high-resolution peripheral quantitative computed tomography. J Clin Endocrinol Metab. 2005;90:6508-6515.
11. Sornay-Rendu E, Boutroy S, Munoz F, Delmas PD. Alterations of cortical and trabecular architecture are associated with fractures in postmenopausal women, partially independent of decreased BMD measured by DXA: the OFELY study. J Bone Miner Res. 2007;22:425-433.
12. Almeida M, Ambrogini E, Han L, Manolagas SC, Jilka RL. Increased lipid oxidation causes oxidative stress, increased PPAR-gamma expression and diminished pro-osteogenic Wnt signaling in the skeleton. J Biol Chem. 2009 August 5. Epub ahead of print.
13. Akune T, Ohba S, Kamekura S, et al. PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest. 2004;113:846-855.
14. Bellantuono I, Aldahmash A, Kassem M. Aging of marrow stromal (skeletal) stem cells and their contribution to age-related bone loss. Biochim Biophys Acta. 2009;1792:364-370.
15. Marie PJ, de Vernejoul MC, Connes D, Hott M. Decreased DNA synthesis by cultured osteoblastic cells in eugonadal osteoporotic men with defective bone formation. J Clin Invest. 1991;88:1167-1172.
16. Nicolas V, Prewett A, Bettica P, et al. Age-related decreases in insulin-like growth factor-I and transforming growth factor-beta in femoral cortical bone from both men and women: implications for bone loss with aging. J Clin Endocrinol Metab. 1994;78:1011-1016.
17. Ahdjoudj S, Lasmoles F, Holy X, Zerath E, Marie PJ. Transforming growth factor beta2 inhibits adipocyte differentiation induced by skeletal unloading in rat bone marrow stroma. J Bone Miner Res. 2002;17:668-677.
18. Kang S, Bennett CN, Gerin I, Rapp LA, Hankenson KD, Macdougald OA. Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein alpha and peroxisome proliferator- activated receptor gamma. J Biol Chem. 2007;282:14515-14524.
19. Rizzoli R, Ammann P, Chevalley T, Bonjour JP. Protein intake and bone disorders in the elderly. Joint Bone Spine. 2001;68:383-392.
20. Canalis E.Mechanisms of glucocorticoid-induced osteoporosis. Curr Opin Rheumatol. 2003;15:454-457.
21. Cummings SR, San Martin J, McClung MR, et al; FREEDOM Trial. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009;361:756-765.
22. Marie PJ. Preclinical aspects of strontium ranelate. In: Reginster JY, Rizzoli R, eds. Innovation in skeletal medicine. Elsevier; 2009:183-192.
23. Arlot ME, Jiang Y, Genant HK, et al. Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated with strontium ranelate. J Bone Miner Res. 2008;23:215-222.
24. Meunier PJ, Roux C, Seeman E, et al. The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N Engl J Med. 2004;350:459-468.
25. Canalis E, Economides AN, Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003;24:218-235.
26. Mueller K, Cortesi R, Modrowski D, Marie PJ. Stimulation of trabecular bone formation by insulin-like growth factor I in adult ovariectomized rats. Am J Physiol. 1994;267(1 pt 1):E1-E6.
27. Machwate M, Zerath E, Holy X, et al. Systemic administration of transforming growth factor-β 2 prevents the impaired bone formation and osteopenia induced by unloading in rats. J Clin Invest. 1995;96:1245-1253.
28. Marie PJ, de Vernejoul MC, Lomri A. Stimulation of bone formation in osteoporosis patients treated with fluoride associated with increased DNA synthesis by osteoblastic cells in vitro. J Bone Miner Res. 1992;7:103-113.
29. Riggs BL, Hodgson SF, O’Fallon WM, et al. Effect of fluoride treatment on the fracture rate in postmenopausal women with osteoporosis. N Engl J Med. 1990;322:802-809.
30. Garrett IR, Mundy GR. The role of statins as potential targets for bone formation. Arthritis Res. 2002;4:237-240.
31. Neer RM, Arnaud CD, Zanchetta JR, et al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med. 2001;344:1434-1441.
32. Jilka RL. Molecular and cellular mechanisms of the anabolic effect of intermittent PTH. Bone. 2007;40:1434-1446.
33. Compston JE. Skeletal actions of intermittent parathyroid hormone: effects on bone remodelling and structure. Bone. 2007;40:1447-1452.
34. Gowen M, Stroup GB, Dodds RA, et al. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats. J Clin Invest. 2000;105:1595-1604.
35. Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. 2006;116:1202-1209.
36. Baron R, Rawadi G, Roman-Roman S. Wnt signaling: a key regulator of bone mass. Curr Top Dev Biol. 2006;76:103-127.
37. Glass DA II, Karsenty G. In vivo analysis of Wnt signaling in bone. Endocrinology. 2007;148:2630-2634.
38. Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, et al. Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem. 2006;281:31720-31728.
39. Manolagas SC, Almeida M. Gone with the Wnts: beta-catenin, T-cell factor, forkhead box O, and oxidative stress in age-dependent diseases of bone, lipid, and glucose metabolism. Mol Endocrinol. 2007;21:2605-2614.
40. Yadav VK, Ryu JH, Suda N, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell. 2008;135:825-837.
41. Baron R. Wnt signaling, LRP5 and gut serotonin: have we been targeting the right pathway for the wrong reasons? IBMS BoneKEy. 2009;6:86-93.
42. Clément-Lacroix P, Ai M, Morvan F, et al. Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice. Proc Natl Acad Sci U S A. 2005;102:17406-17411.
43. Kulkarni NH, Onyia JE, Zeng Q, et al. Orally bioavailable GSK-3alpha/beta dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo. J Bone Miner Res. 2006;21:910-920.
44. Tu X, Joeng KS, Nakayama KI, et al. Noncanonical Wnt signaling through G protein-linked PKC delta activation promotes bone formation. Dev Cell. 2007; 12:113-127.
45. Poole KE, van Bezooijen RL, Loveridge N, et al. Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J. 2005; 19:1842-1844.
46. Li X, Zhang Y, Kang H, et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem. 2005;280:19883-19887.
47. Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem. 2008; 283:5866-5875.
48. Keller H, Kneissel M. SOST is a target gene for PTH in bone. Bone. 2005; 37:148-158.
49. Li X, Ominsky MS, Niu QT, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res. 2008;23:860-869.
50. Li X, Ominsky MS, Warmington KS, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res. 2009;24:578-588.