Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1796–1803, 2013     

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation‐Deficient Mice Through Increased BMP Signaling

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1^?/?) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1^?/? mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1^?/? mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1^?/? mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1^?/? mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.     

Unique regulation of SOST, the sclerosteosis gene, by BMPs and steroid hormones in human osteoblasts

SOST, a novel bone morphogenetic protein (BMP) antagonist and negative regulator of bone formation, is expressed in osteogenic cells. Null mutations in the SOST gene are associated with the sclerosteosis phenotype typified by high bone mass. We sought to delineate the pathways involved in the regulation of SOST expression in human osteoblastic cells. We evaluated the effects of bone growth factors and hormones on the RNA levels of SOST and the BMP antagonists, noggin and gremlin. Parathyroid hormone (PTH), transforming growth factor-beta1 (TGF-beta1), fibroblast growth factors 1 and 2 (FGF1, FGF2), and insulin-like growth factor-1 (IGF-1) had negligible effects on SOST expression in human osteoblasts. In comparison, BMPs-2, 4, and 6 induced the message levels of SOST in a time- and dose-dependent manner. The levels of noggin and, to a lesser extent, gremlin were also increased by BMPs. BMP's stimulatory effects on SOST were further enhanced by retinoic acid or 1,25-dihydroxyvitamin D3. In contrast, dexamethasone (DEX) blocked the effects of the BMPs on SOST and gremlin, but not on noggin. Retinoic acid and 1,25-dihydroxyvitamin D3 did not affect the BMP-enhanced expression of gremlin or noggin. The steroids did not affect the endogenous levels of the BMP antagonists. These findings show that the levels of SOST are modulated by BMPs and the interactions of the BMPs with steroid hormones in human osteoblasts. These effects differed markedly from that of noggin or gremlin, suggesting that there is an exquisite regulation of the expressions of BMP antagonists in cells of the osteoblast lineage.     

Activation of c‐Jun NH2‐terminal kinase 1 increases cellular responsiveness to BMP‐2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

Bone morphogenetic protein 2 (BMP‐2) plays a critical role in the differentiation of precursor cells and has been approved for clinical application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP‐2 have been required to induce bone healing in humans. Thus, enhancing cellular responsiveness to BMP‐2 potentially has critically important clinical implications. BMP responsiveness may be modulated in part by cross‐talk with other signaling pathways, including mitogen‐activated protein kinases (MAPKs). c‐Jun NH₂‐terminal kinase (JNK) is a MAPK that has been reported to be required for late‐stage differentiation of preosteoblasts and BMP‐2‐induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3‐E1‐clone 24 cells (MC‐24) can be induced by BMP‐2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both JNK loss‐of‐function and gain‐of‐function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by BMP‐2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP‐2‐induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor (BMPR‐I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP‐2. Understanding this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost‐effective and safe clinical delivery of these important molecules. © 2011 American Society for Bone and Mineral Research.     

BMP2 Differentially Regulates the Expression of Gremlin1 and Gremlin2, the Negative Regulators of BMP Function, During Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.     

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP‐induced bone formation may lead to the development of more effective BMP‐based therapies. To identify kinases mediating BMP‐induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP‐6‐induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF‐β) generally is considered to antagonize BMP‐induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF‐β receptors displayed reduced BMP‐6‐induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF‐β type I receptor impaired BMP‐induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF‐β further increased expression of osteoblast‐specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF‐β were found to depend on timing and duration of the costimulation. TGF‐β inhibited BMP‐induced activation of a BMP‐Smad‐dependent luciferase reporter, suggesting that the stimulatory effect of TGF‐β is not due to increased BMP‐Smad activity. TGF‐β also inhibited the BMP‐induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF‐β, besides acting as an inhibitor, also can, by dampening the noggin‐mediated negative‐feedback loop, enhance BMP‐induced osteoblast differentiation, which might be beneficial in fracture healing. © 2011 American Society for Bone and Mineral Research.     

Smad6 is essential to limit BMP signaling during cartilage development

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP‐mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6?/? mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6?/? mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6‐deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation. © 2011 American Society for Bone and Mineral Research     

Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins

Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra < 0.4 μm); sandblasted/acid-etched Ti (SLA, Ra = 3.2 μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6 days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment.     

Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development.

In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption. INTRODUCTION: Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology. MATERIALS AND METHODS: We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage. RESULTS: Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen. CONCLUSIONS: Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.     

Sustained RANKL response to parathyroid hormone in oncostatin M receptor-deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo

Parathyroid hormone (PTH) is the only approved anabolic agent for osteoporosis treatment. It acts via osteoblasts to stimulate both osteoclast formation and bone formation, with the balance between these two activities determined by the mode of administration. Oncostatin M (OSM), a gp130‐dependent cytokine expressed by osteoblast lineage cells, has similar effects and similar gene targets in the osteoblast lineage. In this study, we investigated whether OSM might participate in anabolic effects of PTH. Microarray analysis and quantitative real‐time polymerase chain reaction (qPCR) of PTH‐treated murine stromal cells and primary calvarial osteoblasts identified significant regulation of gp130 and gp130‐dependent coreceptors and ligands, including a significant increase in OSM receptor (OSMR) expression. To determine whether OSMR signaling is required for PTH anabolic action, 6‐week‐old male Osmr^?/? mice and wild‐type (WT) littermates were treated with hPTH(1–34) for 3 weeks. In WT mice, PTH increased trabecular bone volume and trabecular thickness. In contrast, the same treatment had a catabolic effect in Osmr^?/? mice, reducing both trabecular bone volume and trabecular number. This was not explained by any alteration in the increased osteoblast formation and mineral apposition rate in response to PTH in Osmr^?/? compared with WT mice. Rather, PTH treatment doubled osteoclast surface in Osmr^?/? mice, an effect not observed in WT mice. Consistent with this finding, when osteoclast precursors were cultured in the presence of osteoblasts, more osteoclasts were formed in response to PTH when Osmr^?/? osteoblasts were used. Neither PTH1R mRNA levels nor cAMP response to PTH were modified in Osmr^?/? osteoblasts. However, RANKL induction in PTH‐treated Osmr^?/? osteoblasts was sustained at least until 24 hours after PTH exposure, an effect not observed in WT osteoblasts. These data indicate that the transient RANKL induction by intermittent PTH administration, which is associated with its anabolic action, is changed to a prolonged induction in OSMR‐deficient osteoblasts, resulting in bone destruction. © 2012 American Society for Bone and Mineral Research.     

Insulin‐like growth factor 2 (IGF‐2) potentiates BMP‐9‐induced osteogenic differentiation and bone formation

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self‐renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP‐9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin‐like growth factor 2 (IGF‐2) on BMP‐9‐induced bone formation. We have found that endogenous IGF‐2 expression is low in MSCs. Expression of IGF‐2 can potentiate BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF‐2 has been shown to augment BMP‐9‐induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF‐2 enhances BMP‐9‐induced endochondral ossification, whereas IGF‐2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF‐2 effect on BMP‐9‐induced ALP activity and matrix mineralization. Mechanistically, IGF‐2 is further shown to enhance the BMP‐9‐induced BMPR‐Smad reporter activity and Smad1/5/8 nuclear translocation. PI3‐kinase (PI3K) inhibitor LY294002 abolishes the IGF‐2 potentiation effect on BMP‐9‐mediated osteogenic signaling and can directly inhibit BMP‐9 activity. These results demonstrate that BMP‐9 crosstalks with IGF‐2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP‐9 and IGF‐2 may be explored as an effective bone‐regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture. © 2010 American Society for Bone and Mineral Research.     

Expression of CD105 and CD34 receptors controls BMP‐induced in vitro mineralization of mouse adipose‐derived stem cells but does not predict their in vivo bone‐forming potential

Adipose‐derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose‐derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone‐forming potential. Four purified populations of mouse ADSCs: CD105⁺CD34⁺, CD105^?CD34^?, CD105⁺CD34^? and CD105^?CD34⁺ were obtained using fluorescence‐activated cell sorting (FACS) and their BMP‐responsiveness was determined in vitro. CD105⁺CD34^? population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP‐responsive phenotype and larger cell size while expression of CD34 correlated with low BMP‐responsive phenotype and smaller cell size. CD105⁺CD34^? population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105⁺CD34^? ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP‐responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:625–632, 2015.

     

L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.     

Relaxin Augments BMP‐2–Induced Osteoblast Differentiation and Bone Formation

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP‐2)‐induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT‐PCR and Western blot during BMP‐2–induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP‐2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro–computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation‐related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP‐2. In vitro, Rln augmented BMP‐2–induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP‐2–induced bone formation in a dose‐dependent manner. Interestingly, Rln synergistically increased and sustained BMP‐2–induced Smad, p38, and transforming growth factor‐β activated kinase (TAK) 1 phosphorylation. BMP‐2–induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP‐2–induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP‐2–induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases. © 2014 American Society for Bone and Mineral Research.