Class I PI‐3‐Kinase Signaling Is Critical for Bone Formation Through Regulation of SMAD1 Activity in Osteoblasts

Bone formation and homeostasis is carried out by osteoblasts, whose differentiation and activity are regulated by osteogenic signaling networks. A central mediator of these inputs is the lipid kinase phosphatidylinositol 3‐kinase (PI3K). However, at present, there are no data on the specific role of distinct class IA PI3K isoforms in bone biology. Here, we performed osteoblast‐specific deletion in mice to show that both p110α and p110β isoforms are required for survival and differentiation and function of osteoblasts and thereby control bone formation and postnatal homeostasis. Impaired osteogenesis arises from increased GSK3 activity and a depletion of SMAD1 protein levels in PI3K‐deficient osteoblasts. Accordingly, pharmacological inhibition of GSK3 activity or ectopic expression of SMAD1 or SMAD5 normalizes bone morphogenetic protein (BMP) transduction and osteoblast differentiation. Together, these results identify the PI3K‐GSK3‐SMAD1 axis as a central node integrating multiple signaling networks that govern bone formation and homeostasis. © 2016 American Society for Bone and Mineral Research.     

BMP9 Reduces Bone Loss in Ovariectomized Mice by Dual Regulation of Bone Remodeling

Bone remodeling is dynamic and is tightly regulated through bone resorption dominated by osteoclasts and bone formation dominated by osteoblasts. Imbalances in this process can cause various pathological conditions, such as osteoporosis. Bone morphogenetic protein 9 (BMP9), a biomolecule produced and secreted by the liver, has many pharmacological effects, including anti-liver fibrosis, antitumor, anti-heart failure, and antidiabetic activities. However, the effects of BMP9 on the regulation of osteoblast and osteoclast functions and the underlying molecular mechanism(s) have not yet been investigated. In this study, BMP9 increased the expression of osteoblastogenic gene markers, such as ALP, Cola1, OCN, RUNX2, and OSX, and ALP activity in MC3T3-E1 cells by upregulating LGR6 and activating the Wnt/β-catenin pathway. BMP9 also suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages (BMMs) by inhibiting the Akt-NF-κB-NFATc1 pathway. More importantly, in an ovariectomy (OVX) mouse model, BMP9 attenuated bone loss and improved bone biomechanical properties in vivo by increasing bone-forming activity and suppressing bone resorption activity. Accordingly, our current work highlights the dual regulatory effects that BMP9 exerts on bone remodeling by promoting bone anabolic activity and inhibiting osteoclast differentiation in OVX mice. © 2020 American Society for Bone and Mineral Research.     

Liver X receptors orchestrate osteoblast/osteoclast crosstalk and counteract pathologic bone loss

Osteoporosis is characterized by enhanced differentiation of bone‐resorbing osteoclasts, resulting in a rapid loss of functional trabecular bone. Bone‐forming osteoblasts and osteoblast‐derived osteocytes perform a key role in the regulation of osteoclast development by providing both the pro‐osteoclastogenic cytokine receptor activator of NF‐κB ligand (RANKL) and its natural decoy receptor osteoprotegerin (OPG). By regulating the RANKL/OPG ratio, osteoblasts hence determine the rate of both osteoclast differentiation and bone turnover. Here, we describe a novel role for liver X receptors (LXRs) during the crosstalk of bone‐forming osteoblasts and bone‐resorbing osteoclasts. By using a system of osteoblast/osteoclast cocultures, we identify LXRs as regulator of RANKL expression and the RANKL/OPG ratio in osteoblasts. Activation of LXRs drastically reduced the RANKL/OPG ratio and interfered with osteoblast‐mediated osteoclast differentiation in vitro. During an ovariectomy (OVX)‐induced model of postmenopausal osteoporosis, the application of an LXR agonist shifted the RANKL/OPG ratio in vivo, ameliorated the enhanced osteoclast differentiation, and provided complete protection from OVX‐induced bone loss. These results reveal an unexpected involvement of LXRs in the regulation of bone turnover and highlight a potential role for LXRs as novel targets in the treatment of osteoporosis and related diseases. © 2012 American Society for Bone and Mineral Research.     

TAK1 regulates cartilage and joint development via the MAPK and BMP signaling pathways

The importance of canonical transforming growth factor β (TGF‐β) and bone morphogenetic protein (BMP) signaling during cartilage and joint development is well established, but the necessity for noncanonical (SMAD‐independent) signaling during these processes is largely unknown. TGF‐β activated kinase 1 (TAK1) is a MAP3K activated by TGF‐β, BMP, and other mitogen‐activated protein kinase (MAPK) signaling components. We set out to define the potential role for noncanonical, TAK1‐mediated signaling in cartilage and joint development via deletion of Tak1 in chondrocytes (Col2Cre;Tak1^f/f) and the developing limb mesenchyme (Prx1Cre;Tak1^f/f). Deletion of Tak1 in chondrocytes resulted in novel embryonic developmental cartilage defects including decreased chondrocyte proliferation, reduced proliferating chondrocyte survival, delayed onset of hypertrophy, reduced Mmp13 expression, and a failure to maintain interzone cells of the elbow joint, which were not observed previously in another Col2Cre;Tak1^f/f model. Deletion of Tak1 in limb mesenchyme resulted in widespread joint fusions likely owing to the differentiation of interzone cells to the chondrocyte lineage. The Prx1Cre;Tak1^f/f model also allowed us to identify novel columnar chondrocyte organization and terminal maturation defects owing to the interplay between chondrocytes and the surrounding mesenchyme. Furthermore, both our in vivo models and in vitro cell culture studies demonstrate that loss of Tak1 results in impaired activation of the downstream MAPK target p38, as well as diminished activation of the BMP/SMAD signaling pathway. Taken together, these data demonstrate that TAK1 is a critical regulator of both MAPK and BMP signaling and is necessary for proper cartilage and joint development. © 2010 American Society for Bone and Mineral Research     

Antagonism of the Met5‐enkephalin‐opioid growth factor receptor‐signaling axis promotes MSC to differentiate into osteoblasts

Chronic opioid therapy is associated with bone loss. This led us to hypothesize that the opioid antagonists, that include naloxone, would stimulate bone formation by regulating MSC differentiation. The opioid growth factor receptor (OGFR) is a non‐canonical opioid receptor that binds naloxone with high affinity whereas the native opioid growth factor, met5‐enkephalin (met5), binds both the OGFR and the canonical delta opioid receptor (OPRD). Naloxone and an shRNA OGFR lentivirus were employed to disrupt the OGFR‐signaling axis in cultured MSC. In parallel, naloxone was administered to bone marrow using a mouse unicortical defect model. OPRD, OGFR, and the met5‐ligand were highly expressed in MSC and osteoblasts. A pulse‐dose of naloxone increased mineral formation in MSC cultures in contrast to MSC treated with continuous naloxone or OGFR deficient MSC. Importantly, SMAD1 and SMAD8/9 expression increased after a pulse dose of naloxone whereas SMAD1, SMAD7, and ID1 were increased in the OGFR deficient MSC. Inhibited OGFR signaling decreased proliferation and increased p21 expression. The addition of naloxone to the unicortical defect resulted in increased bone formation within the defect. Our data suggest that novel mechanism through which signaling through the OGFR regulates osteogenesis via negative regulation of SMAD1 and p21. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1195–1205, 2016.     

Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts

The bone morphogenetic protein (BMP) and Wnt signaling pathways both contribute essential roles in regulating bone mass. However, the molecular interactions between these pathways in osteoblasts are poorly understood. We recently reported that osteoblast‐targeted conditional knockout (cKO) of BMP receptor type IA (BMPRIA) resulted in increased bone mass during embryonic development, where diminished expression of Sost as a downstream effector of BMPRIA resulted in increased Wnt/β‐catenin signaling. Here, we report that Bmpr1a cKO mice exhibit increased bone mass during weanling stages, again with evidence of enhanced Wnt/β‐catenin signaling as assessed by Wnt reporter TOPGAL mice and TOPFLASH luciferase. Consistent with negative regulation of the Wnt pathway by BMPRIA signaling, treatment of osteoblasts with dorsomorphin, an inhibitor of Smad‐dependent BMP signaling, enhanced Wnt signaling. In addition to Sost, Wnt inhibitor Dkk1 also was downregulated in cKO bone. Expression levels of Dkk1and Sost were upregulated by BMP2 treatment and downregulated by Noggin. Moreover, expression of a constitutively active Bmpr1a transgene in mice resulted in the upregulation of both Dkk1 and Sost and partially rescued the Bmpr1a cKO bone phenotype. These effectors are differentially regulated by mitogen‐activated protein kinase (MAPK) p38 because pretreatment of osteoblasts with SB202190 blocked BMP2‐induced Dkk1 expression but not Sost. These results demonstrate that BMPRIA in osteoblasts negatively regulates endogenous bone mass and Wnt/β‐catenin signaling and that this regulation may be mediated by the activities of Sost and Dkk1. This study highlights several interactions between BMP and Wnt signaling cascades in osteoblasts that may be amenable to therapeutic intervention for the modification of bone mass density. © 2010 American Society for Bone and Mineral Research     

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

We identified the neuroprotein collapsing response mediator protein‐4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1–CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain‐of‐function and loss‐of‐function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4‐deficient mice (Crmp4^–/–) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild‐type controls. Increased bone mass in Crmp4^–/– mice was associated with enhanced BMP2 signaling and BMP2‐induced osteoblast differentiation in Crmp4^–/– osteoblasts (OBs). Furthermore, Crmp4^–/– OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4^–/– OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin‐dependent kinase inhibitor 1B, p27^Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.     

Suppression of p38α MAPK Signaling in Osteoblast Lineage Cells Impairs Bone Anabolic Action of Parathyroid Hormone

Intermittent parathyroid hormone administration (iPTH) increases bone mass and strength by stimulating osteoblast number and activity. PTH exerts its anabolic effects through cAMP/protein kinase A (PKA) signaling pathway in mature osteoblasts and osteocytes. Here, we show that inactivation of the p38α MAPK‐encoding gene with the use of an osteocalcin‐cre transgene prevents iPTH bone anabolic action. Indeed, iPTH fails to increase insulin‐like growth factor 1 expression, osteoblast number and activity, and bone formation in mice lacking p38α in osteoblasts and osteocytes. Moreover, iPTH‐induced expression of receptor activator of NF‐κB ligand (RANKL) and subsequent increased bone resorption are suppressed in those mice. Finally, we found that PTH activates p38α MAPK downstream of cAMP/PKA signaling pathway in mature osteoblasts. Our findings identify p38α MAPK as a key component of PTH signaling in osteoblast lineage cells and highlight its requirement in iPTH osteoanabolic activity. © 2015 American Society for Bone and Mineral Research.     

Osteosclerosis owing to Notch gain of function is solely Rbpj‐dependent

Osteosclerosis is a pathologic bone disease characterized by an increase in bone formation over bone resorption. Genetic factors that contribute to the pathogenesis of this disease are poorly understood. Dysregulation or mutation in many components of the Notch signaling pathway results in a wide range of human developmental disorders and cancers, including bone diseases. Our previous study found that activation of the Notch signaling in osteoblasts promotes cell proliferation and inhibits differentiation, leading to an osteosclerotic phenotype in transgenic mice. In this study we report a longer‐lived mouse model that also develops osteosclerosis and a genetic manipulation that completely rescues the phenotype. Conditionally cre‐activated expression of Notch1 intracellular domain (NICD) in vivo exclusively in committed osteoblasts caused massive osteosclerosis with growth retardation and abnormal vertebrae. Importantly, selective deletion of a Notch nuclear effector—Rbpj—in osteoblasts completely suppressed the osteosclerotic and growth‐retardation phenotypes. Furthermore, cellular and molecular analyses of bones from the rescued mice confirmed that NICD‐dependent molecular alterations in osteoblasts were completely reversed by removal of the Rbpj pathway. Together, our observations show that the osteosclerosis owing to activation of Notch signaling in osteoblasts is canonical in nature because it depends solely on Rbpj signaling. As such, it identifies Rbpj as a specific target for manipulating Notch signaling in a cell‐autonomous fashion in osteoblasts in bone diseases where Notch may be dysregulated. © 2010 American Society for Bone and Mineral Research.     

Planar Cell Polarity Aligns Osteoblast Division in Response to Substrate Strain

Exposure of bone to dynamic strain increases the rate of division of osteoblasts and also influences the directional organization of the cellular and molecular structure of the bone tissue that they produce. Here, we report that brief exposure to dynamic substrate strain (sufficient to rapidly stimulate cell division) influences the orientation of osteoblastic cell division. The initial proliferative response to strain involves canonical Wnt signaling and can be blocked by sclerostin. However, the strain‐related orientation of cell division is independently influenced through the noncanonical Wnt/planar cell polarity (PCP) pathway. Blockade of Rho‐associated coiled kinase (ROCK), a component of the PCP pathway, prevents strain‐related orientation of division in osteoblast‐like Saos‐2 cells. Heterozygous loop‐tail mutation of the core PCP component van Gogh‐like 2 (Vangl2) in mouse osteoblasts impairs the orientation of division in response to strain. Examination of bones from Vangl2 loop‐tail heterozygous mice by µCT and scanning electron microscopy reveals altered bone architecture and disorganized bone‐forming surfaces. Hence, in addition to the well‐accepted role of PCP involvement in response to developmental cues during skeletal morphogenesis, our data reveal that this pathway also acts postnatally, in parallel with canonical Wnt signaling, to transduce biomechanical cues into skeletal adaptive responses. The simultaneous and independent actions of these two pathways appear to influence both the rate and orientation of osteoblast division, thus fine‐tuning bone architecture to meet the structural demands of functional loading. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.     

NVP‐BEZ235, a dual pan class I PI3 kinase and mTOR inhibitor,promotes osteogenic differentiation in human mesenchymal stromal cells

Osteoblasts are bone‐forming cells derived from mesenchymal stromal cells (MSCs) that reside within the bone marrow. In response to a variety of factors, MSCs proliferate and differentiate into mature, functional osteoblasts. Several studies have shown previously that suppression of the PI3K and mTOR signaling pathways in these cells strongly promotes osteogenic differentiation, which suggests that inhibitors of these pathways may be useful as anabolic bone agents. In this study we examined the effect of BEZ235, a newly developed dual PI3K and mTOR inhibitor currently in phase I–II clinical trials for advanced solid tumors, on osteogenic differentiation and function using primary MSC cultures. Under osteoinductive conditions, BEZ235 strongly promotes osteogenic differentiation, as evidenced by an increase in mineralized matrix production, an upregulation of genes involved in osteogenesis, including bone morphogenetic proteins (BMP2, ‐4, and ‐6) and transforming growth factor β1 (TGF‐β1) superfamily members (TGFB1, TGFB2, and INHBE), and increased activation of SMAD signaling molecules. In addition, BEZ235 enhances de novo bone formation in calvarial organotypic cultures. Using pharmacologic inhibitors to delineate mechanism, our studies reveal that suppression of mTOR and, to a much lesser extent PI3K p110α, mediates the osteogenic effects of BEZ235. As confirmation, shRNA‐mediated knockdown of mTOR enhances osteogenic differentiation and function in SAOS‐2 osteoblast‐like cells. Taken together, our findings suggest that BEZ235 may be useful in treating PI3K/mTOR‐dependent tumors associated with bone loss, such as the hematologic malignancy multiple myeloma. © 2010 American Society for Bone and Mineral Research.     

Systemic Injection of CK2.3, a Novel Peptide Acting Downstream of Bone Morphogenetic Protein Receptor BMPRIa,Leads to Increased Trabecular Bone Mass

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal‐related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.© 2014 Orthopaedic Research Society. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:208–215, 2015.

     

Estrogen receptor α36 mediates a bone‐sparing effect of 17β‐estrodiol in postmenopausal women

Recently, a membrane‐based estrogen receptor (ER), ER‐α36, was identified and cloned that transduces membrane‐initiated estrogen signaling such as activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signaling pathway. Here we show that the postmenopausal level of estradiol (E2) induces mitogenic, antiapoptotic, and antiosteogenic effects and proapoptotic effects in postmenopausal osteoblasts and osteoclasts with high levels of ER‐α36 expression, respectively. We also found that ER‐α36 mediated the effects of postmenopausal‐level E₂ on proliferation, apoptosis, and differentiation of osteoblasts through transient activation of the MAPK/ERK pathway, whereas ER‐α36‐mediated postmenopausal‐level E₂ induces apoptosis of osteoclasts through prolonged activation of the MAPK/ERK pathway with the involvement of reactive oxygen species. We also show that the levels of ER‐α36 expression in bone are positively associated with bone mineral density but negatively associated with bone biochemical markers in postmenopausal women. Thus the higher levels of ER‐α36 expression are required for preserving bone mass in postmenopausal and menopausal women who become osteoporotic if ER‐α36‐mediated activities are dysregulated. © 2011 American Society for Bone and Mineral Research.     

Origin matters: Differences in embryonic tissue origin and Wnt signaling determine the osteogenic potential and healing capacity of frontal and parietal calvarial bones

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest–derived frontal and paraxial mesodermal–derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest–derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal–derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest–derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF‐2) treatment induces phosphorylation of GSK‐3β and increases the nuclear levels of β‐catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest–derived frontal bone. © 2010 American Society for Bone and Mineral Research     

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.     

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.