Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.     

Increased PLEKHO1 within osteoblasts suppresses Smad‐dependent BMP signaling to inhibit bone formation during aging

Emerging evidence indicates that the dysregulation of protein ubiquitination plays a crucial role in aging‐associated diseases. Smad‐dependent canonical BMP signaling pathway is indispensable for osteoblastic bone formation, which could be disrupted by the ubiquitination and subsequent proteasomal degradation of Smad1/5, the key molecules for BMP signaling transduction. However, whether the dysregulation of Smad1/5 ubiquitination and disrupted BMP signaling pathway is responsible for the age‐related bone formation reduction is still underexplored. Pleckstrin homology domain‐containing family O member 1 (PLEKHO1) is a previously identified ubiquitination‐related molecule that could specifically target the linker region between the WW domains of Smurf1 to promote the ubiquitination of Smad1/5. Here, we found an age‐related increase in the expression of PLEKHO1 in bone specimens from either fractured patients or aging rodents, which was associated with the age‐related reduction in Smad‐dependent BMP signaling and bone formation. By genetic approach, we demonstrated that loss of Plekho1 in osteoblasts could promote the Smad‐dependent BMP signaling and alleviated the age‐related bone formation reduction. In addition, osteoblast‐specific Smad1 overexpression had beneficial effect on bone formation during aging, which could be counteracted after overexpressing Plekho1 within osteoblasts. By pharmacological approach, we showed that osteoblast‐targeted Plekho1 siRNA treatment could enhance Smad‐dependent BMP signaling and promote bone formation in aging rodents. Taken together, it suggests that the increased PLEKHO1 could suppress Smad‐dependent BMP signaling to inhibit bone formation during aging, indicating the translational potential of targeting PLEKHO1 in osteoblast as a novel bone anabolic strategy for reversing established osteoporosis during aging.     

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C₂-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C₂-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.     

Chondrocytes Transdifferentiate into Osteoblasts in Endochondral Bone during Development,Postnatal Growth and Fracture Healing in Mice

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.     

Hedgehog-Gli Activators Direct Osteo-chondrogenic Function of Bone Morphogenetic Protein toward Osteogenesis in the Perichondrium

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1^−/− fetuses, and the phenotype was more severe in Gli1^−/−;Gli2^−/− newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.     

Bone morphogenetic protein receptor IB signaling mediates apoptosis independently of differentiation in osteoblastic cells

Bone morphogenetic protein-2 (BMP-2) is an important regulator of osteoblast differentiation. However, the regulation of osteoblast apoptosis by BMP signaling remains poorly understood. Here we examined the role of type I BMP receptor (BMP-RI) in osteoblast apoptosis promoted by BMP-2. Despite undetectable BMP-RIB expression in OHS4 cells, BMP-2 or BMP-2 overexpression increased osteoblast differentiation similarly as in SaOS2 cells which express BMP-RIB, as shown by alkaline phosphatase and CBFA1/RUNX2 expression. In contrast to SaOS2 cells, however, BMP-2 or BMP-2 overexpression did not increase caspase-9 and caspases-3, -6, and -7 activity and DNA fragmentation in OHS4 cells. Consistently, BMP-2 increased protein kinase C (PKC) activity, and PKC inhibition suppressed BMP-2-induced caspase activity in SaOS2 but not in OHS4 cells that lack BMP-RIB. A dominant negative BMP-RIB inhibited BMP-2-induced caspase activity, whereas wild-type BMP-RIB promoted caspase activity induced by BMP-2 in SaOS2 and MC3T3-E1 cells. Wild-type BMP-RIB rescued the apoptotic response to BMP-2, and a constitutively active BMP-RIB restored the apoptotic signal in OHS4 cells, supporting an essential role for BMP-RIB in osteoblast apoptosis. We also assessed whether BMP-2-induced apoptosis occurred independently of osteoblast differentiation. General inhibition of caspases did not abolish BMP-2-induced alkaline phosphatase and CBFA1/RUNX2 expression in SaOS2 cells. Furthermore, broad caspases inhibition increased matrix mineralization but did not reverse the BMP-2 effect on mineralization in MC3T3-E1 cells. These results indicate that BMP-2-induced apoptosis was mediated by BMP-RIB in osteoblasts and occurred independently of BMP-2-induced osteoblast differentiation, which provides additional insights into the dual mechanism of BMP-2 action on osteoblast fate.     

The TBC (Tre-2/Bub2/Cdc16) domain protein TRE17 regulates plasma membrane-endosomal trafficking through activation of Arf6

TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab family G proteins. While approximately 50 TBC proteins are predicted to exist in humans, little is known about their substrate specificity. Here we show that TRE17 (also called Tre-2 and USP6), a founding member of the TBC family, targets the Arf family GTPase Arf6, which regulates plasma membrane-endosome trafficking. Surprisingly, TRE17 does not function as a GAP for Arf6 but rather promotes its activation in vivo. TRE17 associates directly with Arf6 in its GDP- but not GTP-bound state. Mapping experiments pinpoint the site of interaction to the TBC domain of TRE17. Forced expression of TRE17 promotes the localization of Arf6 to the plasma membrane, leading to Arf6 activation, presumably due to facilitated access to membrane-associated guanine nucleotide exchange factors (GEFs). Furthermore, TRE17 cooperates with Arf6 GEFs to induce GTP loading of Arf6 in vivo. Finally, short interfering RNA-mediated loss of TRE17 leads to attenuated Arf6 activation. These studies identify TRE17 as a novel regulator of the Arf6-regulated plasma membrane recycling system and reveal an unexpected function for TBC domains.     

Bone Morphogenetic Proteins

Bone Morphogenetic Protein (BMP) refers to an activity derived from bone that induces the formation of cartilage and bone in vivo. This activity leads to a series of developmental processes including chemotaxis, proliferation, and differentiation, which result in the transient formation of cartilage and the production of living bone tissue, complete with hematopoietic marrow. The determination of the factor or factors responsible for this activity has clear significance both for our understanding of bone biology and for the clinical application of cartilage and bone replacement. Several newly discovered factors, BMP-1, BMP-2 (BMP-2A), BMP-3 (osteogenin), BMP-4 (BMP-2B), BMP-5, BMP-6, BMP-7, and osteoinductive factor (OIF) have been implicated in the BMP process. BMP-2 through BMP-7 are all in the TGF-β superfamily of molecules, and are closely related to two factors (Vg1 and dpp) which are involved in a variety of developmental processes during embryogenesis. A recently discovered factor, OIF, exhibits BMP activity only in the presence of TGF-β. BMP-2, expressed as a recombinant protein, is the only molecule described to date that has been shown to clearly induce by itself the entire cartilage and bone formation process seen with bone-derived BMP. Evidence is accumulating that the BMP effect is a result of the combined actions of a set of BMP-2-like molecules. Definitive examination of the activities of the other factors will require expression of the recombinant proteins and testing of these in vivo alone and in combinations.     

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3) which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophila aristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp), Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.     

Sympathetic neuron can promote osteoblast differentiation through BMP signaling pathway

Communication between sympathetic neurons and osteoblasts through the adrenergic receptor pathway has already been reported. To investigate whether the sympathetic neurons have a direct effect on osteoblast differentiation, an in vitro Transwell coculture system was established in which osteoblasts were cocultured with sympathetic neurons with no cell-to-cell contact. The expression of osteogenesis-related genes was upregulated in osteoblasts cocultured with sympathetic neurons. Meanwhile, bone morphogenetic protein (BMP) mRNA and protein expressions were detected in sympathetic neurons, and BMP secretion from sympathetic neurons was also confirmed. However, transfection with BMP-2 and/or BMP-6 siRNA in sympathetic neurons caused a down-regulation of osteogenesis-related genes in the cocultured osteoblasts. Sympathetic neurons promoted osteoblast differentiation through BMP signaling pathway, implying that the integrity of sympathetic neurons was important for optimal bone formation and remodeling.