JBJS | BMP3: To Be or Not To Be a BMP

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 1

Scientific Article | March 01, 2001

BMP3: To Be or Not To Be a BMP

Matthew E. Bahamonde, PhD; Karen M Lyons, PhD

Departments of Biological Chemistry, Orthopaedic Surgery, and Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California
Matthew E. Bahamonde, PhD Karen M. Lyons, PhD University of California, Los Angeles, Department of Biological Chemistry, Box 956902, Los Angeles, CA 90095-6902, U.S.A.
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from NIH (NIH AR44528). None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:S56-S62

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Abstract

Background: Bone morphogenetic proteins (BMPs) are osteogenic but also have diverse functions during development. BMP3 is a major component of osteogenin, which has osteogenic activity. However, recombinant BMP3 (rhBMP3) has no apparent osteogenic function, raising the possibility that BMP3 has no bone-inducing activity or that the recombinant material is not properly processed. To resolve this apparent discrepancy, we utilized a retroviral system to study the effects of BMP3 in vitro . In addition, we generated Bmp3 -deficient mice to elucidate the function of BMP3 in vivo .

Methods: Retroviral as well as mammalian expression constructs were utilized to express BMP3 and to create BMP3 conditioned medium. Alkaline phosphatase (ALP) activity and transcriptional response assays were used to monitor the ability of BMP3 to elicit either a BMP-like or a transforming growth factor beta (TGF-ß)/activin-like response in osteoblastic cell lines. Finally, mice deficient in BMP3 were generated to investigate BMP3 function in vivo .

Results: BMP3 was unable to induce an osteogenic response in W-20-17, MC3T3-E1, or C3H10T1/2 cells, although all three cell lines were responsive to BMP2. However, BMP3 inhibited responsiveness to BMP2 in these assays, suggesting that BMP3 antagonizes BMP2 signaling. This inhibition did not occur through inhibition of binding of BMP2 to its receptors. BMP3 activated the TGF-ß/activin-pathway in these cells, suggesting that BMP3 exerts its inhibiting effects by activating a signaling pathway that antagonizes the BMP pathway. To examine the potential functional consequences of BMP3 action, Bmp3^-/- mice, which lack BMP3, were generated. On an outbred genetic background, Bmp3^-/- mice are viable and show no obvious skeletal phenotype as embryos or neonates. However, adult mice exhibit twice as much trabecular bone as do their wild-type littermates. This observation is consistent with our in vitro observations suggesting that BMP3 is an inhibitor of osteogenesis in vitro and of bone formation in vivo .

Conclusions: BMP3 is an inhibitor of osteogenic BMPs and can signal through a TGF-ß/activin pathway. The ability of BMP3 to antagonize BMP2 activity may thus be a consequence of competition for signaling components common to TGF-ß/activin and BMP pathways. BMP3, the most abundant BMP in demineralized bone, may therefore play an essential role as a modulator of the activity of osteogenic BMPs in vivo .

Clinical Relevance: Therapies to accelerate bone healing usually utilize administration of exogenous BMP either in recombinant form or by gene therapy approaches. It is conceivable that the potency of osteogenic BMPs would be increased by inhibiting the activation of antagonistic signaling pathways or by increasing levels of rate-limiting signaling components shared by both BMP and TGF-ß/activin pathways.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

The Bone Morphogenetic Proteins (BMPs) constitute a large subgroup within the transforming growth factor beta (TGF-ß) superfamily. Although they were originally identified by virtue of their ability to induce ectopic bone formation, BMPs are now known to be involved in a myriad of developmental processes. There are approximately 30 different members of the BMP subfamily, with many displaying overlapping expression patterns ⁵ . Do these overlapping patterns signify functional overlap or do different BMPs have fundamentally different signaling properties? A related question is whether different BMPs, in spite of the fact that they bind to common BMP receptors, can elicit different cellular responses as a result of differences in receptor binding affinities. These questions are relevant to BMP3, which accounts for approximately 65% of the total BMP content in demineralized bone ²⁷ . BMP3 is the major component of osteogenin, which is osteoinductive ^17,18 . However, recombinant BMP3 (rhBMP3) has failed to show any biological activity ²⁴ . A number of interpretations may explain this discrepancy. Osteogenin contains trace amounts of osteogenic BMPs that may mediate the biological activity of this material. Moreover, recombinant material may be improperly processed or folded within the cells. Finally, the bioassays employed in previous studies may have not have allowed detection of recombinant protein activity.

Almost all members of the TGF-ß superfamily activate one of two known signaling pathways ¹³ . The majority of family members activate the TGF-ß/activin pathway. The BMPs form a separate subgroup and activate a distinct BMP pathway. For both the TGF-ß/activin and BMP pathways, signaling is activated by the binding of ligand to a receptor complex. The distinction between TGF-ß/activin and BMP signaling is based on the ability of ligands to interact with type I receptors that activate one or the other pathway ¹³ . Activation of the BMP pathway induces osteogenic differentiation in vitro and new bone formation in vivo . The consequences of activating the TGF-ß/activin pathway on osteogenic differentiation are less well defined. Positive and negative effects on osteogenic cell lines in vitro have been reported for TGF-ßs and activin ^7,14 . Although TGF-ß may have synergistic effects with BMPs in ectopic bone formation, members of the TGF-ß or activin subgroups do not induce ectopic bone formation independently. BMP3 is a divergent member of the TGF-ß superfamily and cannot be grouped with the osteoinductive BMPs upon structural considerations (boxed in Fig. 1 ). This observation raises the question of whether BMP3 has signaling properties in common with the osteogenic BMPs. In an attempt to answer this question, we avoided the use of purified BMP3, which contains traces of other BMPs, and rhBMP3, which may not be folded or processed correctly. Rather, we used a retroviral system to produce BMP3 for in vitro studies and to create mice deficient in BMP3 to examine its role in vivo .

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Fig. 1:: An unrooted tree of the transforming growth factor beta (TGF-ß) superfamily. Within the upper box are the TGF-ß/activin molecules, and the lower box contains the osteogenic bone morphogenetic proteins (BMPs). BMP3 is not part of either group. (Adapted from Wozney JM et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528-34.)

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Fig. 2-A:: Figs. 2-A and 2-B Retrovirally produced bone morphogenetic protein 2 (BMP2) can induce ectopic bone formation. A No bone is evident 2 weeks after implantation with pBABEpuro-infected cells.

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Fig. 2-B:: Figs. 2-A and 2-B Retrovirally produced bone morphogenetic protein 2 (BMP2) can induce ectopic bone formation. B Ectopic bone is readily visible 2 weeks after implantation with pBABE-BMP2-infected cells. (Reprinted, with permission, from Engstrand T et al. Transient production of bone morphogenetic protein-2 by allogeneic transplanted transduced cells induces bone formation. Hum Gene Ther. 2000; 11:205-11.)

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Fig. 3-A:: Fig. 3-A Infection of W-20-17 cells with pBABE-bone morphogenetic protein (BMP)-3 reduces alkaline phosphatase activity induced by BMP2. This inhibition can be overcome with increasing amounts of rhBMP2.

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Fig. 3-B:: Fig. 3-B BMP3 inhibits BMP2 induction of a BMP-responsive element. The msx2-Lux reporter construct is activated by BMP2 when transfected into C3H10T1/2 cells. This induction is blocked in the presence of BMP3 conditioned medium.

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Fig. 3-C:: Fig. 3-C BMP3 can activate the transforming growth factor beta (TGF-ß)/activin-responsive element within the reporter construct p3TP-Lux. Induction is comparable with that by activin. (Reprinted, with permission, from Daluiski A et al. Bone morphogenetic protein 3 (BMP3) is a negative regulator of bone density. Nature Genetics. 2001; 27:84-8.)

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Fig. 4:: Histological sections of distal femoral metaphyses stained with modified Goldner's trichrome. Bone stains blue and bone marrow stains pink; cartilage is clear. More bone can be seen in the epiphyseal and metaphyseal regions in the mutant (right panel) as compared with the wild type (left panel). The lower panels are higher magnifications of the upper panels. (Reprinted, with permission, from Daluiski A et al. Bone morphogenetic protein 3 (BMP3) is a negative regulator of bone density. Nature Genetics. 2001; 27:84-8.)

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Fig. 5-A:: Figs. 5-A and 5-B Diagrams showing models of activin antagonism of bone morphogenetic protein 2 (BMP2). Fig. 5-A Signaling by both the activin and the BMP pathways requires the intracellular Smad4 protein. Incorporation of this molecule into activin-specific signaling complexes precludes its use in BMP signaling complexes, effectively inhibiting the BMP signal cascade.

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Fig. 5-B:: Figs. 5-A and 5-B Diagrams showing models of activin antagonism of bone morphogenetic protein 2 (BMP2). Fig. 5-B The activin type II receptor (ActRII) can create active signaling complexes with both activin and BMP7.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Preparation of Retroviral and Expression Constructs

Human BMP2 and BMP3 cDNAs were subcloned into the retroviral vector pBABEpuro to create pBABE-BMP2 and pBABE-BMP3. Replication-defective virus was generated by calcium phosphate cotransfection with a ? (-) packaging vector into 293T cells. Human BMP3 cDNA was also subcloned into the expression vector pCI-Neo to create pCI-BMP3.

Surgical Implantation of Transduced Cells

W-20-17 cells, infected with BMP2 viral supernatant, were implanted into the quadriceps muscles of male CD-1 mice as described previously ⁶ .

Preparation of Conditioned Medium

W-20-17 cells, a bone marrow stromal cell line, were infected with pBABEpuro, pBABE-BMP2, or pBABE-BMP3 viral supernatants. Infected cells were selected by the addition of puromycin. Resistant cells were grown in Dulbecco's modified Eagles medium (DMEM) plus 1% fetal bovine serum (FBS) for 12-16 hours. Conditioned media were concentrated at either 10 or 30-fold relative to the starting volume.

Alkaline Phosphatase Assay (ALP)

ALP assay was performed in 96-well plates as previously described ⁶ .

Transcriptional Response Assay

C3H10T1/2 or MC3T3 cells were seeded at 1 3 10 ⁵ cells per 3.5-cm well 24 hours prior to transfection. Transient cotransfection of reporter constructs, 2 kb Msx2 -Lux or p3TP-Lux, with pCI-BMP3 or pCI-Neo control along with a ß-galactosidase control was performed with use of Superfect transfection reagent as described ⁴ . Three hours after transfection, cells were incubated in conditioned media for inhibition assays or in DMEM plus 10% FBS for activation assays. Luciferase activity was assayed with the Luciferase Assay System (Promega, Madison, WI, U.S.A.) and normalized to ß-galactosidase levels. All experiments were performed in triplicate.

Targeting of the BMP3 Gene

Creation of the BMP3 targeting construct and generation of the homozygous null mice were performed as previously described ⁴ .

Radiography and Histomorphometry of BMP3 Null Mice

Femora from sex and weight-matched littermates were excised, and radiography was performed ⁴ . Histomorphometry was performed on 5-µm sections of femora embedded in methylmethacrylate, stained with modified Goldner's trichrome stain as described ⁴ .

Results

Introduction | Materials and Methods | Results | Discussion | References

To circumvent the use of rhBMP3 and osteogenin, we utilized a retroviral system to produce BMP3. W-20-17 cells infected with pBABE-BMP2 exhibited high levels of ALP activity ⁶ . Implantation of these cells into the quadriceps muscle resulted in ectopic bone formation ( Fig. 2-A , Fig. 2-B ). However, infection of W-20-17 cells with pBABE-BMP3 induced no ALP activity and did not stimulate ectopic bone formation ⁴ (data not shown).

This absence of osteogenic activity led us to investigate the function of BMP3 in the Xenopus embryo mRNA assay system. Over-expression of BMPs 2, 4, and 7 results in an embryo with a ventralized phenotype ^9,27 . On the other hand, antagonism of BMP signaling, or activation of an activin pathway, causes dorsalization ^9,27 . Injection of Xenopus embryos with BMP3 mRNA produced a mild dorsalization ⁴ , demonstrating that BMP3 has signaling properties distinct from those of osteogenic BMPs. This unexpected result is consistent with a role for BMP3 as an antagonist of BMP signaling or as an activator of TGF-ß/activin signaling pathways, or both.

To determine if BMP3 can antagonize osteogenic BMPs, we added exogenous rhBMP2 to cells infected with pBABE-BMP3. No ALP activity was seen in BMP3-producing cells, even though the dose of rhBMP2 given was enough to promote high levels of ALP in cells infected with a control virus ⁴ (data not shown). The antagonistic activity of BMP3 was dose-dependent: higher levels of rhBMP2 were able to saturate this BMP3-mediated inhibition ( Fig. 3-A ). Approximately 10 times more rhBMP2 was required to reach ALP levels comparable with those seen in the absence of BMP3.

As an additional test of BMP3 function, we employed a luciferase reporter, Msx2 -Lux, which contains 2 kb of the Msx2 gene promoter ¹⁶ . The transcription factor Msx2 is a direct target of BMP signaling ¹¹ . rhBMP2 induced an approximate 10-fold increase in luciferase activity, as compared with basal levels seen in the absence of rhBMP2. However, this induction was completely abolished by the presence of BMP3 conditioned medium ( Fig. 3-B ).

Since BMPs are dimers, one mechanism through which BMP3 could antagonize BMP2 is by the formation of inactive heterodimers. For example, activin homodimers are active but activin-inhibin heterodimers antagonize activin signaling ⁷ . However, BMP dimerization occurs intracellularly, and exogenous protein cannot form heterodimers with endogenously produced BMPs ¹⁰ . In these experiments, we added BMP2 exogenously to cells that produced BMP3 or to exogenously added BMP3 conditioned medium. Therefore, the inhibitory activity of BMP3 is unlikely to occur through formation of heterodimers of BMP2 and BMP3. It is also unlikely that BMP2 and BMP3 homodimers form larger complexes that prevent BMP2 binding to its receptors, since co-immunoprecipitation experiments failed to show any complexes of BMP2 and BMP3 (data not shown). Thus, the ability of BMP3 to inhibit BMP2 activity does not occur through regulation of the levels of BMP2 availability.

To test if BMP3 antagonism is the result of competitive, but nonproductive, binding of BMP3 to BMP2 receptors, we tested the ability of BMP3 to inhibit signaling through a constitutively active BMP type I receptor. The Drosophila Thick veins (Tkv) receptor is a type I BMP receptor that has high affinity for BMP2 ^2,21 . A constitutively active version of this receptor (CA-Tkv) can stimulate the activation of Msx2 -Lux in the absence of BMPs ¹² . If BMP3 exerts its antagonistic effects by preventing BMP2 from binding to or signaling through BMP2 receptors, then BMP3 should have no antagonistic effects on the activation of Msx2 -Lux by CA-Tkv. On the other hand, if BMP3 acts through a unique antagonistic pathway, inhibition by BMP3 should be observed in the presence of CA-Tkv. When BMP3 conditioned medium is added to cells transfected with CA-Tkv, Msx2 -Lux induction is abolished ⁴ .

These results suggest that BMP3 antagonizes BMP signaling pathways downstream of BMP2 receptors by activating a distinct signaling pathway. However, although unlikely, it is conceivable that BMP3 is capable of binding to the CA-Tkv and abolishing its constitutive activity. Therefore, to investigate if BMP3 can activate a distinct signaling pathway, we tested the ability of BMP3 to activate the TGF-ß/activin reporter construct p3TP-Lux ¹ . In the osteoblastic cell line MC3T3-E1, BMP3 can stimulate expression of p3TP-Lux whereas BMP2 has no effect ( Fig. 3-C ). Therefore, BMP3 induces the expression of TGF-ß/activin-responsive genes but not BMP-responsive genes.

The antagonistic effect of BMP3 on BMP2-induced osteogenic differentiation in vitro was unexpected, particularly since BMP3 is the most abundant BMP in demineralized bone ²⁷ . The coexpression of BMP3 and osteogenic BMPs in adult bone ²⁶ raises the possibility that this antagonism has important consequences in vivo . To test this hypothesis, we generated mice lacking Bmp3 .

Bmp3 -deficient mice on an outbred genetic background were obtained in Mendelian ratios, demonstrating that BMP3 is not essential for embryonic development on this background. Moreover, no skeletal phenotype was visible in either embryos or neonates, in spite of the expression of Bmp3 in the developing skeletal system. However, examination of adult mutants revealed a skeletal phenotype. Radiographic analysis of 5-6-week-old, sex and weight-matched Bmp3^-/- and control littermates revealed an increase in bone density in the BMP3-deficient femora of the Bmp3^-/- mutant mice ⁴ . Histological and morphometric analyses show that Bmp3^-/- mice exhibit a significant increase in trabecular metaphyseal bone density ( Fig. 4 ). No significant differences in osteoclast or osteoblast numbers were detected between wild-type and mutant littermates, and tetracycline labeling experiments failed to reveal an obvious change in the rates of bone formation ⁴ (data not shown). Therefore, the cellular basis for increased bone density in Bmp3^-/- mutants is currently unknown but may reflect subtle roles of BMP3 in multiple aspects of osteoblast or osteoclast function, or both.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

Osteogenin (BMP3 purified from bovine bone) is osteogenic ^17,18 , but this material contains trace amounts of osteogenic BMPs. On the other hand, rhBMP3 has no function in assays for osteogenic activity, raising the possibility that BMP3 is not osteogenic or that the recombinant material is not properly processed or folded, or both. Our studies circumvented these problems by using retroviral and mammalian expression constructs to produce BMP3.

One advantage of the use of expression vectors for BMPs over application of recombinant protein is that the levels of induction of osteogenic markers such as ALP can be significantly higher from BMP-expressing cells than is seen by treatment with recombinant BMP. This effect is observed despite the fact that protein amounts recovered from infected cells are less than the amount of recombinant protein used to induce bone ⁶ . This is likely due in part to the fact that the biologically active fraction in recombinant protein may make up only a small amount of the total. This would effectively mean that the active dose is much smaller than the actual dose. Moreover, production and secretion of BMPs within osteogenic cells may facilitate the ability of the BMPs to bind to BMP receptors. Finally, a small but steady stream of biologically active protein might induce osteogenesis at consistent levels over a longer time period than can be achieved by a single dose of recombinant protein. In spite of these advantages, we were unable to detect any evidence that BMP3, the most abundant BMP in demineralized bone, has any osteogenic activity. Rather, our experiments indicate that BMP3 antagonizes the ability of BMP2 to induce osteogenic commitment and differentiation in vitro.

We have found that BMP3 is an inhibitor of osteogenic commitment and differentiation in vitro and of bone density in vivo . BMP3 is a highly divergent member of the BMP subfamily ( Fig. 1 ), sharing only 50% identity in amino acid sequence with osteogenic BMPs ^24,26 . BMP3 appears to act through a TGF-ß or activin-like pathway, since BMP3 can induce a TGF-ß/activin-responsive reporter construct but not a BMP-responsive construct. An important caveat of our experiments is that we cannot say anything regarding concentration effects. Many TGF-ß family members are biphasic; TGF-ß inhibits or stimulates osteoblast differentiation and function, depending on the cell system and culture conditions ⁷ . TGF-ßs and BMPs have been demonstrated to have synergistic effects in some circumstances ²³ . It is unknown whether BMP3 acts in a similar biphasic manner, as we are not yet able to purify and quantify the BMP3 present in our conditioned medium. However, in vitro , BMP3 inhibition is seen in multiple osteogenic cell lines such as W-20-17, C3H10T1/2, and MC3T3 over a range of tested amounts of BMP3 conditioned medium. Similarly, TGF-ß has been shown to inhibit osteoblast differentiation in vitro^14,15 .

Our results suggest that regulation of adult bone density involves modulation of BMP signaling output by way of the ability of BMP3 to activate TGF-ß/activin pathways. Activin is known to antagonize BMP signaling by at least two different mechanisms . Both activins and BMPs employ the common downstream element Smad4 as part of their respective signal transduction cascades ( Fig. 5-A ). Competition for the finite pool of Smad4 present in a cell limits signaling output ³ . The activin pathway also shares type II receptors with the BMP pathway. For example, the activin type IIB receptor can bind multiple BMPs and activin ¹³ . Depending on the type I receptor recruited to the complex by either activin or BMP ligands, these type II receptors can transmit either an activin or BMP response, respectively. Thus, activin signaling leads to incorporation of ActRII into activin-responsive receptor complexes, thereby reducing the levels of ActRII available for formation of BMP signaling receptor complexes ( Fig. 5-B ) ²² . Consistent with this interpretation, antagonism between activin and BMP7 can be overcome through over-expression of ActRII ²² . Mice deficient in ActRII have a low penetrance of skeletal and facial abnormalities, which are distinct from the defects seen in mice lacking the ligand activin ¹⁹ . This implies that another ligand, possibly a BMP, functions through the ActRII receptor to specify these structures. The second activin type II receptor, ActRIIB, is responsible for the proper patterning of the vertebrae through the specification of Hox genes. This developmental patterning may be due to BMP signaling through ActRIIB to activate Hox gene expression ²⁰ . Potential differences in bone density have not been reported for these mutant strains.

Our data suggest that this competition between activin and BMP signaling pathways, either at the level of the receptor or downstream at the level of Smad4, may be important for the regulation of bone density in vivo . Osteogenic BMPs are extremely potent and can cause respecification of a variety of mesenchymal cell types to the osteogenic pathway. Therefore, the presence of BMP3 may permit the accumulation of osteogenic BMPs to be utilized for new bone formation during normal remodeling or fracture repair, or both, while preventing inappropriate BMP activity in vivo . Therefore, it is conceivable that TGF-ß/activin and BMP signaling pathways have an antagonistic relationship in some aspects of bone formation and remodeling and synergistic relationships in other aspects.

The cell types responsible for the phenotype of Bmp3^-/- mice are currently unknown. No differences were seen in either osteoclast or osteoblast number. Mineral apposition rates as detected by fluorochrome labeling were similar between mutants and wild types ⁴ . Although TRAP staining revealed no significant difference in osteoclast numbers ⁴ , we do not know if osteoclast function is impaired. Subtle effects on bone turnover may be undetectable in our assays, yet may, on balance, lead to the increased bone density seen in our mutants. These results are similar to those reported for mice expressing an osteoblast-specific dominant-negative TGF-ß type II receptor. These mice experience an age-dependent increase in trabecular bone mass, while the rate of osteoblastic bone formation is unaltered ⁷ . Thus, in this outbred strain, impairment of signaling through the TGF-ß/activin pathway leads to increased bone density. The phenotype of Bmp3^-/- mice is consistent with a role for BMP3 as an activator of TGF-ß/activin signaling pathways in this process in vivo . This line of reasoning is reinforced by the fact that BMP3 can activate a TGF-ß/activin-responsive element in osteoblastic cell lines.

Although BMP3 itself may be of limited value as a therapeutic agent, these results suggest that a modulating signaling output by BMP3 or activin may increase the signaling capacity of the BMP pathway. This could increase the potency of either recombinant or virally produced osteogenic BMPs, allowing for more efficient bone formation by smaller doses of BMP. A more complete understanding of the roles of BMP3 and TGF-ß/activin signaling pathways and their cellular targets in vivo will be required to determine whether this approach can be utilized therapeutically.

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Topics

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DucyP,Karsenty G. The family of bone morphogenetic proteins. Kidney Int,7: 2207-S2214. 2000;72207 2000

HoodlessPA, Haerry T, Abdollah S, Stapleton M, O'Connor MB, Attisano L,Wrana JL. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell,85: 489-S500. 1996;85489 1996 [PubMed]

KawabataM, Imamura T,Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev,9: 49-S61. 1998;949 1998 [PubMed]

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Matthew E. Bahamonde, Karen M Lyons; BMP3: To Be or Not To Be a BMP. The Journal of Bone & Joint Surgery. 2001 Mar;83(1_suppl_1):S56-S62.

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