The signaling and functions of heterodimeric bone morphogenetic proteins

Cytokine & Growth Factor Reviews 23 (2012) 61–67

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Mini review

The signaling and functions of heterodimeric bone morphogenetic proteins Jing Guo a,1, Gang Wu b,* a

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), Research Institute MOVE, VU University and University of Amsterdam, Gustav Mahlerlaan 3004, 1018LA Amsterdam, The Netherlands b Department of Oral Implantology and Prosthetic Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), Research Institute MOVE, VU University and University of Amsterdam, Gustav Mahlerlaan 3004, 1018LA Amsterdam, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history: Available online 13 March 2012

Heterodimeric bone morphogenetic proteins (BMPs) consist of disulfide-linked dimeric monomers derived from different BMP members. Owing to this specific constitution pattern, they bear high affinity to both type I and type II BMP receptors simultaneously. Meanwhile, the antagonism efficiency of extracellular antagonists to heterodimeric BMPs is also significantly lower than that to homodimeric ones. All these specific properties confer heterodimeric BMPs with distinct signaling and bio-functions that are characterized by more speediness, lower concentration/dose threshold and higher efficiency than homodimeric BMPs. Consequently, heterodimeric BMPs bear promising application potential in inducing osteogenesis. In addition, they may play indispensible roles in organogenesis. In this review, we summarize the current knowledge of heterodimeric BMPs in their signaling pathways and bio-functions. ß 2012 Elsevier Ltd. All rights reserved.

Keywords: Heterodimeric Homodimeric Heterodimer Bone morphogenetic protein Bone Signaling

1. Heterodimeric BMPs The discovery of bone morphogenetic proteins (BMPs) in the pioneering work by Urist in 1965 [1] is a landmark in the development of bone tissue engineering. The classical role for BMPs was considered the induction of (ectopic) cartilage and bone formation [1,2]. Owing to the continuous efforts in the past half century, BMPs are currently recognized as a group of metabologens that constitute pivotal morphogenetic signals and orchestrate tissue architecture throughout the body [3]. The BMP family belongs to TGF-b (transforming growth factorb) superfamily and consists of more than 30 members [4] (Table 1). Among them, Dpp, Gbb, and Scw are identified in Drosophila, Univin is found in sea urchin, and Vg1 is found in Xenopus. The others are found in mammals. Some BMP members have different names: next to BMPs, they are called osteogenic proteins (OPs), cartilage-derived morphogenetic proteins (CDMPs), and growth and differentiation factors (GDFs). In human, 19 BMP members are under the designation of BMPs. According to their gene homology, protein structure and functions, the 19 members are further subdivided into 7 subgroups: BMP2/4, BMP3/3b, BMP5/6/7/8/8b, BMP9/10, BMP11/GDF8, BMP12/13/14 and BMP15/GDF9 [5,6] (Table 1).

* Corresponding author. Tel.: +31 020 598 0866; fax: +31 020 598 0333. E-mail addresses: [email protected] (J. Guo), [email protected] (G. Wu). 1 Tel.: +31 020 598 0870; fax: +31 020 598 0333. 1359-6101/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2012.02.001

Most of the mature BMP molecules (except GDF3, 9, 9B [7,8]) consist of two monomers that are covalently linked with a disulphide bond [6]. When the two monomers in one ligand are derived from the same BMP member, the BMP ligand is termed ‘‘homodimeric BMP’’ or BMP homodimer. The present knowledge of BMPs are largely based on these homodimeric BMPs. Heterodimeric BMPs consist of two monomers derived from different BMP members. Heterodimeric BMPs were discovered in 1988 by Wang et al. [9]. From bovine bone, they extracted a BMP with a molecular mass of 30 kDa, which yielded proteins of 30, 18 and 16 kDa upon reduction. This BMP exhibited very high dose efficiency in inducing new bone formation in vivo. Owing to several breakthroughs in the identification of human BMP genes, e.g., BMP1, BMP2A, BMP3 [2] and BMP7 [10], Sampath et al. indicated that this extracted BMP consisted of heterodimerized monomers BMP2 and BMP7 [11]. Thereafter, gene technology was adopted to produce heterodimeric BMPs in large amounts and high efficiency [12–14]. Similar to the naturally occurring proteins, these recombinant heterodimeric BMPs also induced in-vitro and invivo osteogenesis in a significantly higher dose efficiency and lower concentration threshold than the respective homodimeric BMPs [13,14]. A heterodimeric BMP ligand may contain two monomers derived from the same or different subgroups. It seems that the efficiency by which BMPs exert their activity is associated with the homology between the two monomers: the less gene and protein homology the two monomers bear, the higher osteoinductive efficiency the heterodimeric BMP can exhibit [15,16]. Thus, the efficiency of heterodimeric BMPs containing two different monomers derived

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Table 1 Species, suggested subgroups of bone morphogenetic protein (BMP) members (OP: osteogenic protein, CDMP: cartilage-derived morphogenetic protein; GDF: growth and differentiation factor). Species

Subgroups

Mammals

Inhibin b BMP2/4 BMP5/6/7/8/8b CDMP1/2/3 BMP9/10 BMP3/3b BMP11/GDF8 BMP15/GDF9

Drosophila

Xenopus Sea urchin

BMP members GDNF Mullerian inhibiting substance Inhibin a Inhibin bA, Inhibin bB, Inhibin bC, Inhibin bD BMP2, BMP4 BMP5, BMP6, BMP7(OP1), BMP8(OP2), BMP8b(OP3) BMP12 (GDF7/CDMP3), BMP13 (GDF6/CDMP2), BMP14 (GDF5/CDMP1) BMP9 (GDF2), BMP10 BMP3, BMP3b (GDF10) BMP11 (GDF11), GDF8 BMP-15(GDF9b)/GDF9 GDF 3 GDF 1 Nodal Gbb Dpp Scw Vg1 Univin

from the same subgroup (less homology) is higher than that of homodimeric BMPs (full homology). Analogously, the efficiency of heterodimeric BMPs containing two monomers from different subgroups (least homology) is higher than that of heterodimeric BMPs containing two monomers from the same subgroups (less homology). Consequently, most studies in the field of bone tissue engineering focus on the heterodimeric BMPs that consist of two monomers from different subgroups, particularly of BMP2/4 and BMP5/6/7/8/8b subgroups [17,18]. This may be due to the fact that the BMP members from BMP2/4 and BMP5/6/7/8/8b subgroups are highly related to bone regeneration [6]. Heterodimeric BMPs consisting of monomers from the other subgroups do not always exhibit enhanced osteoinductive potency [15]. They may play key roles in the development of other tissues [19]. 2. Synthesis of heterodimeric BMPs Hitherto, four methods have been reported to produce heterodimeric BMPs: (1) extraction from natural bone tissue [9,11], (2) co-transfection of two different BMP genes [14], (3) transfection of a gene that encodes for heterodimeric BMP [20], and (4) heterodimeric refolding of BMP monomers [21]. Similar to homodimeric BMPs, the extraction and purification of heterodimeric BMPs from bone are associated with a very low yield rate [9]. Therefore this approach is not feasible for clinical use. Gene transfection technology that has rapidly developed in the past two decades enables the efficient production of heterodimeric BMPs in a large scale [13]. The production efficiency using viruses is significantly higher than that using mammalian cells [13]. Cotransfection of two BMP genes using a ratio of 1:1 leads to highest yield of heterodimeric BMPs among different ratios [15]. When two BMP genes are co-transfected with the ratio of 1:1, a very high production efficiency (about 80–90%) of heterodimeric BMPs in the total BMPs are obtained regardless of transfection systems [13,22]. This suggests that BMP monomers might preferentially heterodimerize rather than homodimerize [13]. Recently, BMP2/7 fusion gene was constructed by connecting BMP2 cDNA and BMP7 cDNA with a (Gly4Ser)4 linker [20]. This fusion gene was cloned into a plasmid with expression driven by a CMV (cytomegalovirus) promoter (pSCMV-BMP2/7), so that a single transcript encoding BMP2, the linker, and BMP7 would be

expressed. This BMP2/7 fusion gene has already been shown to generate biologically active heterodimeric BMP2/7 [23,24]. Heterodimerization of different BMP monomers is another method to produce heterodimeric BMPs [21]. For this purpose, BMP propeptides are first removed by proteolysis, enabling mature BMP monomers to form active disulfide-linked heterodimers [25]. During the past decades, most of studies are based on heterodimeric BMPs produced by researchers themselves. The differences in the production conditions, makes it difficult to systematically compare the results. Recently, human recombinant heterodimeric BMPs has become commercially available, which may greatly propel the research in heterodimeric BMPs [17]. 3. Signaling pathway of heterodimeric BMPs Heterodimeric BMPs can activate a distinct signaling pattern, that cannot be accomplished by the mixture of homodimeric BMPs. In this section, we try to summarize the principles of their signaling basing on the available evidence derived from human cells, zebra fish and drosophila. 3.1. Ligand–receptor interaction and downstream signaling pathway in human cells Similar to other members in the TGF-b superfamily, BMPs bind to transmembrane serine/threonine kinase receptors on cell surfaces. Thereby, they trigger specific intracellular signaling pathways that activate and influence gene transcription [26]. There are three type I receptors and three type II receptors for BMP signaling. The type I receptors include ActR-IA (Alk-2), BMPR-IA (Alk-3) and BMPR-IB (Alk-6); the type II receptors include BMPR-II, ActR-IIA and ActR-IIB [8,26]. The expression patterns and levels of BMP receptors varies among different cell types [27,28]. Receptors of both types are indispensible to form a functional complex to initiate downstream signaling events [28]. Activated BMP type I receptors phosphorylate Smad1, Smad5, and Smad8 (receptor-regulated Smads, R-Smads), which then assemble into a complex with Smad4 (common-partner Smad, CoSmad) and translocate to the nucleus to regulate the transcription of target genes, such as Runx2 [29]. In addition, the activated BMP receptors can also initiate Smad-independent signaling pathways, resulting in the activation of ERK, p38, and JNK [30–32]. The signaling pathways and modulation mechanisms of homodimeric BMP have already been reviewed elsewhere [8,33,34]. The expression and oligomerization pattern of receptors are one of the main mechanisms for the versatile and specific functions of different BMPs [8]. Different homodimeric BMP ligands exhibit different affinities to either type of receptors [33]. For example, homodimeric BMP2 (from BMP2/4 subgroup) bears a high affinity to type I receptors, but a low affinity to type II receptors [35,36]. While, homodimeric BMP6 and BMP7 (from BMP5/6/7/8/8b subgroup) have a high affinity to type II receptors, and a medium or low affinity to type I receptors [21,37,38]. In contrast, heterodimeric BMP2/6 bears high affinity to both type I and type II receptors [21] (Fig. 1). Since avidity appears to be a major driving force for BMP-receptor complex formation [36], the high affinity of heterodimeric BMPs to both types of receptors is likely to result in more rapid and stable formation of receptor–ligand complexes. This contributes to an earlier and stronger downstream signaling of heterodimeric BMPs in comparison with homodimeric BMPs [21,23] (Fig. 2). Furthermore, heterodimeric BMPs can enhance BMP-receptor complex formation by causing significantly higher expression of BMP receptors than homodimeric BMPs [18] (Fig. 2). As a result, heterodimeric BMPs can trigger significantly higher levels of Smad-dependent signaling (e.g., Smad1/5/8) and thus BMP-target genes (e.g., Inhibitor of differentiation 1), than

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Fig. 1. Schematic graph depicting the different binding affinities of homodimeric BMP2 (from BMP2/4 subgroup), homodimeric BMP6 or BMP7 (from BMP5/6/7/8/8b subgroup) and heterodimeric BMP2/6 or BMP2/7 to two type I receptors (BMPR-IA and BMPR-IB) and two type II receptors (ActR-II and ActR-IIB). Homodimeric BMP2 bears high affinity to BMPR-IA and BMPR-IB, whereas low affinity to ActR-II and ActR-IIB. Homodimeric BMP6 or BMP7 bear low or medium affinity to BMPR-IA and BMPR-IB, whereas high affinity to ActR-II and ActR-IIB. In contrast, heterodimeric BMP2/6 or BMP2/7 bear high affinity to both two type I receptors (BMPR-IA and BMPR-IB) and two type II receptors (ActR-II and ActR-IIB) simultaneously.

homodimeric BMPs [18,39,40] (Fig. 2). For Smad-independent pathway (e.g., ERK, p38), they also exhibit significantly higher promoting efficiency [18]. In fact, the modulation mechanism of BMP signaling is far more complicated. The oligomerization pattern and internalization of receptors can play critical roles in modulating the effects of homodimeric BMPs [41–43]. Homodimeric BMPs are shown to have greatly varied reliance on homodimeric or heterodimeric receptor constitution patterns [27]. The downstream signaling is also modulated by many inhibitors [34] and the crosstalk with other signaling pathways [31,44]. From this knowledge on homodimeric BMPs, it is not unreasonable to extrapolate that the high affinity to both types of receptors may lead to an exclusive receptor constitution and distinct signaling pathways of heterodimeric BMPs [19]. Unfortunately, although continuous efforts have been made, many aspects in heterodimeric BMPs’ signaling remain uncovered. 3.2. Extracellular antagonism Extracellular BMP antagonists, such as noggin [45], chordin and Cerberus, are essential to modulate the specific functions of BMPs both in location and strength [46]. The endogenous expression of extracellular antagonists can be significantly upregulated in response to BMPs as a negative feedback mechanism to suppress and control BMP’s effect. Homodimeric and heterodimeric BMPs react differently to different extracellular antagonists. Noggin can significantly antagonize homodimeric BMP2 and BMP7, whereas it shows no [47] or mild antagonism [40] to heterodimeric BMP2/7. A possible mechanism accounting for this phenomenon is that the structurally symmetrical noggin cannot bind as well to symmetrical homodimeric BMP2 and BMP7 as to asymmetrical heterodimeric BMP2/7 [47]. In addition, this phenomenon may also be partially attributed to the significantly increased avidity of heterodimeric BMP ligand–receptor complexes to compete with noggin. In contrast to noggin, chordin shows a similar affinity for heterodimeric BMP4/7 as for homodimeric BMP4 [48]. Another BMP antagonist CIZ

(casinteracting zinc finger protein) [49] can also partially suppress the heterodimeric BMP2/7-induced Smad signaling [24]. However, significantly lower expression levels of endogenous antagonists (e.g., CIZ and noggin) are detected in response to heterodimeric BMPs than homodimeric ones [24,47] (Fig. 2). This lower negative feedback loop may also contribute to the reduced antagonism, which confer significantly higher osteoinductive dose-efficiency of heterodimeric BMPs than homodimeric ones. 3.3. Signaling pathway of heterodimeric BMPs in embryonic development of zebra fish For the signaling of osteogenesis, the differences between heterodimeric and homodimeric BMPs mostly lie in time and magnitude of signaling and functions. This may be due to an overlap in the usage and functions of the receptors. In contrast, only heterodimeric BMPs can activate the signaling of dorsalventral patterning in zebra fish. The two type I receptors Alk3/6 and Alk8 in zebrafish exhibited non-redundant roles. And the signaling for dorsoventral patterning can only be activated by the receptor complex that contains both Alk3/6 and Alk8. Similar to human, homodimeric BMP2 binds the type I receptors Alk3/6 with high affinity and recruits the type II receptor poorly [50]. Conversely, homodimeric BMP7 possesses high affinity for the type II receptor and thereafter bind Alk8 [51]. Therefore, heterodimeric BMP2/7 possess a high combined affinity for both the type I and type II receptors than homodimeric BMP2 or BMP7. This property facilitates the formation of active signaling complexes that contain Alk3/6 and Alk8 [52]. In contrast, the combination of homodimeric BMP2 and BMP7 could not activate comparable BMP signaling (Fig. 3). BMP antagonism requires only a bimolecular interaction between one ligand and one antagonist molecule, which is thermodynamically more likely than assembly of the fivemolecule ligand–receptor complex required for BMP signaling. Therefore, the signaling will be elicited only from ligands with a robust ability to assemble functional ligand–receptor complexes.

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Fig. 2. Schematic graph depicting the signaling pathways and functional characteristics of heterodimeric BMPs for inducing osteogenesis. In comparison to homodimeric BMPs, heterodimeric BMPs bear high affinity to both types of receptors simultaneously, which enable an enhanced avidity and an accelerated recruitment of receptors. Meanwhile the stability of ligand–receptor complex is also significantly enhanced by the reduced antagonism by extracellular antagonists (e.g., noggin). As a result, heterodimeric BMPs result in significantly higher Smad-dependent (Smad1/5/8) and Smad-independent signaling (p38 and ERK) than homodimeric BMPs. In addition, the signaling of heterodimeric BMPs is also enhanced by higher expression of BMP receptors and lower expression of extracellular antagonists. In consistency with the distinct signaling, heterodimeric BMPs is associated with significantly lower threshold, more rapid effect and higher dose-efficiency when comparing to the respective homodimeric BMPs. (+): promotion; ( ): suppression.

The competition for ligand binding between antagonists and receptors combined with the reduced affinity of homodimers for one type of receptor, possibly in conjunction with reduced affinity of heterodimers for some antagonists, might account for the less potency of homodimers and the exclusive requirement for heterodimers in dorsoventral patterning [52]. 3.4. Signaling pathway of heterodimeric BMPs in embryonic development of drosophila We only briefly summarize the signaling of heterodimeric BMPs in drosophila embryo since the subject has been previously reviewed [53]. Three BMPs are present in Drosophila: Decapentaplegic (Dpp), Glass bottom boat (Gbb), and Screw (Scw) [54]. These BMPs signal through one type II receptor Punt, and two type I receptors Saxophone (Sax) and Thickveins (Tkv) [55]. Heterodimeric BMP (Dpp/Scw) is preferentially transported to the dorsal midline by a complex with Sog and Tsg. There, Dpp/Scw produces optimal output through the synergistic activation of a Sax/Tkv heterodimeric

receptor complex, resulting in the formation of amnioserosa. In contrast, Dpp and Scw homodimers are not efficiently transported as they have a lower affinity for Sog/Tsg. Upon ligand binding, Sax and Tkv phosphorylate Mad, the sole Drosophila BMP Smad. Phosphorylated Mad (pMad) forms a complex with the co-Smad Medea, which then translocates into the nucleus. Similar to BMPs in vertebrates, heterodimeric BMP (Dpp/Scw) also exhibits a tenfold higher signal than an equimolar mixture of homodimeric Dpp and Scw [56]. The increased signaling ability of the heterodimeric BMP (Dpp/Scw) may be due to synergy between the two type I receptors Tkv and Sax [56]. This mechanism is quite similar to signaling of heterodimeric BMPs in embryo patterning of zebra fish (Fig. 3). 4. Functions and applications of heterodimeric BMPs Similar to homodimeric BMPs, an important application of heterodimeric BMPs is to induce osteogenesis. The functional characteristics of heterodimeric BMPs for osteogenesis are highly consistent with their signaling pathways (Fig. 2). Recently,

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Fig. 3. Schematic graph depicting the ligand–receptor complex to activate the signaling for dorsalventral patterning in Drosophila and Zebra fish. Only heterodimeric BMPs (BMP2/7 in Zebra fish or Dpp/Scw in Drosophila) can recruit heterodimeric combination of two type I receptors (Alk3/6&Alk2/8 in Zebra fish or Tkv&Sax in Drosophila), which can activate dorsalventral patterning. In contrast, homodimeric BMPs (BMP2/2 or BMP7/7 in Zebra fish or Dpp/Dpp or Scw/Scw in Drosophila) can recruit homodimeric combination of either type I receptor, which cannot activate dorsalventral patterning.

heterodimeric BMP can also be used to suppress tumorigenesis. Numerous investigations show that the specific functions and activities of heterodimeric BMPs cannot be obtained by an equalmolar mixture of the respective homodimeric BMPs. The findings indicate a promising application potential of heterodimeric BMPs in osteogenesis and other aspects.

unintended areas [68,69]. One approach to solve this problem is to adopt more potent BMPs so that they can be applied in a lower dose. Heterodimeric BMPs exhibit significantly higher dose efficiency in inducing in-vitro osteoblastogenesis and in-vivo osteogenesis [14–16,22]. Therefore, they exhibit very promising application potential in the field of bone tissue engineering.

4.1. Heterodimeric BMPs and osteogenesis

4.1.2. In-vitro functions of heterodimeric BMPs on osteoblastogenesis, chondrogenesis and osteoclastogenesis Heterodimeric BMPs unanimously exhibited significantly higher dose efficiency (from 1.3 to dozens folds) in inducing invitro osteoblastogenesis than the respective homodimeric BMPs [13,16,21]. In line with these findings, a recent time-course and dose-dependent systematic study concluded that heterodimeric BMP2/7 was associated with significantly lower threshold and optimal concentrations, but similar maximum effects in inducing osteoblastogenesis [17]. This data indicated that the advantages of heterodimeric BMPs over the respective homodimeric BMPs were significantly higher at relatively lower concentrations (<100 ng/ ml) and tapered at the increase of BMP concentrations. When they are applied in much higher concentrations (>500 ng/ml), heterodimeric BMPs are not advantageous but even disadvantageous over homodimeric BMPs [70]. Another functional characteristic for heterodimeric BMP is the acceleration of cellular events during osteoblastogenesis [17,22]. Such acceleration can be significant both in a more primitive cell (C2C12) [22] and a more differentiated cell (MC3T3-E1) [17]. Similar to its effect on osteoblastogenesis, heterodimeric BMP2/6 exhibits significantly higher activity in inducing chondrogenesis than homodimeric BMP2 and BMP6 [21]. Consistent with homodimeric BMPs, RANKL is also indispensible for the induction of osteoclastogenesis by heterodimeric

4.1.1. Clinical background The osseous restoration of critical-sized bone defects remains a challenge in the fields of orthopedics, maxillofacial surgery and dental implantology [57,58]. As the ‘‘gold-standard’’ bone-defectfilling material, autologous bone grafts are highly osteoconductive, osteoinductive and osteogenic. However, the use of autologous bone grafts is limited by its intrinsic disadvantages e.g., limited quantity [59], donor site morbidity [60,61], variable resorption rate [62] and difficulties to reproduce the curvature of the local site [63]. Therefore, allografts, xenografts and synthetic materials are widely used in clinic to treat bone defects to substitute for autologous bone grafts. However, most of these materials lack intrinsic osteoinductivity to heal critical-sized bone defects. As an effective approach to this problem, bone morphogenetic proteins (BMPs) are the most widely used cytokines to confer osteoinductivity to these bone-defect-filling materials [64,65]. BMP2 and BMP7 (homodimeric) have already been approved by FDA (Food and Drug Administration) for clinical use. However, the clinical effective doses of these homodimeric BMPs to induce bone formation are extremely high (e.g., up to milligrams) [66]. This results not only in a substantial economic burden to patients but also in a series of potential side effects [67], such as overstimulation of osteoclastic activity and ectopic bone formation in

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BMP2/7 [71]. Heterodimeric BMP2/7 can affect each of the main events related to the formation and activity of osteoclasts: proliferation of precursors, expression of osteoclast-specific genes, morphological characteristics of osteoclasts and calcium phosphate resorption [71]. A relatively low dose (5–50 ng/ml) of heterodimeric BMP2/7 can already significantly enhance osteoclastic function (e.g., calcium phosphate resorption). This suggests heterodimeric BMPs may facilitate a more rapid remodeling of itsinduced new bone. 4.1.3. In-vivo functional characteristic of heterodimeric BMP on osteogenesis Consistent with their in-vitro effects, recombinant heterodimeric BMPs also induced significantly higher in-vivo osteogenesis (bone mineral density) than the respective homodimeric BMPs [14]. This advantage of heterodimeric BMPs is more significant at relatively lower doses. Furthermore, the threshold dose of recombinant heterodimeric BMPs was only one tenth of that of homodimeric BMP. A recent micro-computed tomography study showed that the low-dose (30 ng/mm3) heterodimeric BMP2/7 resulted in significantly higher bone volume fraction, trabecular thickness, trabecular number and significantly lower trabecular separation, structure mode index than the homodimers at 2, 3 and 6 weeks post-operation [72]. This indicated that heterodimeric BMP2/7 could induce bone formation not only with a significantly higher volume but also with a more mature 3-dimensional microarchitectures [72]. All these in-vivo functional characteristics are highly consistent with their in-vitro functions [17]. Apart from the application of recombinant protein, co-transfection with two homodimeric BMP genes also results in a significantly higher osteogenesis (2–3 folds) than with single BMP gene [16,22,39]. 4.2. Heterodimeric BMPs and tumorigenesis Tumorigenesis can be modulated (induced or inhibited) by BMPs [73]. Different BMPs can promote, non-alter or suppress the tumorigenesis breast cancer. Although the exact roles of the different BMPs in these processes is not clear, heterodimeric BMP2/7 was recently shown to be a more efficient stimulator of BMP signaling in breast cancer stem cells (MDA-MB-231) than homodimeric BMP2 or BMP7 [40]. It was shown to effectively reduce TGF-b-driven Smad signaling and invasiveness of these cells. This suggests a promising potential of heterodimeric BMPs to treat cancer. 4.3. Heterodimeric BMPs and neurogenesis As ‘‘body morphogenetic proteins,’’ BMPs also modulate the roof plate-mediated repulsion of commissural axon during spinal cord development. Analysis of gene-knockout mice indicates BMP7 and GDF7 (BMP12) act coordinately, not redundantly, in the roof plate. Furthermore, GDF7 lacks of activity as a chemorepellent in vitro. Taken together, these evidences suggest heterodimeric BMP7/ GDF7 primarily activates plate-mediated repulsion of commissural axon [19]. Besides, recombinant BMP2/6 and BMP2/7 as well as homodimeric BMPs can induce higher levels of differentiation markers in astrocytes, the major glial cell type for neurogenesis [12]. However, the difference between heterodimeric and homodimeric BMPs is not significant. 5. Conclusions Heterodimeric BMPs possess a high affinity to both types of BMP receptors. They also appear to benefit from the lower affinity to BMP antagonists than homodimeric BMPs. These characteristics of heterodimeric BMPs appear to be beneficial since they modulate processes like bone formation at a much lower concentration, thus

enabling a promising application potential in bone tissue engineering. In addition, heterodimeric BMPs play indispensible roles in numerous other processes related to organ formation, such as embryo development. Hopefully, more studies will be performed to further uncover the functional characteristics and signaling pathways of heterodimeric BMPs in near future. Conflict of interest All authors have no conflicts of interest. Acknowledgement The authors thank Prof. Dr Vincent Everts from Academic Centre for Dentistry Amsterdam, Research Institute MOVE, VU University of Amsterdam, University of Amsterdam, the Netherlands for a critical review. References [1] Urist MR. Bone: formation by autoinduction. Science 1965;150:893–9. [2] Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528–34. [3] Reddi AH, Reddi A. Bone morphogenetic proteins (BMPs): from morphogens to metabologens. Cytokine Growth Factor Rev 2009;20:341–2. [4] Ducy P, Karsenty G. The family of bone morphogenetic proteins. Kidney Int 2000;57:2207–14. [5] Reddi AH. BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev 2005;16:249–50. [6] Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic. Part I (basic concepts). J Tissue Eng Regen Med 2008;2:1–13. [7] Liao WX, Moore RK, Otsuka F, Shimasaki S. Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep. J Biol Chem 2003;278:3713–9. [8] Sieber C, Kopf J, Hiepen C, Knaus P. Recent advances in BMP receptor signaling. Cytokine Growth Factor Rev 2009;20:343–55. [9] Wang EA, Rosen V, Cordes P, Hewick RM, Kriz MJ, Luxenberg DP, et al. Purification and characterization of other distinct bone-inducing factors. Proc Natl Acad Sci USA 1988;85:9484–8. [10] Ozkaynak E, Rueger DC, Drier EA, Corbett C, Ridge RJ, Sampath TK, et al. OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J 1990;9:2085–93. [11] Sampath TK, Coughlin JE, Whetstone RM, Banach D, Corbett C, Ridge RJ, et al. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem 1990;265:13198–205. [12] D’Alessandro JS, Yetz-Aldape J, Wang EA. Bone morphogenetic proteins induce differentiation in astrocyte lineage cells. Growth Factors 1994;11:53–69. [13] Hazama M, Aono A, Ueno N, Fujisawa Y. Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system. Biochem Biophys Res Commun 1995;209:859–66. [14] Aono A, Hazama M, Notoya K, Taketomi S, Yamasaki H, Tsukuda R, et al. Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 1995;210:670–7. [15] Israel DI, Nove J, Kerns KM, Kaufman RJ, Rosen V, Cox KA, et al. Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors 1996;13:291–300. [16] Zhao M, Zhao Z, Koh JT, Jin TC, Franceschi RT. Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7. J Cell Biochem 2005;95:1–16. [17] Zheng Y, Wu G, Zhao J, Wang L, Sun P, Gu Z. rhBMP2/7 heterodimer: an osteoblastogenesis inducer of not higher potency but lower effective concentration compared with rhBMP2 and rhBMP7 homodimers. Tissue Eng A 2010;16:879–87. [18] Valera E, Isaacs MJ, Kawakami Y, Izpisua Belmonte JC, Choe S. BMP-2/6 heterodimer is more effective than BMP-2 or BMP-6 homodimers as inductor of differentiation of human embryonic stem cells. PLoS One 2010;5:e11167. [19] Butler SJ, Dodd J. A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons. Neuron 2003;38:389–401. [20] Zhu W, Boachie-Adjei O, Rawlins B, Crystal RG, Hidaka C. A BMP2/7 fusion gene enhances osteoblastic differentiation in mesenchymal cells. Mol Ther 2004;9:S338. [21] Isaacs MJ, Kawakami Y, Allendorph GP, Yoon BH, Belmonte JC, Choe S. Bone morphogenetic protein-2 and -6 heterodimer illustrates the nature of ligand– receptor assembly. Mol Endocrinol 2010;24:1469–77. [22] Zhu W, Rawlins BA, Boachie-Adjei O, Myers ER, Arimizu J, Choi E, et al. Combined bone morphogenetic protein-2 and -7 gene transfer enhances

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[23]

[24]

[25] [26] [27]

[28] [29] [30]

[31] [32]

[33]

[34] [35] [36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

osteoblastic differentiation and spine fusion in a rodent model. J Bone Miner Res 2004;19:2021–32. Pan Q, Yang S, Sun F. Expression of BMP-2/7 heterodimers with potent ability to stimulate osteogenic differentiation in CHO cells. Sheng Wu Gong Cheng Xue Bao 2008;24:473–9. Pan Q, Yang S, Dong Q, Sun F. Relationship between the bioactivity of BMP2/7 heterodimers and its regulation of CIZ expression. China Biotechnol 2007; 27:14–8. Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors 2004;22:233–41. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 2003;425:577–84. Lavery K, Swain P, Falb D, Alaoui-Ismaili MH. BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells. J Biol Chem 2008;283:20948–58. Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. J Biochem 2010;147:35–51. Miyazono K. Signal transduction by bone morphogenetic protein receptors: functional roles of Smad proteins. Bone 1999;25:91–3. Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. J Bone Miner Res 2003;18:2060–8. Massague J. Integration of Smad and MAPK pathways: a link and a linker revisited. Genes Dev 2003;17:2993–7. Hoffmann A, Preobrazhenska O, Wodarczyk C, Medler Y, Winkel A, Shahab S, et al. Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors. J Biol Chem 2005;280:27271–83. Ehrlich M, Horbelt D, Marom B, Knaus P, Henis YI. Homomeric and heteromeric complexes among TGF-beta and BMP receptors and their roles in signaling. Cell Signal 2011;23:1424–32. Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A. Bone morphogenetic proteins: a critical review. Cell Signal 2011;23:609–20. Kirsch T, Nickel J, Sebald W. BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II. EMBO J 2000;19:3314–24. Sebald W, Nickel J, Zhang JL, Mueller TD. Molecular recognition in bone morphogenetic protein (BMP)/receptor interaction. Biol Chem 2004;385:697–710. Allendorph GP, Isaacs MJ, Kawakami Y, Izpisua Belmonte JC, Choe S. BMP-3 and BMP-6 structures illuminate the nature of binding specificity with receptors. Biochemistry 2007;46:12238–47. Saremba S, Nickel J, Seher A, Kotzsch A, Sebald W, Mueller TD. Type I receptor binding of bone morphogenetic protein 6 is dependent on N-glycosylation of the ligand. FEBS J 2008;275:172–83. Koh JT, Zhao Z, Wang Z, Lewis IS, Krebsbach PH, Franceschi RT. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J Dent Res 2008;87:845–9. Buijs JT, van der Horst G, van den Hoogen C, Cheung H, de Rooij B, Kroon J, et al. The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation. Oncogene 2011. doi: 10.1038/onc.2011.400. Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002;277:5330–8. Nohe A, Keating E, Underhill TM, Knaus P, Petersen NO. Effect of the distribution and clustering of the type I A BMP receptor (ALK3) with the type II BMP receptor on the activation of signalling pathways. J Cell Sci 2003;116:3277–84. Heinecke K, Seher A, Schmitz W, Mueller TD, Sebald W, Nickel J. Receptor oligomerization and beyond: a case study in bone morphogenetic proteins. BMC Biol 2009;7:59. Eivers E, Demagny H, De Robertis EM. Integration of BMP and Wnt signaling via vertebrate Smad1/5/8 and Drosophila Mad. Cytokine Growth Factor Rev 2009;20:357–65. Groppe J, Greenwald J, Wiater E, Rodriguez-Leon J, Economides AN, Kwiatkowski W, et al. Structural basis of BMP signalling inhibition by the cystine knot protein Noggin. Nature 2002;420:636–42. Walsh DW, Godson C, Brazil DP, Martin F. Extracellular BMP-antagonist regulation in development and disease: tied up in knots. Trends Cell Biol 2010;20:244–56. Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, et al. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006;39:61–71. Piccolo S, Sasai Y, Lu B, De Robertis EM. Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 1996;86:589–98. Shen ZJ, Nakamoto T, Tsuji K, Nifuji A, Miyazono K, Komori T, et al. Negative regulation of bone morphogenetic protein/Smad signaling by Cas-interacting zinc finger protein in osteoblasts. J Biol Chem 2002;277:29840–46. Graff JM, Thies RS, Song JJ, Celeste AJ, Melton DA. Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell 1994;79:169–79. Macias-Silva M, Hoodless PA, Tang SJ, Buchwald M, Wrana JL. Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J Biol Chem 1998;273:25628–36. Little SC, Mullins MC. Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis. Nat Cell Biol 2009;11:637–43.

67

[53] O’Connor MB, Umulis D, Othmer HG, Blair SS. Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing. Development 2006;133:183–93. [54] Newfeld SJ, Wisotzkey RG, Kumar S. Molecular evolution of a developmental pathway: phylogenetic analyses of transforming growth factor-beta family ligands, receptors and Smad signal transducers. Genetics 1999;152:783–95. [55] Parker L, Stathakis DG, Arora K. Regulation of BMP and activin signaling in Drosophila. Prog Mol Subcell Biol 2004;34:73–101. [56] Shimmi O, Umulis D, Othmer H, O’Connor MB. Facilitated transport of a Dpp/ Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. Cell 2005;120:873–86. [57] Claffey N, Clarke E, Polyzois I, Renvert S. Surgical treatment of peri-implantitis. J Clin Periodontol 2008;35:316–32. [58] Saito N, Takaoka K. New synthetic biodegradable polymers as BMP carriers for bone tissue engineering. Biomaterials 2003;24:2287–93. [59] Kretlow JD, Mikos AG. Review: mineralization of synthetic polymer scaffolds for bone tissue engineering. Tissue Eng 2007;13:927–38. [60] Silber JS, Anderson DG, Daffner SD, Brislin BT, Leland JM, Hilibrand AS, et al. Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine 2003;28:134–9. [61] Heary RF, Schlenk RP, Sacchieri TA, Barone D, Brotea C. Persistent iliac crest donor site pain: independent outcome assessment. Neurosurgery 2002;50: 510–6. discussion 6–7. [62] Smith JD, Abramson M. Membranous vs endochondrial bone autografts. Arch Otolaryngol 1974;99:203–5. [63] Asamura S, Ikada Y, Matsunaga K, Wada M, Isogai N. Treatment of orbital floor fracture using a periosteum–polymer complex. J Craniomaxillofac Surg 2010;38:197–203. [64] Carson JS, Bostrom MP. Synthetic bone scaffolds and fracture repair. Injury 2007;38(Suppl. 1):S33–7. [65] Sokolsky-Papkov M, Agashi K, Olaye A, Shakesheff K, Domb AJ. Polymer carriers for drug delivery in tissue engineering. Adv Drug Deliv Rev 2007;59:187–206. [66] Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine 2002;27:2662–73. [67] Zara JN, Siu RK, Zhang X, Shen J, Ngo R, Lee M, et al. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng A 2011;17:1389–99. [68] Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone 2000;27:479–86. [69] Uludag H, D’Augusta D, Palmer R, Timony G, Wozney J. Characterization of rhBMP-2 pharmacokinetics implanted with biomaterial carriers in the rat ectopic model. J Biomed Mater Res 1999;46:193–202. [70] Carpenter RS, Goodrich LR, Frisbie DD, Kisiday JD, Carbone B, McIlwraith CW, et al. Osteoblastic differentiation of human and equine adult bone marrowderived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone. J Orthop Res 2010;28:1330–7. [71] Zheng Y, Wang L, Zhang X, Gu Z, Wu G. BMP2/7 heterodimer can modulate all cellular events of the in vitro RANKL-mediated osteoclastogenesis respectively in different dose-patterns. Tissue Eng A 2012;18(5–6):621–30. [72] Wang J, Zheng Y, Zhao J, Liu T, Gao L, Gu Z, et al. Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation. J Clin Periodontol 2012;39:98–105. [73] Alarmo EL, Kallioniemi A. Bone morphogenetic proteins in breast cancer: dual role in tumourigenesis? Endocr Relat Cancer 2010;17:R123–39.

Jing Guo obtained her bachelor and master degree in dental science from West China College of Stomatology, Sichuan University in Chengdu, China. She obtained her Ph.D in the same institution in 2011. Meanwhile, she was enrolled in the joint Ph.D program in the Academic Center for Dentistry Amsterdam (ACTA), VU university in the year 2010–2011. Now, she works as a post-doc researcher in the ACTA, VU university. Dr. Guo focuses on the basic research of biological and pathological mineralization process.

Gang Wu obtained his bachelor of dental science (B.D.S) from Tongji University in Shanghai, China in 2003. From 2006, he spent 2 years in University of Bern, Switzerland as a visiting scholar. He obtained M.D. from Zhejiang University, China in 2008 and his Ph.D from VU University in Amsterdam, The Netherlands in 2010. Dr. Wu was appointed as assistant professor in Academic Centre for Dentistry Amsterdam (ACTA), VU University in 2011 after one year of post-doctoral work. Dr. Wu focuses on the basic and clinical research of biomimetic calcium phosphate coatings and bone morphogenetic proteins for bone tissue engineering.