"VSports手机版" The BMP signaling and in vivo bone formation

1. Introduction

BMPs belong to the members of the TGFβ superfamily. The activity of BMPs was discovered in the 1960s (Urist, 1965), but the BMP proteins were purified and sequenced in late 1980s (Luyten et al V体育平台登录. , 1989; Wozney et al. , 1988). After that, recombinant BMP proteins were expressed (Wozney et al. , 1988; Wozney, 1992). To date, over 20 BMP family members have been identified and characterized. BMP signals are mediated by type II and type I serine/threonine kinase receptors. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig et al. , 1994; ten Dijke et al. , 1994; Macias-Silva et al. , 1998). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita et al. , 1995; Nohno et al. , 1995; Rosenzweig et al. , 1995; Kawabata et al. , 1995). Whereas BMPR-IA, IB, and II are specific to BMPs, ActR-IA, II, and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and type II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and type II receptor complex (Moustakas and Heldi, 2002). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. A significant advancement has been achieved in recent years on understanding of BMP signaling mechanism and in vivo functions of BMP ligands, receptors and signaling molecules.

2. BMP signal transduction

Smad proteins play a central role in BMP signaling. Smads 1, 5 and 8 transiently and directly interact with activated type I BMP receptors, which phosphorylate the C-terminal SSXS motif of Smad in a ligand-dependent manner (Hoodless et al. , 1996; Nishimura et al. , 1998; Chen et al. , 1997a). After release from the receptor, the phosphorylated Smad proteins form heteromeric complexes with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription factors. Smads 1 and 5 directly binds to DNA; however, the affinity is relatively low and interaction with sequence-specific DNA binding proteins is critical for the formation of a stable DNA-binding complex (Derynck et al. , 1998). The first demonstration that Smads can directly bind to DNA was reported in Drosophila (Kim et al. , 1997). Vestigial, labial and ultrabithorax (Ubx) are decapentaplegic (dpp)-responsive genes. Mad, a Drosophila homologue of Smad, was shown to directly bind to the enhancer of these genes and GCCGnCGC (GCCG motif) was identified as the consensus binding site. It has been reported that Smads 1 and 5 interact with bone-specific transcription factor Runx2 (Hanai et al. , 1999; Lee et al. , 2000; Zhao et al. , 2003) and activate the transcription of target genes such as COX-2 and type X collagen (Col-X) in osteoblasts or in chondrocytes (Chikazu et al. , 2002; Leboy et al. , 2001). The association of Smad1 with homeodomain-containing proteins suggests another important mechanism of BMP signaling in osteoblasts and in chondrocytes. The Hox homeodomain proteins play important roles in controlling pattern formation of the vertebrate skeleton. It has been shown that Smad1 directly interacts with Hoxc8 to activate the transcription of osteopontin gene (Shi et al VSports注册入口. , 1999; Yang et al. , 2000; Liu et al. , 2004), which is a marker gene for osteoblast and chondrocyte differentiation. In contrast, Smad6 heterodimerizes with Hoxc8 and represses BMP-2-induced gene transcription (Bai et al. , 2000). Smads 1 and 5 are two Smad proteins which have been shown to play an important role in osteoblast differentiation in C2C12 myoblast/osteoblast precursor cells and other osteoblastic cell lines (Yamamoto et al. , 1997; Fujii et al. , 1999). In addition to Smads, BMPs also activate non-Smad signaling pathways such as mitogen-activated protein kinase (MAPK) family of molecules including ERK1/2 and p38 (Guicheux et al. , 2003; Reilly et al. , 2005).

3. Biological functions of BMPs

Physiological roles of BMPs and BMP receptor signaling in normal bone formation have been investigated. Injection of BMP-2 locally over the surface of calvariae of mice induces periosteal bone formation without a prior cartilage phase (Chen et al. , 1997b). Bmpr1a is widely expressed in a variety of tissues during development and in multiple adult tissues (Dewulf et al. , 1995) V体育官网入口. In contrast, expression of Bmpr1b is restricted in early mesenchymal cells and differentiated chondrocytes (Ashique et al. , 2002). Over-expression of a dominant-negative Bmpr1b but not Bmpr1a inhibits chondrocyte differentiation (Enomoto-Iwamoto et al. , 1998) and over-expression of a constitutively active Alk-2 promotes chondrocyte maturation (Zhang et al. , 2003a) in chicken sternal chondrocytes. Over-expression of a dominant-negative Bmpr1b also inhibits osteoblast differentiation in osteoblast precursor cells (Chen et al. , 1998). In the transgenic mice in which expression of a dominant-negative Bmpr1b transgene is targeted to the osteoblast lineage using the osteoblast-specific type I collagen promoter, the postnatal bone formation is reduced (Zhao et al. , 2002). These findings demonstrate that BMP receptor signaling plays a necessary role in normal chondrocyte and osteoblast differentiation and postnatal bone formation.

V体育ios版 - 4. Naturally occurring mutations in BMPs and BMP receptors

Studies of naturally occurring mutations of BMPs and BMP receptors have shown that BMPs play important roles in several inherited diseases. In mice with short ear mutations Bmp5 gene was disrupted. Mutations in the Bmp5 gene are associated with a wide range of skeletal defects, including reductions in long bone width and the size of several vertebral processes and an overall lower body mass (Kingsley et al. , 1992; Mikic et al. , 1995). Mutations in growth/differentiation factor-5 (Gdf5 and CDMP-1) gene result in brachypodism in mice (Storm et al. , 1994) and chondrodysplasia in humans (Thomas et al. , 1996, 1997). Both Bmp5 and Gdf5 genes are localized on chromosome 2 in mice and on chromosome 20 in humans (Storm et al. , 1994) VSports在线直播. GDF5 has been shown to bind BMPR-IB specifically (Nishitoh et al. , 1996) and null mutations in the Bmpr1b gene causes a similar skeletal phenotype as that observed in Gdf5 mutant mice (Yi et al. , 2000). Heterozygous missense mutations in Bmpr1b gene in humans cause brachydactyly type A2 through a dominant-negative effect. The skeletal phenotype of patients is similar to that of acromesomelic chondrodysplasias of Grebe, Hunter–Thompson, and DuPan types caused by homozygous mutations in the gene coding for GDF5 (Lehmann et al. , 2003). In constrast, homozygous mutations in Bmpr1b gene in humans show a severe defect in limb formation, including aplasia of the fibula, severe brachydactyly, ulnar deviation of the hands, and fusion of carpal/tarsal bones (Demirhan et al. , 2005). Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformations of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Ectopic expression of Bmp4 was found in FOP patients (Gannon et al. , 1997; Xu et al. , 2000).

5. Knockout of Bmp, Bmpr and Smad genes

To determine the roles of BMP ligands, receptors and signaling proteins in embryonic development and in postnatal life, null mutations of BMP ligands, receptors and Smad genes have been created and phenotypic changes in these animals have been extensively studied. Mice deficient for Bmp2 and Bmp4 are nonviable. Homozygous Bmp2 mutant embryos die between embryonic day 7. 5 (E7. 5) and 10. 5 (E10. 5) and have defects in cardiac development, manifested by the abnormal development of the heart in the exocoelomic cavity (Zhang and Bradley, 1996). Homozygous Bmp4 mutant embryos die between E6 V体育2025版. 5 and E9. 5 and show little or no mesodermal differentiation (Winnier et al. , 1995). Bmp2/4 conditional knockout (CKO) mice in which Bmp2 and Bmp4 –loxP mice were crossed with Prx1-Cre transgenic mice show severe defects in bone development (Cox et al. , 2004). Similarly Bmp4 CKO mice (Col1-Cre mice were used) also showed defects in bone development and postnatal bone formation (Guo et al. , 2004). In contrast, Bmp3 KO mice show increased bone mass, suggesting that endogenous BMP3 antagonizes BMP signaling in vivo (Daluiski et al. , 2001). Bmp6-deficient mice are viable and fertile and show no overt defects in tissues known to express Bmp6 mRNA (Solloway et al. , 1998). Bmp6 is mainly expressed in hypertrophic cartilage. Since Bmp2 and 6 are co-expressed in this tissue, BMP-2 may functionally compensate the loss of BMP-6 in Bmp6 null mutant mice. Conditional knockout of Bmp2 gene in cartilage in the background Bmp6 null mutation is required to answer this question. Bmp7-deficient mice die shortly after birth because of poor kidney development. Histological analysis of mutant embryos at several stages of development reveals that metanephric mesenchymal cells fail to differentiate, resulting in a virtual absence of glomerulus in newborn kidneys. In addition, Bmp7-deficient mice have eye defects that appear to originate during lens induction. Bmp7-deficient mice have minor defects in the skeleton (Luo et al. , 1995; Dudley et al. , 1995).

Null mutation of the Bmpr1a gene causes embryonic lethality in mice. Animals die at E9. 5. Homozygous mutants with morphological defects are first detected at E7. 5. No mesoderm forms in the mutant embryos, suggesting that Bmpr1a is essential for the inductive events that lead to the formation of mesoderm during gastrulation (Mishina et al. , 1995). Using Cre-loxP CKO approach, the Bmpr1a gene has been deleted in different tissues to investigate its specific functions. Using Mx1-Cre to specifically delete the Bmpr1a gene in bone marrow, a recent report shows that a parallel increase in trabecular osteoblasts and neighboring haematopoietic stem cells (HSCs) is observed in these CKO mice, suggesting that osteoblasts are a major component of the HSC niche within the bone marrow (Zhang et al. , 2003b). Conditional deletion of β-catenin and Bmpr1a gene by Brn4-Cre (Cre activity is restricted to limb ectoderm) shows the interaction between Wnt/β -catenin and BMP signaling during limb development (Soshnikova et al VSports. , 2003). β-catenin acts downstream of the BMP receptor IA in apical ectodermal ridge (AER) induction. β-catenin controls Bmp4 expression in the ectoderm which is responsible for the formation of positive feedback loop. In contrast, β-catenin acts upstream or in parallel to the BMP receptor IA during dorsal–ventral patterning (Soshnikova et al. , 2003).

Mice lacking Bmpr1b are viable and exhibit defects in the appendicular skeleton. In Bmpr1b-deficient mice, proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. In adult mutant mice, the proximal interphalangeal joint is absent and the phalanges are replaced by a single rudimentary element, while the distal phalanges are not affected. The metacarpal and metatarsal bones are reduced although the lengths of the radius, ulna and tibia are normal (Yi et al., 2000). The appendicular defects in Bmpr1b mutant mice resemble those seen in mice homozygous for the Gdf5 null mice (Gdf5^bp-j). Since Gdf5 has been shown to play a critical role in cartilage formation and binds Bmpr1b with high affinity (Nishitoh et al., 1996); these findings suggest that Bmpr1b plays a non-redundant role in cartilage formation in vivo. BMP ligands may utilize multiple type I BMP receptors to mediate their signaling during cartilage and bone formation. In Bmpr1b and Bmp7 double mutant mice, severe appendicular skeletal defects have been observed in the forelimbs and hind limbs. The ulna and radius are severely affected (absent or shortened) (Yi et al., 2000). Since BMP-7 binds efficiently to both Bmpr1b and ActR-IA (Alk2) (Macias-Silva et al., 1998), it is conceivable that Bmpr1b and ActR-IA (Alk2) play important synergistic or overlapping roles in cartilage and bone formation in vivo.

Smad1 null mutant mice die at E10.5 because the mutant embryos fail to connect to the placenta. Smad1 mutant embryos show overgrowth of the posterior visceral endoderm as well as extra-embryonic ectoderm and mesoderm of the chorion. The overgrowth effect on the allantois in Smad1 mutant embryos leads to a dramatic reduction in the size and patterning of this tissue and concomitant failure to form the umbilical connection to the placenta (Tremblay et al., 2001). Deleting the Smad1 gene specifically in osteoblasts (using Col1-Cre mice) causes reduction in bone mass, demonstrating that endogenous BMPs regulate bone mass through Smad1 signaling (Chen et al., 2003). Homozygous Smad5 null mutant mice die between E10.5–11.5 due to defects in angiogenesis. The mutant yolk sacs lack normal vasculature and had irregularly distributed blood cells. The mutant embryos also have enlarged blood vessels surrounded by decreased numbers of vascular smooth muscle cells (Yang et al., 1999). The results suggest that Smad5 may regulate endothelium–mesenchyme interactions during angiogenesis.

6. Negative regulation of BMP signaling

BMPs are potent stimulators on bone formation and on other cellular functions. The activity of BMPs is controlled at different molecular levels: 1) a series of BMP antagonists bind BMP ligands and inhibit BMP functions; 2) Smad6 is a member of the Smad family. It binds type I BMP receptors and prevents the binding and phosphorylation of Smads 1, 5 and 8; 3) Tob is an anti-proliferative protein. It selectively binds Smads 1 and 5 and inhibits BMP signaling in osteoblasts; and 4) Smad ubiquitin regulatory factor 1 (Smurf1) is an E3 ubiquitin ligase. It interacts with Smad1 and 5 and mediates the degradation of these Smad proteins.

Mutations in genes of BMP antagonists have shown the importance of BMP activity in a given system. For example, proximal symphalangism is an autosomal-dominant disorder with ankylosis of the proximal interphalangeal joints, carpal and tarsal bone fusion, and conductive deafness. These symptoms are shared by another disorder of joint morphogenesis, multiple synostoses syndrome. Recently, it was reported that both disorders were caused by heterozygous mutations of the human noggin gene. To date, seven mutations of noggin gene have been identified from unrelated families affected with joint morphogenesis (Gong et al., 1999; Takahashi et al., 2001). Noggin is a secreted polypeptide, which binds and inactivates BMPs 2, 4 and 7. Co-crystal structures of noggin and BMP-7 show that noggin inhibits BMP signaling by blocking the molecular interfaces of the binding epitopes for both type I and type II BMP receptors (Groppe et al., 2002), thus preventing BMP-7 to bind with BMP receptors. This 3D crystal structure clearly shows how noggin specifically inhibits BMP-7 and other BMPs such as BMPs 2 and 4. A transgenic mouse model has recently been established using the osteocalcin promoter (OG2) to drive the noggin transgene. The animals develop osteopenia/osteoporosis. Significant reductions in bone mineral density, bone volume and bone formation rates are observed in adult transgenic mice (Devlin et al., 2003; Wu et al., 2003).

Sclerostosis is a recessive inherited osteosclerotic disorder caused by mutations in the protein sclerostin. Mutations in the SOST gene (encoding sclerostin) in humans cause high bone mass phenotype (Balemans et al., 2001). Recently it was found that sclerostin is related in sequence to the family of secreted BMP antagonists, which includes Noggin, Chordin, Gremlin and Dan. Sclerostin is expressed in osteoblasts and osteocytes and binds BMPs 5, 6 and 7 with high affinity. Expression of sclerostin in multipotent fibroblast C3H10T1/2 cells blocks osteoblast differentiation and over-expression of sclerostin in osteoblasts under the control of the osteocalcin promoter in transgenic mice causes osteoporosis (Winkler et al., 2003).

Taken together, these findings provide evidences that activation of endogenous BMP signaling enhances bone formation and regulation of the BMP activity in postnatal stage is required for normal bone formation. In addition to its inhibitory effect of sclerostin on BMP ligands, a recent report demonstrates that sclerostin also blocks Wnt signaling by binding to Lrp5/6 proteins (Li et al., 2005).

Smad6 is another member of Smad family which plays a negative regulatory role in BMP signaling by stably binding to type I BMP receptors. Smad6 interrupts the phosphorylation of Smads 1 and 5 proteins and the subsequent heteromerization with Smad4 (Imamura et al., 1997). Expression of Smad6 is regulated by BMPs. In mouse Smad6 promoter, four overlapping copies of the GCCGnCGC-like motif, which is the binding element for Smads 1 and 5, have been identified (Ishida et al., 2000). These findings establish a negative feedback regulation mechanism for BMP signaling. Smad6 knock-in mice show that expression of Smad6 is largely restricted to the heart and blood vessels. Smad6 mutant mice have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract separation defects indicate that Smad6 plays an important function in the regulation of endocardial cushion transformation. The development of aortic ossification and elevated blood pressure in Smad6 mutant mice demonstrate that Smad6 also plays a role in homeostasis of adult cardiovascular system (Galvin et al., 2000).

Tob is a member of a novel anti-proliferative protein family. Tob inhibits BMP-induced, Smad-dependent transcription in osteoblasts through its association with Smads 1 and 5 proteins (Yoshida et al., 2000). In Tob knockout mice, BMP-2 signaling is enhanced and the effects of BMP-2 on osteoblast proliferation and differentiation are increased. In long bones, bone volume and bone formation rates are increased (Yoshida et al., 2000). BMP-2-induced local bone formation is also enhanced in Tob knockout mice (Usui et al., 2002).

7. Ubiquitin–proteasome degradation of BMP signaling proteins

Another important regulatory mechanism by which the activity of BMP signaling proteins is modulated involves ubiquitin-mediated proteasomal degradation. The ubiquitin–proteasome proteolytic pathway is essential for various important biological processes including cell-cycle progression, gene transcription, and signal transduction (Hershko and Ciechanover, 1998; Weissman, 2001). The formation of ubiquitin–protein conjugates requires three enzymes that participate in a cascade of ubiquitin transfer reactions: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes (Hershko, 1983; Ciechanover et al., 2000).

Smurf1 was identified by the yeast two-hybrid assay by its ability to interact with Smads 1 and 5 (Zhu et al., 1999) and later immunoprecipitation studies show that Smurf1 has much higher affinity to bind Smads 6 and 7 (Ebisawa et al., 2001). Smurf1 is a member of the Hect domain family of E3 ubiquitin ligases. Hect domain proteins represent a major subclass of E3 ligases and contain a conserved cysteine, located at the carboxyl terminal of the Hect domain, which is capable of forming a thioester bond with ubiquitin (Hochetrasser, 1996; Zhu et al., 1999). Mutation of this conserved cysteine residue to an alanine (C710A) abolished the ubiquitination and degradation activity of Smurf1 (Zhu et al., 1999). Another motif often found in the Hect domain family of E3 ligase is the WW domain, which contains two highly conserved tryptophans and a conserved proline in an approximately 30-amino acid region (Rotin, 1998; Zhu et al., 1999). The WW domains have a preference for binding to small proline-rich sequences, PPXY motifs, and different WW domains possess different substrate specificity. Mutations of the PY motif of Smads 1 and 5 proteins prevent them to interact with Smurf1 and inhibit the degradation of these Smad proteins (Zhu et al., 1999). Smurf1 is located in the nucleus and it is exported to the cell membrane and cytoplasm when it binds Smad6 or 7 and induces the degradation of type I TGFβ and BMP receptors and Smads 1 and 5 (Ebisawa et al., 2001; Suzuki et al., 2002; Murakami et al., 2003). Several lines of evidence suggest that Smurf1 may have synergistic effect with Smad6 and inhibit BMP signaling (Murakami et al., 2003; Horiki et al., 2004). Recent report showed that the E3 ligase complex SCF^{β– TrCP1} is responsible for Smad4 ubiquitination and degradation (Wan et al., 2004).

Since bone-specific transcription factor Runx2 interacts with Smad1 protein (Hanai et al., 1999; Lee et al., 2000) whose degradation is mediated by Smurf1 (Zhu et al., 1999), the effect of Smurf1 on Runx2 degradation was examined in myoblast/osteoblast precursor C2C12 cells. Smurf1 induces Runx2 degradation in a ubiquitin–proteasome-dependent manner (Zhao et al., 2003). Similar findings are also reported when Smurf1 is transfected into osteoblastic OB-6 cells (Bellido et al., 2003). Smurf1 may interact with Runx2 in a direct or indirect mechanism. The conserved PY motif has been identified in all Runx family members. It has been reported that Smurf1 mediates Runx3 degradation in 293 cells and Runx3 with the PY motif mutation is resistant to Smurf1-induced degradation (Jin et al., 2004).

The role of Smurf1 in bone formation in vivo was examined by generating Col1 (2.3 kb)–Smurf1 transgenic mice in which expression of a Smurf1 transgene is targeted to osteoblasts. In Col1–Smurf1 transgenic mice, trabecular bone volume and bone formation rates are decreased and osteoblast proliferation and differentiation are inhibited (Zhao et al., 2004). Recent studies also demonstrate that bone mass is increased in Smurf1 null mutant mice (Yamashita et al., 2005). These findings demonstrate that regulation of BMP signaling proteins may also play an important physiological role in bone formation in vivo.

Although a significant achievement has been made in recent years in understanding the role of BMP signaling in vivo (Table 1), tissue-specific knockouts of the individual BMP ligands, receptors and signaling molecules are required to further determine the specific roles of BMP signaling in a particular tissue since null mutations of most of BMP ligands, receptors and signaling molecules produce lethal phenotype perinatally. Generation of tissue-specific and inducible conditional knockout alleles for BMP ligands, receptors and signaling molecules would allow us to gain further information about physiological functions of BMP signaling in postnatal and adult animals.