Craniofacial defects in mice lacking BMP type I receptor Alk2 in neural crest cells

Neural crest cells (NCCs) are pluripotent migratory cells that contribute to the development of various craniofacial structures. Many signaling molecules have been implicated in the formation, migration and differentiation of NCCs including bone morphogenetic proteins (BMPs). BMPs signal through a receptor complex composed of type I and type II receptors. Type I receptors (Alk2, Alk3 and Alk6) are the primary determinants of signaling specificity and therefore understanding their function is important in revealing the developmental roles of molecular pathways regulated by BMPs. Here we used a Cre/loxP system for neural crest specific deletion of Alk2. Our results show that mice lacking Alk2 in the neural crest display multiple craniofacial defects including cleft palate and a hypotrophic mandible. Based on the present results we conclude that signaling via Alk2 receptors is non-redundant and regulates normal development of a restricted set of structures derived from the cranial neural crest.     

Bone Morphogenetic Protein Type I Receptor Antagonists Decrease Growth and Induce Cell Death of Lung Cancer Cell Lines

Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.     

BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of growth factors and are used clinically to induce new bone formation. The purpose of this study was to evaluate receptor utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal stem cells (hMSC), a physiologically relevant cell type that probably mediates the in vivo effects of BMPs. RNA interference-mediated gene knockdown revealed that osteoinductive BMP activities in hMSC are elicited through the type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2. BMPR1B and ACVR2B were expressed at low levels and were not found to play a significant role in signaling by any of the BMPs evaluated in this study. Type II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7, whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4. Significant differences were also observed for the type I receptors. Although BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to form homodimers and heterodimers was confirmed by bioluminescence resonance energy transfer analyses. These results suggest different mechanisms for BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Structure of the Alk1 Extracellular Domain and Characterization of Its Bone Morphogenetic Protein (BMP) Binding Properties

Bone morphogenetic proteins (BMPs) are secreted signaling proteins - they transduce their signals by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homologue Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the β-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the β4-β5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the β4-β5 loop, and to lesser extent β1-β2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9/Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3 but is rotated by 40 deg. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the β4-β5 loop that contribute significantly to binding and are nonconservatively substituted relative to the corresponding residues in Alk3.     

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.     

Expression of Bmp ligands and receptors in the developing Xenopus retina

Bone morphogenetic proteins (BMPs) act repeatedly in the development of nervous system tissues. While BMP signaling is critical for the early growth and patterning of the eye, we are interested in possible later functions of BMPs in the morphological development of retinal neurons and formation of synaptic connections. Therefore, we conducted an in situ hybridization analysis of the mRNA expression for the ligands Bmp2, -4 and 7 and the type Ia, Ib and II receptors (BmprIa, BmprIb and BmprII) during development of the retina of Xenopus laevis. Bmp4 mRNA is expressed in the dorsal retina and Bmp7 in the distal peripheral retina during the period of cell differentiation, while Bmp2 is not present in the eye. The type I receptors are expressed predominantly ventrally, from the optic vesicle stage until at least stage 35/36, after most cells have differentiated and many synaptic connections have formed. BmprII mRNA, however, is distributed evenly across the dorsoventral axis, with highest expression in retinal ganglion cell and inner nuclear layers.     

Osteogenic protein-1 differentially regulates the mRNA expression of bone morphogenetic proteins and their receptors in primary cultures of osteoblasts

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in long-term primary cultures of fetal rat calvaria (FRC) cells were examined by Northern analysis. Their temporal orders of expression were correlated with those of several biochemical markers characteristic of osteoblastic cell differentiation. Distinct temporal patterns of expression of BMPs and BMPRs during osteoblastic cell differentiation were observed. BMP-2 and BMP-7 mRNA levels did not change significantly. BMP-4 mRNA expression increased and reached a peak prior to matrix formation. BMP-5 mRNA expression increased during the mineralization phase and BMP-6 mRNA expression increased throughout all phases of cell differentiation. Effects of BMP-7 (Osteogenic Protein-1; OP-1) on the expression patterns of several other members of the BMP family and the receptors were also studied. OP-1 downregulated the BMP-4, -5, and -6 mRNA levels by a maximal of 2-fold, 1.5-fold, and 6-fold, respectively. OP-1 did not change significantly the OP-1 and BMP-2 mRNA expression. Of the three type I BMPR examined, OP-1 upregulated ActR-I and BMPR-IA mRNA expression slightly but with statistical significance. OP-1 downregulated BMPR-IB mRNA expression slightly. OP-1 upregulated BMPR-II mRNA expression by a maximum of 2-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and their receptors in osteoblasts. The observations suggest that OP-1 action on osteoblastic cells involves a complex regulation of gene expression of related members of the BMP family and their receptors in a cell differentiation stage dependent manner.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Mechanism of osteogenic induction by FK506 via BMP/Smad pathways

FK506 is an immunosuppressant that exerts effects by binding to FK506-binding protein 12 (FKBP12). Recently, FK506 has also been reported to promote osteogenic differentiation when administered locally or in vitro in combination with bone morphogenetic proteins (BMPs), although the underlying mechanism remains unclarified. The present study initially showed that FK506 alone at a higher concentration (1muM) induced osteogenic differentiation of mesenchymal cell lines, which was suppressed by adenoviral introduction of Smad6. FK506 rapidly activates the BMP-dependent Smads in the absence of BMPs, and the activation was blocked by Smad6. Overexpression of FKBP12, which was reported to block the ligand-independent activation of BMP type I receptor A (BMPRIA), suppressed Smad signaling induced by FK506, but not that induced by BMP2. BMPRIA and FKBP12 bound to each other, and this binding was suppressed by FK506. These data suggest that FK506 promotes osteogenic differentiation by activating BMP receptors through interacting with FKBP12.     

Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins

Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.     

BMPs and BMPRs in chicken ovary and effects of BMP-4 and -7 on granulosa cell proliferation and progesterone production in vitro

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are now known to have important roles in mammalian ovarian folliculogenesis. This study determined the expression of the mRNA encoding for BMPs and their receptors in the chicken ovary and explored possible roles for them. The expression of the mRNA for BMP-2, -4, -6, -7, and BMPR-IA, -IB, and -II was determined and quantified by a semiquantitative RT-PCR. The mRNAs for all the BMPs and receptors determined were present in both the granulosa (G) and theca (T) cells of the F1, F2, and F3 follicles. All BMP mRNAs increased in G cells with follicular development, whereas only BMP-7 mRNA had this trend in the T cells. BMP-2, -4, and -6 mRNAs in T were similar between follicles. BMPR-IA mRNA was similar in F2G and F3G but lower in F1G. BMPR-IB mRNA was similar in G of all follicles, and BMPR-II mRNA increased with development. In the T, each receptor subtype showed equal distribution between follicles. mRNA levels for BMPR-IB and -II were higher in G than in T, suggesting that the G is a major target for BMPs. BMP-4 and -7 stimulated basal, IGF-I-, and gonadotropin-stimulated progesterone production by cultured G cells, with differential responses between cells from the F1 and F3/4. This suggests involvement in follicular differentiation. BMP-4 and -7 reversed the inhibitory effects of transforming growth factor (TGF)-alpha on basal and gonadotropin-stimulated G cell progesterone production, with greater effect in the F1 than in the F3/4. This effect suggests an important role for BMPs interacting with TGF-alpha in modulating the effects of gonadotropins and IGF-I on follicular differentiation. Finally, BMP-7 stimulated G cell proliferation, but BMP-4 inhibited TGF-alpha+ IGF-I- and/or FSH-stimulated G cell proliferation, suggesting a role in the control of follicular growth during development. These effects of BMP-4 and -7 on the G cell function showed relationships with the expression levels of the BMPs and the BMPR-II.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

The BMP7/ActRII extracellular domain complex provides new insights into the cooperative nature of receptor assembly

Activins and bone morphogenetic proteins (BMPs) elicit diverse biological responses by signaling through two pairs of structurally related type I and type II receptors. Here we report the crystal structure of BMP7 in complex with the extracellular domain (ECD) of the activin type II receptor. Our structure produces a compelling four-receptor model, revealing that the types I and II receptor ECDs make no direct contacts. Nevertheless, we find that truncated receptors lacking their cytoplasmic domain retain the ability to cooperatively assemble in the cell membrane. Also, the affinity of BMP7 for its low-affinity type I receptor ECD increases 5-fold in the presence of its type II receptor ECD. Taken together, our results provide a view of the ligand-mediated cooperative assembly of BMP and activin receptors that does not rely on receptor-receptor contacts.