Functions Of Bone Morphogenetic Proteins Research Articles

Calmodulin-dependent protein kinase IV mediated antagonism of BMP signaling regulates lineage and survival of hematopoietic progenitors

In the current study, we show that bone morphogenetic proteins (BMPs) play a role in hematopoiesis that is independent of their function in specifying ventral mesodermal fate. When BMP activity is upregulated or inhibited in Xenopus embryos hematopoietic precursors are specified properly but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of BMP activity induces erythroid precursors to undergo apoptotic cell death, whereas constitutive activation of BMPs causes an increase in commitment of hematopoietic progenitors to myeloid differentiation and a concomitant decrease in erythrocytes that is not due to enhanced apoptosis. These blood defects are observed even when BMP activity is misregulated solely in non-hematopoietic (ectodermal) cells, demonstrating that BMPs generate extrinsic signals that regulate hematopoiesis independent of mesodermal patterning. Further analysis revealed that endogenous calmodulin-dependent protein kinase IV (CaM KIV) is required to negatively modulate hematopoietic functions of BMPs downstream of receptor activation. Our data are consistent with a model in which CaM KIV inhibits BMP signals by activating a substrate, possibly cAMP-response element-binding protein (CREB), that recruits limiting amounts of CREB binding protein (CBP) away from transcriptional complexes functioning downstream of BMPs.

The specification of noradrenergic locus coeruleus (LC) neurones depends on bone morphogenetic proteins (BMPs).

The role of BMPs in the development of the major noradrenergic centre of the brain, the locus coeruleus (LC), was investigated. LC generation is reflected by initial expression of the transcription factors Phox2a and Phox2b in dorsal rhombomere1 (r1), followed by expression of dopamine-beta-hydroxylase and tyrosine hydroxylase. Bmp5 is expressed in the dorsal neuroepithelium in proximity to Phox2-expressing cells. BMP inhibition in stage 10 chick embryos resulted in the lack of LC neurones or in their generation at the dorsal midline, and loss of roof plate and rhombic lip, but it did not affect neural crest development. These results reveal late essential BMP functions in the specification of dorsal neuronal phenotypes in r1, including LC neurones, and in the development of dorsal midline structures.

Bone Morphogenetic Protein (BMP)-2 Induces Apoptosis in Human Myeloma Cells

BMPs (bone morphogenetic proteins), members of the transforming growth factor (TGF)- β superfamily, are a group of related proteins which are capable of inducing the formation of cartilage and bone, but are now regarded as multifunctional cytokines. However, little is known about their role in hematopoiesis. Recently, we found a novel function of BMPs to hematopoietic cells in that BMP-2 induces apoptosis not only in human myeloma cell lines, but also in primary samples from patients with multiple myeloma in vitro. BMP-2 caused cell cycle arrest in the G1 phase which was associated with accumulation of p21 CIP1/WAF1 and p27 KIP1, and the subsequent apoptosis of myeloma cells. Further analysis showed that BMP-2 induced down-regulation of Bcl-x L through the inactivation of STAT3, resulting in the induction of apoptosis in myeloma cells. We conclude that BMP-2 may have the potential to be one of the novel therapeutic agents for treatment in patients with multiple myeloma because of the beneficial effects on both myeloma cells and bone diseases. In this review, we summarize data concerning BMPs and BMP-2-induced apoptosis of myeloma cells including our own recent experimental data.

BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb.

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.

Zic Gene expression marks anteroposterior pattern in the presumptive neurectoderm of the zebrafish gastrula.

Anteroposterior (A/P) patterning of the vertebrate neurectoderm begins early during development. In vitro explant assays have been used to show that an A/P subdivision of the zebrafish neurectoderm is in place by early gastrulation. However, no direct markers of this subdivision had been described. We report isolation of two members of the zic gene family, zic2 and zic3, which mark distinct regions in the developing nervous system. zic3 is expressed in early gastrula embryos in a posterior domain, directly demonstrating that A/P pattern is present in the future neurectoderm by this stage. Analysis of mutants in genes encoding bone morphogenetic proteins (BMPs) shows that dorsal restriction of zic3 expression requires intact BMP function, and that posterior restriction of zic3 expression is regulated independently of BMP signaling. These data show an early subdivision of the A/P axis in the presumptive neurectoderm.

Neural induction takes a transcriptional twist.

Over the past decade, several molecules have been identified that influence neural cell fate in vertebrate embryos during gastrulation. The first neural inducers studied were proteins produced by dorsal mesoderm (the Spemann organizer); most of these proteins act by directly binding to and antagonizing the function of bone morphogenetic proteins (BMPs). Recent experiments have suggested that other secreted signals, such as Wnt and FGF, may neuralize ectoderm before organizer function by a different mechanism. Neural effector genes that mediate the response of ectoderm to secreted neuralizing signals have also been discovered. Interestingly, most of these newly identified neuralizing pathways continue the theme of BMP antagonism, but rather than antagonizing BMP protein function, they may neuralize tissue by suppressing Bmp expression. Down-regulation of Bmp expression in the prospective neural plate during gastrulation seems to be a shared feature of neural induction in vertebrate embryos. However, the signals used to accomplish this task seem to vary among vertebrates. Here, we will discuss the role of the recently identified secreted signals and neural effector genes in vertebrate neurogenesis.

Perspectives in the biological function, the technical and therapeutic application of bone morphogenetic proteins.

The bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of growth and differentiation factors and have been characterized by their ability to induce new bone formation in ectopic (non-skeletal) sites. BMPs are secreted molecules and are key regulators in early embryogenesis and organogenesis. One of the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in vertebrates. This osteo-inductive capacity of BMPs has long been considered very promising for applications in bone repair, in the treatment of skeletal diseases, and in oral applications such as dentiogenesis and cementogenesis during regeneration of periodontal wounds. We discuss here biological roles of the BMPs in the organism and their signaling cascades leading to bone and cartilage formation in particular. It is also the aim of this review to evaluate the potential and the problems of BMPs in skeletal tissue engineering for the regeneration of bone damaged by disease or trauma and to serve as therapeutic agents for periodontal defects.

Suppression of Tumor Necrosis Factor-mediated Apoptosis by Nuclear Factor κB-independent Bone Morphogenetic Protein/Smad Signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.

Effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat.

We have previously established the presence of a functional bone morphogenetic protein (BMP) system in the ovary by demonstrating the expression of BMP ligands and receptors as well as novel cellular functions. Specifically, BMP-4 and BMP-7 are expressed in theca cells, and their receptors by granulosa cells. These BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. To investigate the underlying mechanism of the differential regulation, we analyzed mRNA levels for key regulators in the steroid biosynthetic pathways by RNase protection assay. BMP-7 enhanced P450 aromatase (P450(arom)) but suppressed steroidogenic acute regulatory protein (StAR) mRNAs induced by FSH, whereas mRNAs encoding further-downstream steroidogenic enzymes, including P450 side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase, were not significantly altered. These findings suggest that BMP-7 stimulation and inhibition of P450(arom) and StAR mRNA expression, respectively, may play a role in the mechanisms underlying the differential regulation of estradiol and progesterone production. To establish the physiological relevance of BMP functions, we investigated the in vivo effects of injections of recombinant BMP-7 into the ovarian bursa of rats. Ovaries treated with BMP-7 had decreased numbers of primordial follicles, yet had increased numbers of primary, preantral, and antral follicles, suggesting that BMP-7 may act to facilitate the transition of follicles from the primordial stage to the pool of primary, preantral, and antral follicles. In this regard, we have also found that BMP-7 caused an increase in DNA synthesis and proliferation of granulosa cells from small antral follicles in vitro. In contrast to the stimulatory activity, BMP-7 exhibited pronounced inhibitory effects on ovulation rate and serum progesterone levels. These findings establish important new biological activities of BMP-7 in the context of ovarian physiology, including folliculogenesis and ovulation.

Twisted gastrulation can function as a BMP antagonist.

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal-ventral development. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG), modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal-ventral patterning, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.

Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes.

To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.

Comparison of bone morphogenetic protein receptors expression in the fetal and adult skin.

Wounds on fetal skin can be repaired without leaving scars until the second trimester, but after this period, skin wounds leave scars as in adults. It's known that certain growth factors such as TGF-beta, and bFGF are present at a very low levels during wound repair in fetal skin. These low levels of growth factors minimize inflammatory response and fibroblast proliferation at the wound site, which in turn inhibit collagen synthesis, and thus, allows scarless wound healing. Recently bone morphogenetic proteins (BMPs), one of the TGF-beta superfamily members, have been studied in the wound healing process. According to several studies, BMPs are related to the differentiation and growth of epithelial and mesenchymal cells, but the precise functions of BMPs and of BMP receptors on skin wound healing have not been elucidated. In this study, we investigated the expression pattern of BMP receptors in fetal skin during the second trimester and in adult skin by immunohistochemical staining and RT-PCR. BMP receptors were detected on the suprabasal epithelial cells and in the hair follicles in adult skin, but were not defected in the fetal skin except for the hair follicles. This was confirmed by confirming mRNA levels of BMP receptors by RT-PCR in both adult and fetal skins. In conclusion, BMPs and BMP receptors seem to be related to fetal and adult wound healing, and low levels of BMPs and BMP receptors during the second trimester seem to contribute to scarless wound healing in the fetus, as is TGF-beta during the second trimester.

Head and trunk in zebrafish arise via coinhibition of BMP signaling by bozozok and chordino.

Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double loss-of-function mutations in BMP inhibitors have less dramatic effects. We report that combining mutations in the zebrafish genes bozozok and chordino causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk.

Evidence for an evolutionary conserved role of bone morphogenetic protein growth factors and phox2 transcription factors during noradrenergic differentiation of sympathetic neurons. Induction of a putative synexpression group of neurotransmitter-synthesizing enzymes.

The noradrenergic transmitter phenotype in postganglionic sympathetic neurons is induced early during embryonic development in avian and mammalian primary sympathetic ganglia. The simultaneous expression of tyrosine hydroxylase and dopamine beta-hydroxylase, enzymes of the noradrenaline biosynthesis pathway, indicates that different genes contributing to the noradrenergic transmitter phenotype are regulated as a synexpression group. This conclusion is supported by the demonstration of bone morphogenetic protein (BMP) growth factors and Phox2 transcription factors being necessary for the expression of both tyrosine hydroxylase and dopamine beta-hydroxylase in differentiating sympathetic neurons. The close similarity in the expression patterns of the relevant genes as well as in the function of BMPs and Phox2s between avian and mammalian embryos strongly suggests that noradrenergic induction occurs along a conserved signalling pathway in these vertebrate classes.

Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3

Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2-induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21(CIP1/WAF1) and p27(KIP1)) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-x(L); however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-x(L) and by cell-cycle arrest through the up-regulation of p21(CIP1/WAF1) and p27(KIP1) and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

The transcription factor dHAND is a downstream effector of BMPs in sympathetic neuron specification.

The dHAND basic helix-loop-helix transcription factor is expressed in neurons of sympathetic ganglia and has previously been shown to induce the differentiation of catecholaminergic neurons in avian neural crest cultures. We now demonstrate that dHAND expression is sufficient to elicit the generation of ectopic sympathetic neurons in vivo. The expression of the dHAND gene is controlled by bone morphogenetic proteins (BMPs), as suggested by BMP4 overexpression in vivo and in vitro, and by noggin-mediated inhibition of BMP function in vivo. The timing of dHAND expression in sympathetic ganglion primordia, together with the induction of dHAND expression in response to Phox2b implicate a role for dHAND as transcriptional regulator downstream of Phox2b in BMP-induced sympathetic neuron differentiation.

Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development.

Transforming growth factor beta (TGFbeta) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFbeta receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFbeta signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFbeta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2(delta)exon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2(delta)exon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2(delta)exon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGF(delta) superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho-Smad2/Smad2(delta)exon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2(delta)exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo.

The family of bone morphogenetic proteins

The family of bone morphogenetic proteins. Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The first BMPs were originally identified by their ability to induce ectopic bone formation when implanted under the skin of rodents. In this ectopic overexpression assay, there was a recapitulation of all the events occurring during skeletogenesis. This latter aspect indicated that these molecules could play important roles during development. More than 30 BMPs have been identified to date. The study of their expression pattern as well as the analysis of spontaneously mutated or genetically depleted mice have demonstrated a much broader range of function. These activities are mainly localized at sites of epithelial-mesenchymal interactions, including but not restricted to the skeleton. This review presents our current knowledge about the functions of BMPs during skeleton development as well as in many other biologic processes.

Bone morphogenetic proteins and bone morphogenetic protein gene therapy in neurological surgery: a review.

To review the uses of bone morphogenetic proteins (BMPs) and BMP gene therapy for the treatment of neurosurgical disorders. Literature review. BMPs are members of the transforming growth factor beta superfamily, and they play an important role in the growth and development of numerous tissues, including bone, brain, and spinal cord. Although the majority of previous studies have focused on the regulatory functions of BMPs in the normal growth and differentiation of the skeletal system, BMPs also seem to be exquisitely involved in the regulation of cellular proliferation, survival, differentiation, apoptosis, and lineage commitment in the central nervous system. When specific BMPs are delivered on biological matrices, they have the capacity to induce bone, cartilage, ligament, and tendon at both heterotopic and orthotopic sites, suggesting that they may play a major role in the future treatment of spinal and craniofacial pathology. For example, recent studies have clearly demonstrated the usefulness of BMPs and BMP gene therapy for the induction of spinal arthrodesis in several animal models. In addition, several BMPs have been shown to have a neuroprotective effect in animal models of head injury, cerebral ischemia, and Parkinson's disease and may therefore have direct clinical applications for the treatment of central nervous system disorders. As the physiological activity of BMPs in the development and pathology of the central nervous system and spine are more fully elucidated, BMP therapeutics and gene therapy will probably have numerous applications in neurological surgery.

Protein Kinase A Is a Negative Regulator of Renal Branching Morphogenesis and Modulates Inhibitory and Stimulatory Bone Morphogenetic Proteins

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961-F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.