Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Novel contact-dependent bone morphogenetic protein (BMP) signaling mediated by heparan sulfate proteoglycans

We previously proposed a model that DALLY, a Drosophila glypican, acts as a trans co-receptor to regulate BMP signaling in the germ line stem cell niche. To investigate the molecular mechanisms of contact-dependent BMP signaling, we developed novel in vitro assay systems to monitor trans signaling using Drosophila S2 cells. Using immunoblot-based as well as single-cell assay systems, we present evidence that Drosophila glypicans indeed enhance BMP signaling in trans in a contact-dependent manner in vitro. Our analysis showed that heparan sulfate modification is required for the trans co-receptor activity of DALLY. Two BMP-like molecules, Decapentaplegic (DPP) and Glass bottom boat, can mediate trans signaling through a heparan sulfate proteoglycan co-receptor in S2 cells. The in vitro systems reflect the molecular characteristics of heparan sulfate proteoglycan functions observed previously in vivo, such as ligand specificity and biphasic activity dependent on the ligand dosage. In addition, experiments using a DALLY-coated surface suggested that DALLY regulates DPP signaling in trans by its effect on the stability of DPP protein on the surface of the contacting cells. Our findings provide the molecular foundation for novel contact-dependent signaling, which defines the physical space of the stem cell niche in vivo.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.     

Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.     

The Short Stature Homeobox 2 (Shox2)-bone Morphogenetic Protein (BMP) Pathway Regulates Dorsal Mesenchymal Protrusion Development and Its Temporary Function as a Pacemaker during Cardiogenesis

The atrioventricular (AV) junction plays a critical role in chamber septation and transmission of cardiac conduction pulses. It consists of structures that develop from embryonic dorsal mesenchymal protrusion (DMP) and the embryonic AV canal. Despite extensive studies on AV junction development, the genetic regulation of DMP development remains poorly understood. In this study we present evidence that Shox2 is expressed in the developing DMP. Intriguingly, this Shox2-expressing domain possesses a pacemaker-specific genetic profile including Hcn4 and Tbx3. This genetic profile leads to nodal-like electrophysiological properties, which is gradually silenced as the AV node becomes matured. Phenotypic analyses of Shox2^−/− mice revealed a hypoplastic and defectively differentiated DMP, likely attributed to increased apoptosis, accompanied by dramatically reduced expression of Bmp4 and Hcn4, ectopic activation of Cx40, and an aberrant pattern of action potentials. Interestingly, conditional deletion of Bmp4 or inhibition of BMP signaling by overexpression of Noggin using a Shox2-Cre allele led to a similar DMP hypoplasia and down-regulation of Hcn4, whereas activation of a transgenic Bmp4 allele in Shox2^−/− background attenuated DMP defects. Moreover, the lack of Hcn4 expression in the DMP of mice carrying Smad4 conditional deletion and direct binding of pSmad1/5/8 to the Hcn4 regulatory region further confirm the Shox2-BMP genetic cascade in the regulation of DMP development. Our results reveal that Shox2 regulates DMP fate and development by controlling BMP signaling through the Smad-dependent pathway to drive tissue growth and to induce Hcn4 expression and suggest a temporal pacemaking function for the DMP during early cardiogenesis.     

An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.     

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.     

SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

Lung tumor-associated osteoblast-derived bone morphogenetic protein-2 increased epithelial-to-mesenchymal transition of cancer by Runx2/Snail signaling pathway

Bone is a frequent target of lung cancer metastasis and is associated with significant morbidity and a dismal prognosis. Interaction between cancer cells and the bone microenvironment causes a vicious cycle of tumor progression and bone destruction. This study analyzed the soluble factors secreted by lung tumor-associated osteoblast (TAOB), which are responsible for increasing cancer progression. The addition of bone morphogenetic protein-2 (BMP-2), present in large amounts in TAOB conditioned medium (TAOB-CM) and lung cancer patient sera, mimicked the inductive effect of TAOB-CM on lung cancer migration, invasion, and epithelial-to-mesenchymal transition. In contrast, inhibition of BMP by noggin decreases the inductive properties of TAOB-CM and lung cancer patient sera on cancer progression. Induction of lung cancer migration by BMP-2 is associated with increased ERK and p38 activation and the up-regulation of Runx2 and Snail. Blocking ERK and p38 by a specific inhibitor significantly decreases cancer cell migration by inhibiting Runx2 up-regulation and subsequently attenuating the expression of Snail. Enhancement of Runx2 facilitates Rux2 to recruit p300, which in turn enhances histone acetylation, increases Snail expression, and decreases E-cadherin. Furthermore, inhibiting Runx2 by siRNA also suppresses BMP-2-induced Snail up-regulation and cell migration. Our findings provide novel evidence that inhibition of BMP-2 or BMP-2-mediated MAPK/Runx2/Snail signaling is an attractive therapeutic target for osteolytic bone metastases in lung cancer patients.