Modulating Bone Morphogenetic Protein Signaling Research Articles

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Complementary modulation of BMP signaling improves bone healing efficiency

The bone morphogenetic protein (BMP) signaling pathway plays a crucial role in bone development and regeneration. While BMP-2 is widely used as an alternative to autograft, its clinical application has raised concerns about adverse side effects and deteriorated bone quality. Therefore, there is a need to develop more sophisticated approaches to regulate BMP signaling and promote bone regeneration. Here, we present a novel complementary strategy that targets both BMP antagonist noggin and agonist Trb3 to enhance bone defect repair without the application of exogenous BMP-2. In vitro studies showed that overexpression of Trb3 with simultaneous noggin suppression significantly promotes osteogenic differentiation of mesenchymal stem cells. This was accompanied by increased BMP/Smad signaling. We also developed sterosome nanocarriers, a non-phospholipid liposomal system, to achieve non-viral mediated noggin suppression and Trb3 overexpression. The gene-loaded sterosomes were integrated onto an apatite-coated polymer scaffold for in vivo calvarial defect implantation, resulting in robust bone healing compared to BMP-2 treatments. Our work provides a promising alternative for high-quality bone formation by regulating expression of BMP agonists and antagonists.

A stress-responsive miRNA regulates BMP signaling to maintain tissue homeostasis

Adult organisms must sense and adapt to environmental fluctuations. In high-turnover tissues such as the intestine, these adaptive responses require rapid changes in gene expression that, in turn, likely involve posttranscriptional gene control. However, intestinal-tissue-specific microRNA (miRNA)-mediated regulatory pathways remain unexplored. Here, we report the role of an intestinal-specific miRNA, miR-958, that non-cell autonomously regulates stem cell numbers during tissue homeostasis and regeneration in the Drosophila adult midgut. We identify its downstream target cabut, the Drosophila ortholog of mammalian KLF10/11 transcription factors, which mediates this miR-958 function by promoting paracrine enterocyte-to-stem-cell bone morphogenetic protein (BMP) signaling. We also show that mature miR-958 levels transiently decrease in response to stress and that this decrease is required for proper stem cell expansion during tissue regeneration. In summary, we have identified a posttranscriptional mechanism that modulates BMP signaling activity within Drosophila adult intestinal tissue during both normal homeostasis and tissue regeneration to regulate intestinal stem cell numbers.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β-thalassemia. YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. Invitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?

Osteochondroprogenitors are crucial for embryonic bone development and postnatal processes such as bone repair in response to fracture injury, and their dysfunction may contribute to insufficient repair of structural damage in inflammatory arthritides. In the fracture healing, the early inflammatory phase is crucial for normal callus development and new bone formation. This process involves a complex interplay of many molecules and cell types, responsible for recruitment, expansion and differentiation of osteochondroprogenitor populations. In inflammatory arthritides, inflammation induces bone resorption and causes insufficient bone formation, which leads to local and systemic bone loss. While bone loss is a predominant feature in rheumatoid arthritis, inflammation also induces pathologic bone formation at enthesial sites in seronegative spondyloarthropathies. Bone morphogenetic proteins (BMP) are involved in cell proliferation, differentiation and apoptosis, and have fundamental roles in maintenance of postnatal bone homeostasis. They are crucial regulators of the osteochondroprogenitor pool and drive their proliferation, differentiation, and lifespan during bone regeneration. In this review, we summarize the effects of inflammation on osteochondroprogenitor populations during fracture repair and in inflammatory arthritides, with special focus on inflammation-mediated modulation of BMP signaling. We also present data in which we describe a population of murine synovial osteochondroprogenitor cells, which are reduced in arthritis, and characterize their expression of genes involved in regulation of bone homeostasis, emphasizing the up-regulation of BMP pathways in early progenitor subset. Based on the presented data, it may be concluded that during an inflammatory response, innate immune cells induce osteochondroprogenitors by providing signals for their recruitment, by producing BMPs and other osteogenic factors for paracrine effects, and by secreting inflammatory cytokines that may positively regulate osteogenic pathways. On the other hand, inflammatory cells may secrete cytokines that interfere with osteogenic pathways, proapoptotic factors that reduce the pool of osteochondroprogenitor cells, as well as BMP and Wnt antagonists. The net effect is strongly context-dependent and influenced by the local milieu of cells, cytokines, and growth factors. Further elucidation of the interplay between inflammatory signals and BMP-mediated bone formation may provide valuable tools for therapeutic targeting.

Marcksb plays a key role in the secretory pathway of zebrafish Bmp2b.

During vertebrate early embryogenesis, the ventral development is directed by the ventral-to-dorsal activity gradient of the bone morphogenetic protein (BMP) signaling. As secreted ligands, the extracellular traffic of BMP has been extensively studied. However, it remains poorly understood that how BMP ligands are secreted from BMP-producing cells. In this work, we show the dominant role of Marcksb controlling the secretory process of Bmp2b via interaction with Hsp70 in vivo. We firstly carefully characterized the role of Marcksb in promoting BMP signaling during dorsoventral axis formation through knockdown approach. We then showed that Marcksb cell autonomously regulates the trafficking of Bmp2b from producing cell to the extracellular space and both the total and the extracellular Bmp2b was decreased in Marcksb-deficient embryos. However, neither the zygotic mutant of marcksb (Zmarcksb) nor the maternal zygotic mutant of marcksb (MZmarcksb) showed any defects of dorsalization. In contrast, the MZmarcksb embryos even showed increased BMP signaling activity as measured by expression of BMP targets, phosphorylated Smad1/5/9 levels and imaging of Bmp2b, suggesting that a phenomenon of “genetic over-compensation” arose. Finally, we revealed that the over-compensation effects of BMP signaling in MZmarcksb was achieved through a sequential up-regulation of MARCKS-family members Marcksa, Marcksl1a and Marcksl1b, and MARCKS-interacting protein Hsp70.3. We concluded that the Marcksb modulates BMP signaling through regulating the secretory pathway of Bmp2b.

Upregulated BMP-Smad signaling activity in the glucuronyl C5-epimerase knock out MEF cells

Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce, in mice resulted in neonatal lethality with varied defects in organ development. To understand the underlying molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activitywhen cultured in osteogenic medium. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.

UNC-5 netrin receptor B mediates osteogenic differentiation by modulating bone morphogenetic protein signaling in human adipose-derived stem cells

UNC-5 netrin receptor B (UNC5B) is a dependence receptor of netrin-1 that plays an essential role in mediating angiogenesis and tumorigenesis. Despite its significant roles, there is limited knowledge about the role played by UNC5B in osteogenesis. In the present study, we first demonstrated that UNC5B was required for osteogenic differentiation of human adipose-derived stem cells (hASCs), both in vitro and in vivo. We also found that mechanistically, UNC5B promotes osteogenic differentiation by activating bone morphogenetic protein signaling. These findings point to a new important function of UNC5B and provide a potential basis for hASCs-mediated bone regeneration.

Hoxa2 Inhibits Bone Morphogenetic Protein Signaling during Osteogenic Differentiation of the Palatal Mesenchyme.

Cleft palate is one of the most common congenital birth defects worldwide. The homeobox (Hox) family of genes are key regulators of embryogenesis, with Hoxa2 having a direct role in secondary palate development. Hoxa2−/− mice exhibit cleft palate; however, the cellular and molecular mechanisms leading to cleft palate in Hoxa2−/− mice is largely unknown. Addressing this issue, we found that Hoxa2 regulates spatial and temporal programs of osteogenic differentiation in the developing palate by inhibiting bone morphogenetic protein (BMP) signaling dependent osteoblast markers. Expression of osteoblast markers, including Runx2, Sp7, and AlpI were increased in Hoxa2−/− palatal shelves at embryonic day (E) 13.5 and E15.5. Hoxa2−/− mouse embryonic palatal mesenchyme (MEPM) cells exhibited increased bone matrix deposition and mineralization in vitro. Moreover, loss of Hoxa2 resulted in increased osteoprogenitor cell proliferation and osteogenic commitment during early stages of palate development at E13.5. Consistent with upregulation of osteoblast markers, Hoxa2−/− palatal shelves displayed higher expression of canonical BMP signaling in vivo. Blocking BMP signaling in Hoxa2−/− primary MEPM cells using dorsomorphin restored cell proliferation and osteogenic differentiation to wild-type levels. Collectively, these data demonstrate for the first time that Hoxa2 may regulate palate development by inhibiting osteogenic differentiation of palatal mesenchyme via modulating BMP signaling.

Neuroligin 4 regulates synaptic growth via the bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction

The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1–3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. Here, we report that the Drosophila neuroligin 4 (DNlg4) is different from DNlg1–3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, we looked for genetic interactions and found connections with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and Western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor thickvein (Tkv) abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. Our study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv.

Bone Morphogenetic Protein-Based Therapeutic Approaches.

Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the transforming growth factor (TGF)-β family of ligands and exert most of their effects through the canonical effectors Smad1, 5, and 8. Appropriate regulation of BMP signaling is critical for the development and homeostasis of numerous human organ systems. Aberrations in BMP pathways or their regulation are increasingly associated with diverse human pathologies, and there is an urgent and growing need to develop effective approaches to modulate BMP signaling in the clinic. In this review, we provide a wide perspective on diseases and/or conditions associated with dysregulated BMP signal transduction, outline the current strategies available to modulate BMP pathways, highlight emerging second-generation technologies, and postulate prospective avenues for future investigation.

A Survey of Strategies to Modulate the Bone Morphogenetic Protein Signaling Pathway: Current and Future Perspectives.

Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the TGF-β family of ligands and are unequivocally involved in regulating stem cell behavior. Appropriate regulation of canonical BMP signaling is critical for the development and homeostasis of numerous human organ systems, as aberrations in the BMP pathway or its regulation are increasingly associated with diverse human pathologies. In this review, we provide a wide-perspective on strategies that increase or decrease BMP signaling. We briefly outline the current FDA-approved approaches, highlight emerging next-generation technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways.

Fly LMBR1/LIMR-type protein Lilipod promotes germ-line stem cell self-renewal by enhancing BMP signaling

Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. However, their biological function is unknown. Here, we show that the fly family member Lilipod (Lili) is required for germ-line stem cell (GSC) self-renewal in the Drosophila ovary where it enhances bone morphogenetic protein (BMP) signaling. lili mutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factor bam. Conversely, overexpressed Lili induces supernumerary pMad-positive bamP-GFP-negative GSCs. Interestingly, differentiation of lili mutant GSCs is bam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing a bam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first, to our knowledge, in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.

RGM co-receptors add complexity to BMP signaling.

Ten years ago, the repulsive guidance molecules (RGMs), a family of three glycosylphosphatidylinositol–anchored glycoproteins, were identified as highly specific co-receptors of the bone morphogenetic proteins (BMPs). Newly reported crystal structures provide exciting insights into how RGM co-receptors may modulate BMP signaling.

Roles of bone morphogenetic protein signaling in osteosarcoma.

Since the original extraction of bone morphogenetic proteins (BMPs) from bovine bone, research interest and clinical use has increased exponentially. With this, a concomitant analysis of BMP expression in bone tumours has been performed. BMP ligands, receptors, and signaling activity have been observed in diverse benign and malignant bone tumours. However, the reported expression, function, and importance of BMPs in bone tumours, and specifically osteosarcomas, have been far from uniform. This review highlights recent advances in understanding the role of BMP signaling in osteosarcoma biology, focusing on the sometimes divergent findings by various researchers and the challenges inherent in the study of osteosarcoma. We performed a literature review of all studies examining BMP signaling in osteosarcoma. Overall, multiple BMP ligands and receptors are expressed in most osteosarcoma cell lines and subtypes, although BMP signaling may be reduced in comparison with benign bone-forming tumours. Studies suggest that osteosarcomas with different lineages of differentiation may have differential expression of BMP ligands. Although significant disagreement in the literature exists, the presence of BMP signaling in osteosarcoma may impart a worse prognosis. On the cellular level, BMP signaling appears to mediate promigratory effects in osteosarcoma and chondrosarcoma cell types, possibly via interaction and activation of Integrin β1. BMP signaling has clear biologic importance in osteosarcoma, although it is not yet fully understood. Future questions for study include assessing the utility of BMP signaling in prognostication of osteosarcoma and the potential modulation of BMP signaling for inhibition of osteosarcomagenesis, growth and invasion.

Link-N Exerts Its Growth Factor-Like Effects through Activation of Smad 1/5 Signaling in Human IVD Cells

Introduction Back pain is a fairly common problem, which affects a large portion of the population and has an impact on quality of life. Intervertebral disc (IVD) degeneration is one of the most common causes of back pain. Link-N peptide represents the 16 amino acid sequence (DHLSDNYTLDHDRAIH) from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and IVDs. We have previously shown that Link-N can stimulate collagen and proteoglycan synthesis in IVD cells in vitro1 and in intact human IVDs ex vivo,2 as well as increase disc height in a rabbit model of disc degeneration.3 Recently, BMPII receptor has been shown to interact with Link-N in rabbit IVD cells.4 The purpose of this study was to determine the mechanism by which Link-N exerts its beneficial effects by promoting matrix production in human IVD cells. Materials and Methods Human lumbar spines were retrieved through the Transplant Quebec organ donation program. IVD cells were isolated from nucleus pulposus (NP) and annulus fibrosus (AF) regions. The cells were cultured to 90% confluence in NP Cell Medium (NPCM, Cat. No.4801, ScienCell). Then the cells were incubated overnight in serum-free DMEM followed by treatment with Link-N (1 μg/mL), TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) for 10 minutes to 4 hours. TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) was used as positive controls. Cell extracts were prepared using NP40 lysis buffer. Total protein in the cell extracts was determined by the bicinchoninic acid assay (BCA). Protein expression was analyzed by immunoblotting using specific antibodies. Western blot images were quantified using ImageJ (NIH) software. For gene expression studies, cells were incubated for 24 hours with Link-N (1 μg/mL) in the presence or absence of human recombinant noggin (100 ng/mL) and total RNA was isolated using Trizol reagent and relative gene expression was determined by RT PCR. For receptor-binding studies, AF and NP cells were treated with biotinylated Link-N (1 μg/mL) with or without untagged Link-N (10 μg/mL) for competitive displacement. Scrambled biotinylated Link-N (1 μg/mL) was used as a specificity control. After treatments, cells were fixed in 2% paraformaldehyde. Fixed cells were treated with fluorescent-dye conjugated streptavidin (Alexa Fluor 700 conjugate, streptavidin). We assessed whether noggin directly binds with biotinylated Link-N by dot-blot technique. Results Link-N addition to human IVD cells did not promote the phosphorylation of Smads 2 and 3. This suggested that Link-N signaling does not function in a fashion similar to TGF-β, which stimulates the phosphorylation/activation of Smads 2 and 3. Further, similar to BMP-2, Link-N promoted the phosphorylation of Smad ? (Fig. 1). These results suggest that Link-N exerts its growth factor-like effects via a BMP-signaling mechanism rather than TGF-β. Noggin is a known physiological negative modulator of BMP signaling and noggin levels are elevated in disease conditions. As we observed that Link-N signaling is similar to BMP, it is essential to assess if noggin can interfere with the function of Link-N. Dot-blot results showed that noggin does not directly bind with Link-N. Therefore, noggin inhibition of Link-N signaling is unlikely because of noggin binding with Link-N. This suggests that Link-N may have an added advantage as a therapeutic agent over BMPs. In addition, Link-N increased BMP 4 expression significantly in both AF and NP cells, with the increase in AF cells being significantly higher than that in NP cells. Specific binding (also internalization) of biotinylated Link-N was found both in AF and NP cells, as this binding was competitively displaced by nonbiotinylated Link-N. No binding was seen with scrambled biotinylated Link-N. Conclusion Link-N represents a potential economic growth factor with beneficial effects on disc repair and the identification of the intracellular signaling pathways that it transduces is a step further toward clinical trials on NP repair in the degenerate human IVD. Disclosure of Interest None declared References Mwale F, Demers CN, Petit A, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem 2003;88(6):1202–1213 Gawri R, Antoniou J, Ouellet J, et al. Best paper NASS 2013: link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs. Eur Cell Mater 2013;26:107-119, discussion 119 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120 Wang Z, Weitzmann MN, Sangadala S, Hutton WC, Yoon ST. Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J Biol Chem 2013;288(39):28243–28253

The clip-segment of the von Willebrand domain 1 of the BMP modulator protein Crossveinless 2 is preformed.

Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.

Abstract 3759: SMURF1 modulates BMP signaling and maintenance of HNSCC cancer stem cells.

Abstract Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal and differentiation. In addition, they are highly implicated in drug resistance and tumor recurrence, which are associated with treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Bone morphogenetic protein (BMP) signaling pathways are involved in orchestrating diverse biological functions, as well as regulation of embryogenesis, cell differentiation, proliferation, migration, and apoptosis. BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), which has been implicated in invasiveness in several malignancies. The roles of BMP signaling and SMURF1 regulation in HNSCC CSC phenotypes and chemoresistance are not known. Objectives: In this study, our objectives were to enrich CSC populations from HNSCC cell lines and to characterize molecular mechanisms promoting CSC survival and drug resistance. Methods: Enrichment of HNSCC CSCs was performed using long-term anchorage-independent sphere forming growth under serum-free and low oxygen conditions. Sphere-forming cells were then characterized for CSC-like phenotypes using qRT-PCR and immunoblotting for known CSC markers. Sphere cells also were tested for CSC-like characteristics, including long-term survival and self-renewal using a sphere reformation assay, ability to undergo adipogenesis, and chemoresistance. Results: We determined that sphere-forming cells mimic characteristics previously attributed to CSCs, showing increased expression of CD44 and BMI1. Sphere cells also showed markedly higher SMURF1 expression than their monolayer counterparts cultured under standard anchorage-dependent differentiating growth conditions, suggesting that these sphere-forming cells may have reduced BMP signaling, differentiation, and growth. Consistently, BMP receptor downstream signal transduction was inactivated, as indicated by decreased phosphorylation of SMAD1/5/8 and down-regulation of downstream BMP target genes, including ID1 and ID2. Other BMP signaling inhibitors, BMPER and BAMBI, were upregulated in sphere cells. Resistance to the anti-cancer agent, cisplatin, was also higher in sphere cells. Knockdown of SMURF1 expression by shRNA did not affect initial sphere formation and resistance to cisplatin, but it reduced long-term sphere renewal (or maintenance) and enhanced adipogenesis. Sphere cells with high SMURF1 expression showed low or no adipogenic differentiation. Conclusion: Our data suggest that SMURF1 inhibits BMP signaling in HNSCC to maintain the CSC niche by controlling stemness and quiescence of the CSC population, but it has no direct effect on chemoresistance. Targeting SMURF1 may therefore provide a long-term benefit for HNSCC treatment to reduce resistant CSC populations and disease recurrence. Citation Format: Ali Khammanivong, Miranda J. Mulligan, Nicholas J. Brady, Raj Gopalakrishnan, Erin B. Dickerson. SMURF1 modulates BMP signaling and maintenance of HNSCC cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3759. doi:10.1158/1538-7445.AM2013-3759

Comparative Genomics Identifies The Mouse Bmp3 Promoter and an Upstream Evolutionary Conserved Region (ECR) in Mammals

The Bone Morphogenetic Protein (BMP) pathway is an ancient, multi‐member signaling cascade whose basic components are found in all animals. One member, BMP3, which is found only in deuterostomes, serves a unique role as an antagonist to both the canonical BMP and Activin pathways. However, the mechanisms and cis‐regulatory regions controlling BMP3 expression remain poorly defined. With this in mind, we sought to identify the Bmp3 promoter in mouse through functional and comparative genomic analyses. We found that the minimal promoter required for expression resides within 0.8 kb upstream of Bmp3 in a region that is highly conserved with rat. We also found that an upstream region abutting the minimal promoter acts as a repressor of the minimal promoter. Strikingly, a portion of this region is conserved among all available eutherian mammal genomes (47/47), but not in any non‐eutherian animal (0/136). We also identified multiple conserved transcription factor binding sites in the Bmp3 upstream ECR, suggesting that this region may preserve common cis‐regulatory elements that govern Bmp3 expression across eutherian mammals. Since dysregulation of BMP signaling appears to play a role in human health and disease, our findings may have application in the development of novel therapeutics aimed at modulating BMP signaling in humans. This work was supported by an American Recovery & Reinvestment Act Award.