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Sample records for morphogenetic protein receptors

  1. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  2. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  3. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  4. Bone Morphogenetic Proteins.

    PubMed

    Katagiri, Takenobu; Watabe, Tetsuro

    2016-01-01

    Bone morphogenetic proteins (BMPs), originally identified as osteoinductive components in extracts derived from bone, are now known to play important roles in a wide array of processes during formation and maintenance of various organs including bone, cartilage, muscle, kidney, and blood vessels. BMPs and the related "growth and differentiation factors" (GDFs) are members of the transforming growth factor β (TGF-β) family, and transduce their signals through type I and type II serine-threonine kinase receptors and their intracellular downstream effectors, including Smad proteins. Furthermore, BMP signals are finely tuned by various agonists and antagonists. Because deregulation of the BMP activity at multiple steps in signal transduction is linked to a wide variety of human diseases, therapeutic use of activators and inhibitors of BMP signaling will provide potential avenues for the treatment of the human disorders that are caused by hypo- and hyperactivation of BMP signals, respectively. PMID:27252362

  5. The biological function of type I receptors of bone morphogenetic protein in bone

    PubMed Central

    Lin, Shuxian; Svoboda, Kathy K H; Feng, Jian Q; Jiang, Xinquan

    2016-01-01

    Bone morphogenetic proteins (BMPs) have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors (BMPRI and BMPRII). In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 (ALK2, also called type IA activin receptor), activin-like kinase 3 (ALK3, also called BMPRIA), and activin-like kinase 6 (ALK6, also called BMPRIB). The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future. PMID:27088043

  6. Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling

    PubMed Central

    Vogt, Janis; Dingwell, Kevin S.; Herhaus, Lina; Gourlay, Robert; Macartney, Thomas; Campbell, David; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway. PMID:24554596

  7. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. PMID:26475222

  8. Bone morphogenetic protein

    SciTech Connect

    Xiao Yongtao; Xiang Lixin; Shao Jianzhong

    2007-10-26

    Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.

  9. Long-Range Communication Network in the Type 1B Bone Morphogenetic Protein Receptor.

    PubMed

    Evangelista, Wilfredo; Yeh, Lee-Chuan C; Gmyrek, Aleksandra; Lee, J Ching; Lee, John C

    2015-12-01

    Protein-protein interactions are recognized as a fundamental phenomenon that is intimately associated with biological functions and thus are ideal targets for developing modulators for regulating biological functions. A challenge is to identify a site that is situated away from but functionally connected to the protein-protein interface. We employed bone morphogenetic proteins (BMPs) and their receptors as a model system to develop a strategy for identifying such a network of communication. Accordingly, using computational analyses with the COREX/BEST algorithm, we uncovered an overall pattern connecting various regions of BMPR-1B ectodomain, including the four conserved residues in the protein-protein interface. In preparation for testing the long-range effects of mutations of distal residues for future studies, we examined the extent of measurable perturbation of the four conserved residues by determination of the conformation and relative affinities of these BMPR-1B mutants for ligands BMP-2, -6, and -7 and GDF-5. Results suggest no significant structural changes in the receptor but do suggest that the four residues play different roles in defining ligand affinity and both intra- and intermolecular interactions play a role in defining ligand affinity. Thus, these results established two primary but necessary goals: (1) the baseline knowledge of perturbation of conserved interfacial residues for future reference and (2) the ability of the computational approach to identify the distal residues connecting to the interfacial residues. The data presented here provide the foundation for future experiments to identify the effects of distal residues that affect the specificity and affinity of BMP recognition. Protein-protein interactions are integral reactions in essentially all biological activities such as gene regulation and age-related development. Often, diseases are consequences of the alteration of these intermacromolecular interactions, which are thus recognized

  10. Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis

    PubMed Central

    Robinson, Linda J.; Moore, Christy S.; Blackwell, Thomas R.; Gladson, Santhi; Penner, Niki L.; Burman, Ankita; McClellan, Lucas J.; Polosukhin, Vasiliy V.; Tanjore, Harikrishna; McConaha, Melinda E.; Gleaves, Linda A.; Talati, Megha A.; Hemnes, Anna R.; Fessel, Joshua P.; Lawson, William E.; Blackwell, Timothy S.; West, James D.

    2015-01-01

    Abstract Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis. PMID:26697175

  11. MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a

    PubMed Central

    Man, Xiao-Fei; Tan, Shu-Wen; Tang, Hao-Neng; Guo, Yue; Tang, Chen-Yi; Tang, Jun; Zhou, Ci-La; Zhou, Hou-De

    2016-01-01

    Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy. PMID:27398155

  12. Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice

    PubMed Central

    Chou, J.; Harvey, B. K.; Ebendal, T.; Hoffer, B.; Wang, Y.

    2008-01-01

    Summary Background We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. Methods Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. Results BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. Conclusions Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. PMID:18642641

  13. Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

    PubMed Central

    De Gottardi, Andrea; Seijo, Susana; Milá, Montserrat; Alvarez, M Isabel; Bruguera, Miquel; Abraldes, Juan G; Bosch, Jaime; García-Pagán, Juan-Carlos

    2012-01-01

    In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH. PMID:22129439

  14. The classic: Bone morphogenetic protein.

    PubMed

    Urist, Marshall R; Strates, Basil S

    2009-12-01

    This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406. PMID:19727989

  15. Anti-wrinkle effect of bone morphogenetic protein receptor 1a-extracellular domain (BMPR1a-ECD)

    PubMed Central

    Yoon, Byung-Hak; Jeon, Yun-Hui; Hwang, Byunghee; Kwon, Hyuknam; Choe, Senyon; Yang, Zungyoon

    2013-01-01

    Bone morphogenetic proteins (BMPs) have diverse and important roles in the proliferation and differentiation of adult stem cells in our tissues. Especially, BMPs are well known to be the main inducers of bone formation, by facilitating both proliferation and differentiation of bone stem cells. Interestingly, in skin stem cells, BMPs repress their proliferation but are indispensable for the proper differentiation into several lineages of skin cells. Here, we tested whether BMP antagonists have an effect on the prevention of wrinkle formation. For this study we used an in vivo wrinkle-induced mouse model. As a positive control, retinoic acid, one of the top anti-wrinkle effectors, showed a 44% improvement compared to the non-treated control. Surprisingly, bone morphogenetic protein receptor 1a extracellular domain (BMPR1a-ECD) exhibited an anti-wrinkle effect which was 6-fold greater than that of retinoic acid. Our results indicate that BMP antagonists will be good targets for skin or hair diseases. [BMB Reports 2013; 46(9): 465-470] PMID:24064062

  16. Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia Ossificans Progressiva

    PubMed Central

    Kaplan, Frederick S.; Seemann, Petra; Haupt, Julia; Xu, Meiqi; Lounev, Vitali Y.; Mullins, Mary; Shore, Eileen M.

    2016-01-01

    Bone morphogenetic protein (BMP) type I receptors are serine-threonine kinase transmembrane signal transduction proteins that regulate a vast array of ligand-dependent cell-fate decisions with temporal and spatial fidelity during development and postnatal life. A recent discovery identified a recurrent activating heterozygous missense mutation in a BMP type I receptor [Activin receptor IA/activin-like kinase 2 (ACVR1; also known as ALK2)] in patients with the disabling genetic disorder fibrodysplasia ossificans progressiva (FOP). Individuals with FOP experience episodes of tissue metamorphosis that convert soft connective tissue such as skeletal muscle into a highly ramified and disabling second skeleton of heterotopic bone. The single nucleotide ACVR1/ALK2 mutation that causes FOP is one of the most specific disease-causing mutations in the human genome and to date the only known inherited activating mutation of a BMP receptor that causes a human disease. Thus, the study of FOP provides the basis for understanding the clinically relevant effects of activating mutations in the BMP signaling pathway. Here we briefly review methodologies that we have applied to studying activated BMP signaling in FOP. PMID:21036241

  17. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2

    PubMed Central

    Mediero, Aránzazu; Wilder, Tuere; Perez-Aso, Miguel; Cronstein, Bruce N.

    2015-01-01

    Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase-positive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.—Mediero, A., Wilder, T., Perez-Aso, M., Cronstein, B. N. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. PMID:25573752

  18. Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus.

    PubMed

    Nett, Philipp C; Ortmann, Jana; Celeiro, Jennifer; Haas, Elvira; Hofmann-Lehmann, Regina; Tornillo, Luigi; Terraciano, Luigi M; Barton, Matthias

    2006-04-01

    Endothelin (ET) and bone morphogenic proteins (BMP) have been implicated in the development of micro- and macrovascular complications of type 2 diabetes mellitus due to atherosclerosis. This study investigated vascular BMP-expression during early development of experimental autoimmune diabetes mellitus and whether ET(A) receptors are involved in its regulation, using the selective ET(A) receptor antagonist BSF461314. Specificity of BSF461314 was confirmed through ET-mediated p44/42 mitogen-activated protein kinase (ERK1/2) phosphorylation experiments. For animal studies, non-obese diabetic (NOD) and control mice at 16 weeks of age were treated with BSF461314 for 6 weeks. Plasma glucose levels were measured before and after treatment and vascular gene expression of BMP-2, BMP-7, and BMP-type II receptor was determined in the aorta by quantitative real-time polymerase chain reaction analysis. At the beginning of the study in all animals, plasma glucose levels were within the normal range. After 6 weeks gene expression of vascular BMP-2, BMP-7 and BMP-type II receptor was almost doubled in NOD mice compared with non-diabetic controls (p < 0.05). Concomitant treatment with BSF461314 significantly reduced expression of all BMPs and lowered plasma glucose levels in NOD mice close to controls (all p < 0.05 versus untreated). In conclusion, vascular BMP-2, BMP-7, and BMP-type II receptor expression is upregulated in early stages of autoimmune diabetes mellitus. The data further indicate that ET(A) receptors inhibit diabetes-associated activation of vascular BMPs and regulate plasma glucose levels suggesting that ET(A) receptors might provide a new therapeutic target to interfere with the early development of atherosclerosis in patients with type 1 diabetes mellitus. PMID:16300798

  19. A bone morphogenetic protein ligand and receptors in mud crab: A potential role in the ovarian development.

    PubMed

    Shu, Ling; Yang, Yanan; Huang, Huiyang; Ye, Haihui

    2016-10-15

    In vertebrates, bone morphogenetic proteins (BMPs) play an important role in various biological processes. However, the function of BMPs in crustaceans is still unknown. In our study, a ligand (BMP7) and two receptors (Sp-BMPRIB and Sp-BMPRII) are cloned firstly in the mud crab, Scylla paramamosain. The qRT-PCR demonstrated that both ligand and receptors were expressed in various tissues, especially in ovary. The expression of BMPRs mRNA increased along the ovarian development, while BMP7 had an opposite tendency. In-situ hybridization revealed that Sp-BMPRIB and Sp-BMPRII were expressed in both oocytes and follicle cells, whereas Sp-BMP7 was exclusively localized in follicle cells. RNAi experiments showed that the expression levels of Smad1 and vitellogenin receptor declined rapidly after BMPRs were silenced. Based on these data, we hypothesized that in S. paramamosain, BMP7 and BMPRs had impact on the ovarian development, presumably via the autocrine/paracrine way. PMID:27345242

  20. The Bone Morphogenetic Protein Type Ib Receptor Is a Major Mediator of Glial Differentiation and Cell Survival in Adult Hippocampal Progenitor Cell Culture

    PubMed Central

    Brederlau, A.; Faigle, R.; Elmi, M.; Zarebski, A.; Sjöberg, S.; Fujii, M.; Miyazono, K.; Funa, K.

    2004-01-01

    Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture. PMID:15194807

  1. Sequence analysis of bone morphogenetic protein receptor type II mRNA from ascitic and nonascitic commercial broilers.

    PubMed

    Cisar, C R; Balog, J M; Anthony, N B; Donoghue, A M

    2003-10-01

    Ascites syndrome, also known as pulmonary hypertension syndrome (PHS), is a common metabolic disorder in rapidly growing meat-type chickens. Environmental factors, such as cold, altitude, and diet, play significant roles in development of the disease, but there is also an important genetic component to PHS susceptibility. The human disease familial primary pulmonary hypertension (FPPH) is similar to PHS in broilers both genetically and physiologically. Several recent studies have shown that mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are a cause of FPPH in humans. To determine whether mutations in the chicken BMPR2 gene play a similar role in PHS susceptibility, BMPR-II mRNA from ascitic and nonascitic commercial broilers were sequenced and compared with the published Leghorn chicken BMPR-II mRNA sequence. Fourteen single nucleotide polymorphisms (SNP) were identified in the commercial broiler BMPR-II mRNA. No mutations unique to ascites-susceptible broilers were present in the coding, 5' untranslated or 3' untranslated regions of BMPR-II mRNA. The twelve SNP present within the coding region of BMPR-II mRNA were synonymous substitutions and did not alter the BMPR-II protein sequence. In addition, analysis of BMPR2 gene expression by reverse transcriptase-PCR indicated that there were no differences in BMPR-II mRNA levels in ascitic and nonascitic birds. Therefore, it appears unlikely that mutations in the BMPR2 gene were responsible for susceptibility to PHS in these commercial broilers. PMID:14601724

  2. Bone Morphogenetic Protein Receptor Type II Deficiency and Increased Inflammatory Cytokine Production. A Gateway to Pulmonary Arterial Hypertension

    PubMed Central

    Soon, Elaine; Crosby, Alexi; Southwood, Mark; Yang, Peiran; Tajsic, Tamara; Toshner, Mark; Appleby, Sarah; Shanahan, Catherine M.; Bloch, Kenneth D.; Pepke-Zaba, Joanna; Upton, Paul

    2015-01-01

    Rationale: Mutations in bone morphogenetic protein receptor type II (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (PAH). However, disease penetrance is only 20–30%, suggesting a requirement for additional triggers. Inflammation is emerging as a key disease-related factor in PAH, but to date there is no clear mechanism linking BMPR-II deficiency and inflammation. Objectives: To establish a direct link between BMPR-II deficiency, a consequentially heightened inflammatory response, and development of PAH. Methods: We used pulmonary artery smooth muscle cells from Bmpr2+/− mice and patients with BMPR2 mutations and compared them with wild-type controls. For the in vivo model, we used mice heterozygous for a null allele in Bmpr2 (Bmpr2+/−) and wild-type littermates. Measurements and Main Results: Acute exposure to LPS increased lung and circulating IL-6 and KC (IL-8 analog) levels in Bmpr2+/− mice to a greater extent than in wild-type controls. Similarly, pulmonary artery smooth muscle cells from Bmpr2+/− mice and patients with BMPR2 mutations produced higher levels of IL-6 and KC/IL-8 after lipopolysaccharide stimulation compared with controls. BMPR-II deficiency in mouse and human pulmonary artery smooth muscle cells was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase. Chronic lipopolysaccharide administration caused pulmonary hypertension in Bmpr2+/− mice but not in wild-type littermates. Coadministration of tempol, a superoxide dismutase mimetic, ameliorated the exaggerated inflammatory response and prevented development of PAH. Conclusions: This study demonstrates that BMPR-II deficiency promotes an exaggerated inflammatory response in vitro and in vivo, which can instigate development of pulmonary hypertension. PMID:26073741

  3. Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

    PubMed Central

    Wang, Cheng; Roy, Shyamal K.

    2009-01-01

    To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors. PMID:19074578

  4. Systemic injection of CK2.3, a novel peptide acting downstream of Bone Morphogenetic Protein receptor BMPRIa, leads to increased trabecular bone mass

    PubMed Central

    Akkiraju, Hemanth; Bonor, Jeremy; Olli, Kristine; Bowen, Chris; Bragdon, Beth; Coombs, Harold; Donahue, Leah Rae; Duncan, Randall; Nohe, Anja

    2014-01-01

    Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal-related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation. PMID:25331517

  5. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    NASA Astrophysics Data System (ADS)

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-04-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds.

  6. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    PubMed Central

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-01-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds. PMID:27075233

  7. Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4.

    PubMed

    Su, Xin; Ao, Lihua; Shi, Yi; Johnson, Thomas R; Fullerton, David A; Meng, Xianzhong

    2011-04-01

    Vascular calcification is a common complication in atherosclerosis. Bone morphogenetic protein-2 (BMP-2) plays an important role in atherosclerotic vascular calcification. The aim of this study was to determine the effect of oxidized low density lipoprotein (oxLDL) on BMP-2 protein expression in human coronary artery endothelial cells (CAECs), the roles of Toll-like receptor (TLR) 2 and TLR4 in oxLDL-induced BMP-2 expression, and the signaling pathways involved. Human CAECs were stimulated with oxLDL. The roles of TLR2 and TLR4 in oxLDL-induced BMP-2 expression were determined by pretreatment with neutralizing antibody, siRNA, and overexpression. Stimulation with oxLDL increased cellular BMP-2 protein levels in a dose-dependent manner (40-160 μg/ml). Pretreatment with neutralizing antibodies against TLR2 and TLR4 or silencing of these two receptors reduced oxLDL-induced BMP-2 expression. Overexpression of TLR2 and TLR4 enhanced the cellular BMP-2 response to oxLDL. Furthermore, oxLDL was co-localized with TLR2 and TLR4. BMP-2 expression was associated with activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK)1/2. Inhibition of NF-κB and ERK1/2 reduced BMP-2 expression whereas inhibition of p38 MAPK had no effect. In conclusion, oxLDL induces BMP-2 expression through TLR2 and TLR4 in human CAECs. The NF-κB and ERK1/2 pathways are involved in the signaling mechanism. These findings underscore an important role for TLR2 and TLR4 in mediating the BMP-2 response to oxLDL in human CAECs and indicate that these two immunoreceptors contribute to the mechanisms underlying atherosclerotic vascular calcification. PMID:21325271

  8. Synergy between IL-6 and soluble IL-6 receptor enhances bone morphogenetic protein-2/absorbable collagen sponge-induced bone regeneration via regulation of BMPRIA distribution and degradation.

    PubMed

    Huang, Ru-Lin; Chen, Gang; Wang, Wenjin; Herller, Tanja; Xie, Yun; Gu, Bin; Li, Qingfeng

    2015-10-01

    Bone morphogenetic protein-2/absorbable collagen sponge (BMP-2/ACS) implants have been approved for clinical use to induce bone regeneration. We previously showed that exaggerated inflammation characterized by elevated level of inflammatory cytokines including TNF-α, IL-1β, and IL-6 has been shown to inhibit BMP-2/ACS-induced bone regeneration. Furthermore, unlike the negative effects of TNF-α and IL-1β, IL-6 seemed not to affect BMP-2-induced osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). We hypothesized that there may be a regulatory loop between IL-6 and BMP-2 singling to affect BMP-2/ACS-induced bone regeneration. Here, we established a BMP-2/ACS-induced ectopic bone formation model in rats and fund that IL-6 injection significantly increased BMP-2/ACS-induced bone mass. Consistent with this animal model, an in vitro study demonstrated that synergy between IL-6 and soluble IL-6 receptor (IL-6/sIL-6R) promotes BMP-2-induced osteoblastic differentiation of human BMSCs through amplification of BMP/Smad signaling. Strikingly, IL-6 injection did not activate osteoclast-mediated bone resorption in the ectopic bone formation model, and IL-6/sIL-6R treatment did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastic differentiation of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, IL-6/sIL-6R treatment did not affect expression of BMP receptors, but enhanced the cell surface translocation of BMP receptor IA (BMPRIA) and inhibited the degradation of BMPRIA. Collectively, these findings indicate that synergy between IL-6 and sIL-6R promotes the cell surface translocation of BMPRIA and maintains the stability of BMPRIA expression, leading to enhanced BMP-2/ACS-induced bone regeneration. PMID:26232880

  9. Dynamin-dependent endocytosis of Bone Morphogenetic Protein2 (BMP2) and its receptors is dispensable for the initiation of Smad signaling.

    PubMed

    Paarmann, Pia; Dörpholz, Gina; Fiebig, Juliane; Amsalem, Ayelet R; Ehrlich, Marcelo; Henis, Yoav I; Müller, Thomas; Knaus, Petra

    2016-07-01

    Bone Morphogenetic Protein (BMP) signal transduction via the canonical Smad158 pathway has previously been linked to dynamin-dependent endocytosis, since the application of chemical inhibitors of clathrin or dynamin in functional cell culture based assays negatively affects initiation and propagation of the Smad response. More recent studies, however, demonstrated efficient Smad signaling by non-internalizable BMP2. The role of endocytosis in BMP signal transduction thus remained controversial. In our study we aimed to refine cell biological assays and to apply novel tools, including a new site-directed fluorescently labeled BMP2 ligand, to revisit key steps in BMP Smad signaling. We found that dynamin2 function was required for BMP2 uptake but was dispensable for C-terminal phosphorylation, nuclear translocation and transcriptional activity of BMP-dependent Smads. Furthermore, we demonstrated a role of dynamin2 in the regulation of steady-state and surface BMP receptor levels, as well as an impact on Smad1 protein level. Thus, dynamin2 allows for modulation of basal and ligand-dependent Smad signaling capacity. High levels of functional dynamin2 enhanced the myogenic differentiation of precursor cells. From our study we conclude that dynamin-dependent endocytosis serves as a regulatory mechanism to fine-tune Smad signaling, but it is not a prerequisite for signal initiation and propagation. Our findings contribute to the understanding of fundamental mechanisms of BMP signaling and thus provide important information for future consideration in the context of therapeutic applications of BMPs. PMID:27113717

  10. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor.

    PubMed

    Amsalem, Ayelet R; Marom, Barak; Shapira, Keren E; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I; Ehrlich, Marcelo

    2016-02-15

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  11. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  12. Role of growth differentiation factor-5 and bone morphogenetic protein type II receptor in the development of lumbar intervertebral disc degeneration

    PubMed Central

    Li, Yi-Fan; Tang, Xian-Zhong; Liang, Chao-Ge; Hui, Yao-Ming; Ji, Yun-Han; Xu, Wei; Qiu, WenJun; Cheng, Li-Ming

    2015-01-01

    The present study was designed to evaluate the role of growth differentiation factor-5 (GDF-5) and bone morphogenetic protein type II receptor (BMPR-II) in the development of lumbar intervertebral disc degeneration (IDD). A total of 24 patients with lumbar IDD (experiment group) and 6 patients with lumbar vertebral fracture (control group) were enrolled in the study. Tissue samples of IVD from the experiment group and control group were obtained during lumbar fusion operation, respectively. Fixation and decalcification of IVD tissue were performed, and then HE staining was carried out to observe the morphological changes of the lumbar IVD tissues. The expression of GDF-5 and BMPRII in human lumbar IVD was detected by immunohistochemical staining. HE staining results showed that non- and minimal degeneration was found in 11 cases (score range, 0-3), moderate degeneration in 12 cases (score range, 4-8), and severe degeneration in 7 cases (score range, 9-12). According to the immunohistochemical results, the positive expression rates of GDF-5 and BMPRII in NP were higher than those in AF of the non- and minimal degeneration group, moderate degeneration group and severe degeneration group (all P < 0.05). However, no significant difference in GDF-5 or BMPRII positive expression was observed among the normal, non- and minimal, moderate and severe degeneration groups in neither NP area nor AF area (all P > 0.05). In conclusion, our results showed that GDF-5 and BMPRII expressed both in normal and degenerated IVD tissues, and GDF-5 might have an inhibition effect on degenerated lumbar IVD, suggesting that gene therapy may be a useful approach in producing physiological effects during early- and late-phase of lumbar IDD. PMID:25755766

  13. The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells

    PubMed Central

    Gao, Zhenya; Huo, Lijun; Cui, Dongmei; Yang, Xiao; Zeng, Junwen

    2016-01-01

    Purpose All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells. Methods The effects of ATRA (concentrations from 10−9 to 10−5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10−9 to 10−5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ. Results RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10−9 to 10−5 mol/l) with a maximum effect observed at 10−6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10−6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135. Conclusion ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated

  14. Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

    PubMed

    Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

    2016-07-01

    In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. c

  15. Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy

    PubMed Central

    Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

    2016-01-01

    In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. c

  16. SM22α-Targeted Deletion of Bone Morphogenetic Protein Receptor IA in Mice Impairs Cardiac and Vascular Development and Influences Organogenesis

    PubMed Central

    El-Bizri, Nesrine; Guignabert, Christophe; Wang, Lingli; Cheng, Alexander; Stankunas, Kryn; Chang, Ching-Pin; Mishina, Yuji; Rabinovitch, Marlene

    2009-01-01

    Summary Expression of bone morphogenetic protein receptor 1a (Bmpr1a) is attenuated in lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We therefore ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMC) by breeding mice with a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22α-Cre mice. SM22α-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22α-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMC that was also related to reduced proliferation. SM22α-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP)-2 and -9 were reduced in E9.5 and E10.5 SM22α-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of Bmpr1a by RNA interference in human pulmonary artery SMC reduced MMP-2 and MMP-9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP-2 or MMP-9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of Bmpr1a reduced MMP-2 activity and knock-down of either Bmpr1a or MMP-2 caused resistance to apoptosis. Thus loss of Bmpr1a, by decreasing MMP-2 and/or MMP-9 activity, can account for vascular dilatation and persistence of brain microvessels leading to impaired organogenesis documented in the brain. PMID:18667463

  17. Hepatoregenerative role of bone morphogenetic protein-9

    PubMed Central

    Sosa, Ivan; Cvijanovic, Olga; Celic, Tanja; Cuculic, Drazen; Crncevic-Orlic, Zeljka; Vukelic, Lucian; Cvek, Sanja Zoricic; Dudaric, Luka; Bosnar, Alan; Bobinac, Dragica

    2011-01-01

    Summary Bone morphogenetic protein-9 (BMP-9) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines, which regulate cell growth and differentiation during embryogenesis. Apart of that, the hypoglycemic potential of BMP-9 is of great interest. It has been confirmed that BMP-9, like insulin, improves glycemia in diabetic mice and regulates directional glucose metabolism in hepatocytes; therefore it is proposed to be a candidate hepatic insulin-sensitizing substance (HISS). In liver fibrosis, due to the portocaval shunt, insulin bypasses the organ and the liver undergoes atrophy. Parenteral administration of insulin reverses atrophy by stimulating mitogenic activity of the hepatocytes. Because BMP-9 has a signaling pathway similar to other BMPs and insulin, it is to be expected that BMP-9 has a certain regenerative role in the liver, supporting the above-mentioned is evidence of BMP-9 expression in Dissè’s spaces and BMP-7’s mitogenic activity in mucosal cells. However, further studies are needed to confirm the possible regenerative role of BMP-9. PMID:22129908

  18. Hemojuvelin and bone morphogenetic protein (BMP) signaling in iron homeostasis.

    PubMed

    Core, Amanda B; Canali, Susanna; Babitt, Jodie L

    2014-01-01

    Mutations in hemojuvelin (HJV) are the most common cause of the juvenile-onset form of the iron overload disorder hereditary hemochromatosis. The discovery that HJV functions as a co-receptor for the bone morphogenetic protein (BMP) family of signaling molecules helped to identify this signaling pathway as a central regulator of the key iron hormone hepcidin in the control of systemic iron homeostasis. This review highlights recent work uncovering the mechanism of action of HJV and the BMP-SMAD signaling pathway in regulating hepcidin expression in the liver, as well as additional studies investigating possible extra-hepatic functions of HJV. This review also explores the interaction between HJV, the BMP-SMAD signaling pathway and other regulators of hepcidin expression in systemic iron balance. PMID:24860505

  19. Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells*

    PubMed Central

    Wang, Zili; Weitzmann, M. Neale; Sangadala, Sreedhara; Hutton, William C.; Yoon, S. Tim

    2013-01-01

    Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain. PMID:23940040

  20. Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.

    PubMed

    Ding, Sai; Zhang, Jing; Tian, Yu; Huang, Baolin; Yuan, Yuan; Liu, Changsheng

    2016-09-01

    Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration. PMID:27156155

  1. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  2. Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

    PubMed Central

    Szymczak, Lindsey C.; Aydin, Taner; Yun, Sijung; Constas, Katharine; Schaeffer, Arielle; Ranjan, Sinthu; Kubba, Saad; Alam, Emad; McMahon, Devin E.; He, Jingpeng; Shwartz, Neta; Tian, Chenxi; Plavskin, Yevgeniy; Lindy, Amanda; Dad, Nimra Amir; Sheth, Sunny; Amin, Nirav M.; Zimmerman, Stephanie; Liu, Dennis; Schwarz, Erich M.; Smith, Harold; Krause, Michael W.; Liu, Jun

    2015-01-01

    Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development. PMID:25978409

  3. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  4. Recombinant human bone morphogenetic protein induces bone formation.

    PubMed Central

    Wang, E A; Rosen, V; D'Alessandro, J S; Bauduy, M; Cordes, P; Harada, T; Israel, D I; Hewick, R M; Kerns, K M; LaPan, P

    1990-01-01

    We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans. Images PMID:2315314

  5. Bone Morphogenetic Protein (BMP) signaling in development and human diseases

    PubMed Central

    Wang, Richard N.; Green, Jordan; Wang, Zhongliang; Deng, Youlin; Qiao, Min; Peabody, Michael; Zhang, Qian; Ye, Jixing; Yan, Zhengjian; Denduluri, Sahitya; Idowu, Olumuyiwa; Li, Melissa; Shen, Christine; Hu, Alan; Haydon, Rex C.; Kang, Richard; Mok, James; Lee, Michael J.; Luu, Hue L.; Shi, Lewis L.

    2014-01-01

    Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are important in embryogenesis and development, and also in maintenance of adult tissue homeostasis. Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects, highlighting the essential functions of BMPs. In this review, we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development. A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling. PMID:25401122

  6. Studies of Bone Morphogenetic Protein based Surgical Repair

    PubMed Central

    Lo, Kevin W.-H.; Ulery, Bret D.; Ashe, Keshia M.; Laurencin, Cato T.

    2012-01-01

    Over the past several decades, recombinant human bone morphogenetic proteins (rhBMPs) have been the most extensively studied and widely used osteoinductive agents for clinical bone repair. Since rhBMP-2 and rhBMP-7 were approved by the U.S. Food and Drug Administration for certain clinical uses, millions of patients worldwide have been treated with rhBMPs for various musculoskeletal disorders. Current clinical applications include treatment of long bone fracture non-unions, spinal surgeries, and oral maxillofacial surgeries. Considering the growing number of recent publications related to clincal research of rhBMPs, there exists enormous promise for these proteins to be used in bone regenerative medicine. The authors take this opportunity to review the rhBMP literature paying specific attention to the current applications of rhBMPs in bone repair and spine surgery. The prospective future of rhBMPs delivered in combination with tissue engineered scaffolds is also reviewed. PMID:22512928

  7. The role of human bone morphogenetic proteins in spinal fusion.

    PubMed

    Zlotolow, D A; Vaccaro, A R; Salamon, M L; Albert, T J

    2000-01-01

    The attainment of a stable arthrodesis is critical to the successful management of some types of spinal disorders. Autologous iliac-crest bone graft has been the most commonly utilized substance associated with predictable healing in spinal fusion applications. Although alternative graft substances exist, these have not been shown to be as uniformly effective in achieving spinal fusion. Because of the morbidity associated with bone autograft harvest, there is increasing interest in alternative graft substances and especially in the osteoinductive abilities of bone morphogenetic proteins (BMPs). Several animal models have demonstrated that BMP-containing allograft or synthetic carrier medium is as effective as or superior to autograft bone in promoting spinal fusion. Furthermore, the limited number of human trials utilizing BMPs to treat nonunions in the appendicular skeleton indicate that the results found in animal models will be reproducible in the clinical setting. PMID:10666648

  8. Bone morphogenetic protein-2: a potential regulator in scleral remodeling

    PubMed Central

    Hu, Jianmin; Cui, Dongmei; Yang, Xiao; Wang, Shaowei; Hu, Shoulong; Li, Chuanxu

    2008-01-01

    Purpose Bone morphogenetic protein 2 (BMP-2) is a member of the main subgroup of bone morphogenetic proteins within the transforming growth factor-β superfamily. BMP-2 is involved in numerous cellular functions including development, cell proliferation, apoptosis, and extracellular matrix synthesis. We examined BMP-2 expression in human scleral fibroblasts (HSF) and assessed the effects of recombinant human BMP-2 (rhBMP-2) on HSF proliferation, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Methods We used confocal fluorescence microscopy (CFM) to study BMP-2 distribution in HSF cells and frozen human scleral sections. The influence of rhBMP-2 on cell proliferation at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml) was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effects of rhBMP-2 on the cell cycle were investigated with flow cytometric analysis. Reverse transcription polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine MMP-2 and TIMP-2 mRNAs and secreted proteins in HSF that were incubated with rhBMP-2. Results BMP-2 protein expression from human sclera was confirmed by CFM. Cell proliferation was significantly increased with 100 ng/ml rhBMP-2 in a time-dependent manner (p<0.05). The HSF cell cycle moved to the S and S+G2M phases after rhBMP-2 stimulation at 100 ng/ml compared to normal cells (p<0.05). TIMP-2 mRNA levels were significantly increased in HSF incubated for 24 h with 100 ng/ml rhBMP-2 (p<0.01). A 48 h incubation with 10 ng/ml or 100 ng/ml rhBMP-2 resulted in significantly increased TIMP-2 mRNA and protein expression and significantly decreased MMP-2 mRNA expression (p<0.01) while MMP-2 protein expression significantly decreased at 100 ng/ml rhBMP-2 (p<0.01). Conclusions Human sclera fibroblasts expressed BMP-2, which promoted cell proliferation, and elicited changes in MMP-2 and TIMP-2

  9. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    PubMed

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  10. The history and histology of bone morphogenetic protein.

    PubMed

    Murray, Samuel S; Brochmann Murray, Elsa J; Wang, Jeffrey C; Duarte, Maria Eugenia Leite

    2016-07-01

    Bone morphogenetic proteins are a group of structurally related proteins within the TGF-β superfamily of proteins with a diverse repertoire of functions in embryonic and adult organisms. As is apparent from the name, the members first characterized participate in bone growth, development, and remodeling. The "morphogenic" activity per se is defined as the induction of a recapitulation of endochondral bone formation by appropriate stem cells. The regenerative capacity of bone has been recognized since ancient times. The mechanism, applications, and conceptual basis of bone transplantation, bone implantation, ectopic bone formation, and exogenously induced bone formation have been studied by many investigators for more than a century. This review examines the efforts to characterize this activity in the European and American literature over approximately the last century. Because of the inherently complex nature of the process induced by these molecules (inflammation, stem cell proliferation, cartilage differentiation, replacement of cartilage with bone) it is important to evaluate previous investigations through a histological perspective. The cellular basis of the contemporary bioassay for BMP activity is illustrated and discussed from the histological point of view. PMID:26907674

  11. Role of bone morphogenetic protein-7 in renal fibrosis

    PubMed Central

    Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

    2015-01-01

    Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

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

    PubMed

    Martinovic, Snjezana; Mazic, Sanja; Kisic, Veronika; Basic, Nikolina; Jakic-Razumovic, Jasminka; Borovecki, Fran; Batinic, Drago; Simic, Petra; Grgurevic, Lovorka; Labar, Boris; Vukicevic, Slobodan

    2004-09-01

    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. PMID:15314083

  13. Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts

    PubMed Central

    Bobacz, K; Ullrich, R; Amoyo, L; Erlacher, L; Smolen, J S; Graninger, W B

    2006-01-01

    Objective To investigate effects of cartilage derived morphogenetic protein‐1 and ‐2 (CDMP‐1, CDMP‐2), bone morphogenetic protein (BMP)‐7 and BMP‐6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. Methods Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6. Expression of the indicated growth factors was assessed by RT‐PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT‐PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [35S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. Results CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [35S]sulphate incorporation. Toluidine blue staining showed cartilage‐specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. Conclusion CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair. PMID:15975973

  14. Heparan sulfate is required for bone morphogenetic protein-7 signaling.

    PubMed

    Irie, Atsushi; Habuchi, Hiroko; Kimata, Koji; Sanai, Yutaka

    2003-09-01

    Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling. PMID:12927798

  15. Sustained release emphasizing recombinant human bone morphogenetic protein-2.

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

    Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems. PMID:10837631

  16. Bone morphogenetic protein 4 mediates human embryonic germ cell derivation.

    PubMed

    Hiller, Marc; Liu, Cyndi; Blumenthal, Paul D; Gearhart, John D; Kerr, Candace L

    2011-02-01

    Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency. PMID:20486775

  17. Role of morphogenetic proteins in skeletal tissue engineering and regeneration.

    PubMed

    Reddi, A H

    1998-03-01

    Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints. PMID:9528003

  18. A glass polyalkenoate cement carrier for bone morphogenetic proteins.

    PubMed

    Alhalawani, Adel M F; Rodriguez, Omar; Curran, Declan J; Co, Russell; Kieran, Sean; Arshad, Saad; Keenan, Timothy J; Wren, Anthony W; Crasto, Gazelle; Peel, Sean A F; Towler, Mark R

    2015-03-01

    This work considers a glass polyalkenoate cement (GPC)-based carrier for the effective delivery of bone morphogenetic proteins (BMPs) at an implantation site. A 0.12 CaO-0.04 SrO-0.36 ZnO-0.48 SiO2 based glass and poly(acrylic acid) (PAA, Mw 213,000) were employed for the fabrication of the GPC. The media used for the water source in the GPC reaction was altered to produce a series of GPCs. The GPC liquid media was either 100 % distilled water with additions of albumin at 0, 2, 5 and 8 wt% of the glass content, 100 % formulation buffer (IFB), and 100 % BMP (150 µg rhBMP-2/ml IFB). Rheological properties, compressive strength, ion release profiles and BMP release were evaluated. Working times (Tw) of the formulated GPCs significantly increased with the addition of 2 % albumin and remained constant with further increases in albumin content or IFB solutions. Setting time (Ts) experienced an increase with 2 and 5 % albumin content, but a decrease with 8 % albumin. Changing the liquid source to IFB containing 5 % albumin had no significant effect on Ts compared to the 8 % albumin-containing BT101. Replacing the albumin with IFB/BMP-2 did not significantly affect Tw. However, Ts increased for the BT101_BMP-2 containing GPCs, compared to all other samples. The compressive strength evaluated 1 day post cement mixing was not affected significantly by the incorporation of BMPs, but the ion release did increase from the cements, particularly for Zn and Sr. The GPCs released BMP after the first day, which decreased in content during the following 6 days. This study has proven that BMPs can be immobilized into GPCs and may result in novel materials for clinical applications. PMID:25773232

  19. Bone morphogenetic protein-7 accelerates fracture healing in osteoporotic rats

    PubMed Central

    Diwan, Ashish D; Leong, Anthony; Appleyard, Richard; Bhargav, Divya; Fang, Zhi Ming; Wei, Aiqun

    2013-01-01

    Background: Osteoporosis is characterized by low bone mass, bone fragility and increased susceptibility to fracture. Fracture healing in osteoporosis is delayed and rates of implant failure are high with few biological treatment options available. This study aimed to determine whether a single dose of bone morphogenetic protein-7 (BMP-7) in a collagen/carboxy-methyl cellulose (CMC) composite enhanced fracture healing in an osteoporotic rat model. Materials and Methods: An open femoral midshaft osteotomy was performed in female rats 3 months post-ovarectomy. Rats were randomized to receive either BMP-7 composite (n = 30) or composite alone (n = 30) at the fracture site during surgery. Thereafter calluses were collected on days 12, 20 and 31. Callus cross-sectional area, bone mineral density, biomechanical stiffness and maximum torque, radiographic bony union and histological callus maturity were evaluated at each time point. Results: There were statistically significant increases in bone mineral density and callus cross-section area at all time points in the BMP-7 group as compared to controls and biomechanical readings showed stronger bones at day 31 in the BMP-7 group. Histological and radiographic evaluation indicated significant acceleration of bony union in the BMP-7 group as compared to controls. Conclusion: This study demonstrated that BMP-7 accelerates fracture healing in an oestrogen-deficient environment in a rat femoral fracture healing model to scientific relevance level I. The use of BMP-7 composite could offer orthopedic surgeons an advantage over oestrogen therapy, enhancing osteoporotic fracture healing with a single, locally applied dose at the time of surgery, potentially overcoming delays in healing caused by the osteoporotic state. PMID:24379457

  20. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  1. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition

    PubMed Central

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  2. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

    PubMed

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  3. Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

    PubMed

    Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-09-01

    Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. PMID:27208502

  4. [Bone morphogenetic proteins (BMP): clinical application for reconstruction of bone defects].

    PubMed

    Sierra-García, Gerardo Daniel; Castro-Ríos, Rocío; Gónzalez-Horta, Azucena; Lara-Arias, Jorge; Chávez-Montes, Abelardo

    2016-01-01

    Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results. PMID:27335195

  5. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development

    PubMed Central

    Holtzhausen, Alisha; Golzio, Christelle; How, Tam; Lee, Yong-Hun; Schiemann, William P.; Katsanis, Nicholas; Blobe, Gerard C.

    2014-01-01

    The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor β (TGF-β)-responsive Smad2 and Smad3. BMP-induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP-binding TGF-β superfamily receptors, activin receptor-like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP-mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF-β superfamily receptors are broader than previously appreciated.—Holtzhausen, A., Golzio, C., How, T., Lee, Y.-H., Schiemann, W. P., Katsanis, N., Blobe, G. C. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development. PMID:24308972

  6. Transgenic overexpression of bone morphogenetic protein 11 propeptide in skeleton enhances bone formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bone morphogenetic protein 11 (BMP11) is a key regulatory protein in skeletal development. BMP11 propeptide has been shown to antagonize GDF11 activity in vitro. To explore the role of BMP11 propeptide in skeletal formation in vivo, we generated transgenic mice with skeleton-specific overexpression...

  7. Availability of chemosensory receptors is down-regulated by habituation of larvae to a morphogenetic signal.

    PubMed Central

    Trapido-Rosenthal, H G; Morse, D E

    1986-01-01

    Larvae of the gastropod mollusc Haliotis rufescens are induced to settle from the plankton and metamorphose in response to exogenous gamma-aminobutyric acid (GABA) and a number of GABA-mimetic compounds, including a GABA-mimetic inducer uniquely associated with the surfaces of the naturally recruiting algae. Previous evidence has shown that recognition of these inducers is mediated by specialized chemosensory receptors on the larval epithelium and that transduction of the morphogenetic signal then is mediated by cAMP and excitatory depolarization. We demonstrate here the specific and saturable labeling of a population of larval receptors with the GABA analog beta-(p-chlorophenyl)-[3H]GABA ([3H]baclofen); identification of these labeled receptors with those controlling metamorphosis is suggested by four independent criteria: the effectiveness of GABA and its close structural analogs to induce metamorphosis is closely correlated with the effectiveness of these compounds to compete for binding to this receptor; the natural inducer purified from the recruiting algae competes for binding to this receptor; (-)-[3H]baclofen specifically bound to the receptors is shed from the larvae after approximately 20 hr, at the time corresponding to the metamorphic abscission and shedding of sensory cilia and other structures from the larvae; and the availability of the receptors for labeling and the ability of the larvae to respond to GABA and GABA analogs can be down-regulated in parallel by habituation of the larvae early in their development. These down-regulated larvae are fully capable of settlement and metamorphosis in response to agents that elevate intracellular cAMP or depolarize the chemosensory membrane, confirming that down-regulation is confined to the receptors, with no effect on the postreceptor pathway. The results reported here thus suggest that the sensitivity of marine invertebrate larvae to morphogenetic stimuli from the environment can be down-regulated by

  8. Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

    PubMed Central

    2011-01-01

    Background Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. Results We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. Conclusions Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway. PMID:22085733

  9. Bone morphogenetic protein 15 may promote follicle selection in the hen.

    PubMed

    Stephens, C S; Johnson, P A

    2016-09-01

    In the hen, optimal ovulation rate depends on selection of a single follicle into the pre-ovulatory hierarchy. Follicle selection is associated with increased oocyte growth and changes in gene expression in granulosa cells surrounding the oocyte, in preparation for ovulation. This study investigated the expression, function and regulation of bone morphogenetic protein-15 (BMP15) during follicle development in the hen. BMP15 mRNA expression was analyzed in the ooplasm and granulosa cells of 3mm follicles and was confirmed to be primarily in the ooplasm. BMP15 was detected by immunoblotting in 6 and 8mm follicles near the time of follicle selection. Expression of mRNA for BMP15 receptors (BMPR1B and BMPR2) in granulosa cells increased with follicle size, indicating that BMP15 may play an important role around follicle selection. The function of BMP15 was examined by culturing granulosa cells from 3-5mm and 6-8mm follicles with recombinant human BMP15 (rhBMP15). BMP15 increased expression of follicle stimulating hormone receptor (FSHR) mRNA and decreased anti-Müllerian hormone (AMH) mRNA and occludin (OCLN), factors associated with follicle maturation and growth in the hen. Hormonal regulation of BMP15 was assessed by whole follicle culture with estradiol (E2) which increased BMP15 mRNA expression. The distinct expression pattern of BMP15 and its receptors, coupled with the effects of BMP15 to increase FSHR mRNA and decrease AMH mRNA and OCLN mRNA and protein expression suggest that the oocyte may have a role in follicle selection in the chicken. PMID:27340039

  10. Bone Morphogenetic Proteins Regulate Enteric Gliogenesis by Modulating ErbB3 Signaling

    PubMed Central

    Chalazonitis, Alcmène; D’Autréaux, Fabien; Pham, Tuan D.; Kessler, John A.; Gershon, Michael D.

    2010-01-01

    The neural crest-derived cell population that colonizes the bowel (ENCDC) contains proliferating neural/glial progenitors. We tested the hypothesis that bone morphogenetic proteins (BMPs 2 and 4), which are known to promote enteric neuronal differentiation at the expense of proliferation, function similarly in gliogenesis. Enteric gliogenesis was analyzed in mice that overexpress the BMP antagonist, noggin, or BMP4 in the primordial ENS. Noggin-induced loss-of-function decreased, while BMP4-induced gain-of-function increased the glial density and glia/neuron ratio. When added to immunoisolated ENCDC, BMPs provoked nuclear translocation of phosphorylated SMAD proteins and enhanced both glial differentiation and expression of the neuregulin receptor ErbB3. ErbB3 transcripts were detected in E12 rat gut, before glial markers are expressed; moreover, expression of the ErbB3 ligand, glial growth factor 2 (GGF2) escalated rapidly after its first detection at E14. ErbB3-immunoreactive cells were located in the ENS of fetal and adult mice. GGF2 stimulated gliogenesis and proliferation and inhibited glial cell derived neurotrophic factor (GDNF)-promoted neurogenesis. Enhanced glial apoptosis occurred following GGF2 withdrawal; BMPs intensified this GGF2-dependence and reduced GGF2-stimulated proliferation. These observations support the hypotheses that BMPs are required for enteric gliogenesis and act by promoting responsiveness of ENCDC to ErbB3 ligands such as GGF2. PMID:21094638

  11. Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages.

    PubMed

    Rocher, Crystal; Singla, Reetu; Singal, Pawan K; Parthasarathy, Sampath; Singla, Dinender K

    2012-07-01

    It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis. PMID:22720873

  12. Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Jao Yiu; Yu Le; Cho, C.H.

    2008-06-27

    Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21{sup Waf1/Cip1} mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21{sup Waf1/Cip1} induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

  13. Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins.

    PubMed

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Haithcock, David A; Cundiff, Caitlin A; Schwartz, Zvi; Boyan, Barbara D

    2015-04-01

    Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4 μm); sandblasted/acid-etched Ti (SLA, Ra=3.2 μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment. PMID:25554602

  14. Coordinated Regulation of Mesenchymal Stem Cell Differentiation on Microstructured Titanium Surfaces by Endogenous Bone Morphogenetic Proteins

    PubMed Central

    Olivares-Navarrete, Rene; Hyzy, Sharon L.; Haithcock, David A.; Cundiff, Caitlin A.; Schwartz, Zvi; Boyan, Barbara D.

    2015-01-01

    Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4μm); sandblasted/acid-etched Ti (SLA, Ra=3.2μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6 days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment. PMID:25554602

  15. Protein serine/threonine phosphatase PPM1A dephosphorylates Smad1 in the bone morphogenetic protein signaling pathway.

    PubMed

    Duan, Xueyan; Liang, Yao-Yun; Feng, Xin-Hua; Lin, Xia

    2006-12-01

    Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation. PMID:16931515

  16. Does bone morphogenetic protein 6 (BMP6) affect female fertility in the mouse?

    PubMed

    Sugiura, Koji; Su, You-Qiang; Eppig, John J

    2010-12-01

    Bone morphogenetic protein 6 (BMP6) is a transforming growth factor beta superfamily member produced by mammalian oocytes as well as other cell types. Despite well-characterized effects of recombinant BMP6 on granulosa cells in vitro, the function of BMP6 in vivo has been ill-defined. Therefore, the effects of genetic deletion of the Bmp6 gene on female mouse fertility were assessed. The mean litter size of Bmp6(-/-) females was reduced by 22% (P < 0.05) compared to Bmp6(+/+) controls. Not only did Bmp6(-/-) females naturally ovulate 24% fewer eggs, but competence of in vitro-matured oocytes to complete preimplantation development after fertilization in vitro was decreased by 50%. No apparent effect of Bmp6 deletion on either the morphology or the dynamics of follicular development was apparent. Nevertheless, levels of luteinizing hormone (LH)/human chorionic gonadotropin (hCG)-induced transcripts, which encode proteins required for cumulus expansion (HAS2, PTGS2, PTX3, and TNFAIP6), and of epidermal growth factor-like peptides (AREG, BTC, and EREG) were lower in Bmp6(-/-) mice than in controls after administration of a reduced dose of hCG (1 IU) in vivo. LH receptor (Lhcgr) transcript levels were not significantly lower in Bmp6(-/-) granulosa cells, suggesting that BMP6 is required for processes downstream of LH receptors. To assess whether another oocyte-derived BMP, BMP15, could have BMP6-redundant functions in vivo, the fertility of Bmp15/Bmp6 double mutants was assessed. Fertility was not significantly reduced in double-homozygous mutants compared with that in double-heterozygous controls. Therefore, BMP6 promotes normal fertility in female mice, at least in part, by enabling appropriate responses to LH and normal oocyte quality. Thus, Bmp6 probably is part of the complex genetic network that determines female fertility. PMID:20702851

  17. Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

    PubMed Central

    Boswell, Bruce A.; Musil, Linda S.

    2015-01-01

    Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency—fiber cell differentiation and gap junction–mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function. PMID:25947138

  18. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats.

    PubMed

    Casarin, R C; Casati, M Z; Pimentel, S P; Cirano, F R; Algayer, M; Pires, P R; Ghiraldini, B; Duarte, P M; Ribeiro, F V

    2014-07-01

    This study investigated the effect of resveratrol on bone healing and its influence on the gene expression of osteogenic markers. Two calvarial defects were created and one screw-shaped titanium implant was inserted in the tibia of rats that were assigned to daily administration of placebo (control group, n=15) or 10mg/kg of resveratrol (RESV group, n=15) for 30 days. The animals were then sacrificed. One of the calvarial defects was processed for histomorphometric analysis and the tissue relative to the other was collected for mRNA quantification of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin (OPN), bone sialoprotein (BSP), osteoprotegrin (OPG), and receptor activator of NF-κB ligand (RANKL). Implants were removed by applying a counter-torque force. Histomorphometric analysis revealed higher remaining defect in the calvarial defects of the control group than the RESV group (P=0.026). Resveratrol increased the counter-torque values of implant removal when compared to control therapy (P=0.031). Gene expression analysis showed a higher expression of BMP-2 (P=0.011), BMP-7 (P=0.049), and OPN (P=0.002) genes in the RESV group than in the control group. In conclusion, resveratrol improved the repair of critical-sized bone defects and the biomechanical retention of implants. Indeed, this natural agent may up-regulate the gene expression of important osteogenic markers. PMID:24530035

  19. Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1.

    PubMed

    Wohl, Alexander P; Troilo, Helen; Collins, Richard F; Baldock, Clair; Sengle, Gerhard

    2016-06-10

    Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor. PMID:27059954

  20. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse

    PubMed Central

    Plas, Daniel T.; Dhande, Onkar; Lopez, Joshua E.; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C.

    2009-01-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  1. Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1*

    PubMed Central

    Wohl, Alexander P.; Troilo, Helen; Collins, Richard F.; Baldock, Clair

    2016-01-01

    Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor. PMID:27059954

  2. Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

    PubMed

    Marques, Cátia L; Fernández, Ignacio; Viegas, Michael N; Cox, Cymon J; Martel, Paulo; Rosa, Joana; Cancela, M Leonor; Laizé, Vincent

    2016-02-01

    BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events. PMID:26341094

  3. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

    PubMed

    Hirai, Takao; Tanaka, Kenjiro; Togari, Akifumi

    2014-06-13

    Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts. PMID:24794868

  4. Elafin Reverses Pulmonary Hypertension via Caveolin-1–Dependent Bone Morphogenetic Protein Signaling

    PubMed Central

    Nickel, Nils P.; Spiekerkoetter, Edda; Gu, Mingxia; Li, Caiyun G.; Li, Hai; Kaschwich, Mark; Diebold, Isabel; Hennigs, Jan K.; Kim, Ki-Yoon; Miyagawa, Kazuya; Wang, Lingli; Cao, Aiqin; Sa, Silin; Jiang, Xinguo; Stockstill, Raymond W.; Nicolls, Mark R.; Zamanian, Roham T.; Bland, Richard D.

    2015-01-01

    Rationale: Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. Objectives: To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Methods: Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. Measurements and Main Results: In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via

  5. N-methyl pyrrolidone as a potent bone morphogenetic protein enhancer for bone tissue regeneration.

    PubMed

    Miguel, Blanca San; Ghayor, Chafik; Ehrbar, Martin; Jung, Ronald E; Zwahlen, Roger A; Hortschansky, Peter; Schmoekel, Hugo G; Weber, Franz E

    2009-10-01

    In medicine, N-methyl pyrrolidone (NMP) has a long track record as a constituent in medical devices approved by the Food and Drug Administration and thus can be considered as a safe and biologically inactive small chemical. In the present study, we report on the newly discovered pharmaceutical property of NMP in enhancing bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with bone morphogenetic protein (BMP)-2, because NMP increased early and late markers for maturation of preosteoblasts and human bone marrow-derived stem cells in vitro. When we used the multipotent cell line C2C12 without autologous BMP expression, NMP alone had no effect on alkaline phosphatase activity, a marker for osteogenic transdifferentiation. Nevertheless, in combination with low BMP-2 doses, alkaline phosphatase activity was more than eight times as great. Thus, the pharmaceutical NMP mode of action is that of an enhancer of BMP activity. The dependency of the effects of NMP on BMP was confirmed in preosteoblasts because noggin, an extracellular BMP inhibitor, suppressed NMP-induced increases in early markers for osteoblast maturation in vitro. At the molecular level, NMP was shown to have no effect on the binding of BMP-2 to the ectodomain of the high-affinity BMP receptor IA. However, NMP further increased the phosphorylation of p38 and Smad1,5,8 induced by BMP-2. Thus, the small chemical NMP enhances BMP activity by increasing the kinase activity of the BMP receptor complex for Smad1,5,8 and p38 and could be employed as a potent drug for bone tissue regeneration and engineering. PMID:19320543

  6. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury. PMID:26765958

  7. A fucoidan from Nemacystus decipiens disrupts angiogenesis through targeting bone morphogenetic protein 4.

    PubMed

    Wang, Wucheng; Chen, Huanjun; Zhang, Lei; Qin, Yi; Cong, Qifei; Wang, Peipei; Ding, Kan

    2016-06-25

    A sulfated and acetylated fucoidan, named NDH01, was extracted from seaweed Nemacystus decipiens. NDH01 was composed of mannose, glucuronic acid, fucose, sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-d-1,2-Manp and β-d-1,4-GlcpA disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-d-1,2-Manp. The sidechain was composed of α-l-1,3,4-Fucp, α-l-1,4-Fucp, α-l-1,3-Fucp and α-l-1,4-GlcpA. The sulfate group was linked to C4 of α-l-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-l-1,2,3-Fucp. NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells. Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4 expression. The results suggested that NDH01 might be an angiogenesis inhibitor through targeting bone morphogenetic protein 4. PMID:27083822

  8. Cartilage-derived morphogenetic proteins enhance the osteogenic protein-1-induced osteoblastic cell differentiation of C2C12 cells.

    PubMed

    Yeh, Lee-Chuan C; Tsai, Alicia D; Zavala, Michelle C; Lee, John C

    2004-12-01

    Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) induces differentiation of the pluripotent mesenchymal cell line C2C12 into osteoblastic cells. OP-1 also alters the steady-state levels of messenger RNA (mRNA) encoding for the cartilage-derived morphogenetic proteins (CDMPs) in C2C12 cells. In the present study, the effects of exogenous CDMPs on bone cell differentiation induced by OP-1 in C2C12 cells were examined. Exogenous CDMP-1, -2, and -3 synergistically and dose-dependently enhanced OP-1 action in stimulating alkaline phosphatase (AP) activity and osteocalcin (OC) mRNA expression. AP staining studies revealed that the combination of OP-1 and CDMP enhanced OP-1 action by stimulating those cells that had responded to OP-1 and not by activating additional cells. The combination did not change the mRNA expression of the BMPs and their receptors. CDMP-1 enhanced the suppression of the OP-1-induced expression of the myogeneic differentiation regulator MyoD. CDMP-1 and OP-1 alone stimulated Smad5 protein expression, but the combination of OP-1 and CDMP-1 stimulated synergistically Smad5 protein expression. Thus, one mechanism of the observed synergy involved enhancement of the induced Smad5 protein expression. At the same protein concentration, CDMP-1 is most potent in enhancing OP-1 activity in inducing osteoblastic cell differentiation of C2C12 cells. CDMP-3 is about 80% as potent as CDMP-1, and CDMP-2 is the least potent (about 50% of CDMP-1). PMID:15389555

  9. Effect of recombinant human bone morphogenetic protein-2 on bisphosphonate-treated osteoblasts

    PubMed Central

    Kwon, Taek-Kyun; Song, Jae-Min; Kim, In-Ryoung; Park, Bong-Soo; Kim, Chul-Hoon; Cheong, In-Kyo

    2014-01-01

    Objectives Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. Materials and Methods Human fetal osteoblast cells (hFOB 1.19) were treated with 100 µM alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results Cell viability was decreased to 82.75%±1.00% by alendronate and then increased to 110.43%±1.35% after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. Conclusion rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression. PMID:25551094

  10. Inhibition of bone morphogenetic protein signaling reduces vascular calcification and atherosclerosis

    PubMed Central

    Derwall, Matthias; Malhotra, Rajeev; Lai, Carol S; Beppu, Yuko; Aikawa, Elena; Seehra, Jasbir S.; Zapol, Warren M; Bloch, Kenneth D.; Yu, Paul B.

    2012-01-01

    Objective The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. While genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes. Methods and Results We tested the impact of pharmacologic BMP inhibition upon atherosclerosis and calcification in low density lipoprotein receptor-deficient (LDLR−/−) mice. LDLR−/− mice fed a high-fat diet developed abundant vascular calcification within twenty weeks. Prolonged treatment of LDLR−/− mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the anti-atherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species (ROS) induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis. Conclusions These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification. PMID:22223731

  11. The Direct Interaction between Two Morphogenetic Proteins Is Essential for Spore Coat Formation in Bacillus subtilis

    PubMed Central

    Isticato, Rachele; Sirec, Teja; Vecchione, Stefano; Crispino, Anna; Saggese, Anella; Baccigalupi, Loredana; Notomista, Eugenio; Driks, Adam; Ricca, Ezio

    2015-01-01

    In Bacillus subtilis the protective layers that surround the mature spore are formed by over seventy different proteins. Some of those proteins have a regulatory role on the assembly of other coat proteins and are referred to as morphogenetic factors. CotE is a major morphogenetic factor, known to form a ring around the forming spore and organize the deposition of the outer surface layers. CotH is a CotE-dependent protein known to control the assembly of at least nine other coat proteins. We report that CotH also controls the assembly of CotE and that this mutual dependency is due to a direct interaction between the two proteins. The C-terminal end of CotE is essential for this direct interaction and CotH cannot bind to mutant CotE deleted of six or nine C-terminal amino acids. However, addition of a negatively charged amino acid to those deleted versions of CotE rescues the interaction. PMID:26484546

  12. ATP-Driven Self-Assembly of a Morphogenetic Protein in Bacillus subtilis

    PubMed Central

    Ramamurthi, Kumaran S.; Losick, Richard

    2008-01-01

    SUMMARY A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of over fifty proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  13. ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis.

    PubMed

    Ramamurthi, Kumaran S; Losick, Richard

    2008-08-01

    A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of more than 50 proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  14. Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation

    PubMed Central

    Shibasaki, Seiji; Kitano, Sachie; Karasaki, Miki; Tsunemi, Sachi; Sano, Hajime; Iwasaki, Tsuyoshi

    2015-01-01

    We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3

  15. Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.

    PubMed

    Shibasaki, Seiji; Kitano, Sachie; Karasaki, Miki; Tsunemi, Sachi; Sano, Hajime; Iwasaki, Tsuyoshi

    2015-01-01

    We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3

  16. Bone morphogenetic protein signaling in vertebrate motor neurons and neuromuscular communication

    PubMed Central

    Osses, Nelson; Henríquez, Juan P.

    2015-01-01

    An accurate communication between motor neurons and skeletal muscle fibers is required for the proper assembly, growth and maintenance of neuromuscular junctions (NMJs). Several signaling and extracellular matrix molecules play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in Drosophila have revealed crucial functions for early morphogens, such as members of the Wnt and Bone Morphogenetic Proteins (BMP) signaling pathways, during the assembly and maturation of the NMJ. Here, we bring together recent findings that led us to propose that BMPs also work in vertebrate organisms as diffusible cues to communicate motor neurons and skeletal muscles. PMID:25674047

  17. Bone Morphogenetic Protein 4 Signalling in Neural Stem and Progenitor Cells during Development and after Injury

    PubMed Central

    Cole, Alistair E.; Murray, Simon S.; Xiao, Junhua

    2016-01-01

    Substantial progress has been made in identifying the extracellular signalling pathways that regulate neural stem and precursor cell biology in the central nervous system (CNS). The bone morphogenetic proteins (BMPs), in particular BMP4, are key players regulating neuronal and glial cell development from neural precursor cells in the embryonic, postnatal, and injured CNS. Here we review recent studies on BMP4 signalling in the generation of neurons, astrocytes, and oligodendroglial cells in the CNS. We also discuss putative mechanisms that BMP4 may utilise to influence glial cell development following CNS injury and highlight some questions for further research. PMID:27293450

  18. Functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

    PubMed Central

    Glister, Claire; Satchell, Leanne; Bathgate, Ross A. D.; Wade, John D.; Dai, Yanzhenzi; Ivell, Richard; Anand-Ivell, Ravinder; Rodgers, Raymond J.; Knight, Philip G.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>twofold; P < 0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (−43-fold) with cytochrome P450, subfamily XVII (CYP17A1) and other key steroidogenic transcripts including steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11, subfamily A1 (CYP11A1) and 3 beta-hydroxysteroid dehydrogenase type 1 (HSD3B1) also down-regulated. BMP6 also reduced expression of nuclear receptor subfamily 5A1 (NR5A1) known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA (75%) and protein (94%) level and elicited a 77% reduction in CYP17A1 mRNA and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 expression (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ∼twofold. The CYP17A1 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling. PMID:23530236

  19. The role of the bone morphogenetic proteins in leukaemic stem cell persistence.

    PubMed

    Toofan, Parto; Irvine, David; Hopcroft, Lisa; Copland, Mhairi; Wheadon, Helen

    2014-08-01

    CML (chronic myeloid leukaemia) is characterized by the presence of the oncogenic tyrosine kinase fusion protein BCR (breakpoint cluster region)-Abl, responsible for driving the disease. Current TKI (tyrosine kinase inhibitor) therapies effectively inhibit BCR-Abl to control CML in the majority of patients, but do not eliminate the LSC (leukaemic stem cell) population, which becomes quiescent following treatment. Patients require long-term treatment to sustain remission; alternative strategies are therefore required, either alone or in combination with TKIs to eliminate the LSCs and provide a cure. The embryonic morphogenetic pathways play a key role in haemopoiesis with recent evidence suggesting LSCs are more dependent on these signals following chemotherapy than normal HSCs (haemopoietic stem cells). Recent evidence in the literature and from our group has revealed that the BMP (bone morphogenetic protein) pathway is differentially expressed in CML patients compared with normal donors. In the present review, we explore the role that BMP signalling plays in oesteoblast differentiation, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM (bone marrow) niche. Overall, we highlight the BMP pathway as a potential target for developing LSC-directed therapies in CML in the future. PMID:25109962

  20. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25508424

  1. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25464793

  2. Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification.

    PubMed

    Borodinsky, Laura N; Belgacem, Yesser H

    2016-04-01

    Morphogenetic proteins are responsible for patterning the embryonic nervous system by enabling cell proliferation that will populate all the neural structures and by specifying neural progenitors that imprint different identities in differentiating neurons. The adoption of specific neurotransmitter phenotypes is crucial for the progression of neuronal differentiation, enabling neurons to connect with each other and with target tissues. Preliminary neurotransmitter specification originates from morphogen-driven neural progenitor specification through the combinatorial expression of transcription factors according to morphogen concentration gradients, which progressively restrict the identity that born neurons adopt. However, neurotransmitter phenotype is not immutable, instead trophic factors released from target tissues and environmental stimuli change expression of neurotransmitter-synthesizing enzymes and specific vesicular transporters modifying neuronal neurotransmitter identity. Here we review studies identifying the mechanisms of catecholaminergic, GABAergic, glutamatergic, cholinergic and serotonergic early specification and of the plasticity of these neurotransmitter phenotypes during development and in the adult nervous system. The emergence of spontaneous electrical activity in developing neurons recruits morphogenetic proteins in the process of neurotransmitter phenotype plasticity, which ultimately equips the nervous system and the whole organism with adaptability for optimal performance in a changing environment. PMID:26686293

  3. The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development.

    PubMed

    Kumar, J P; Moses, K

    2001-07-01

    The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium. PMID:11526075

  4. Additive Effects of Retinoic Acid (RA) and Bone Morphogenetic Protein 4 (BMP-4) Apoptosis Signaling in Retinoblastoma Cell Lines

    PubMed Central

    Müller, Patrick; Doliva, Rebekka; Busch, Maike; Philippeit, Claudia; Stephan, Harald; Dünker, Nicole

    2015-01-01

    Retinoids have been shown to serve promising therapeutic agents for human cancers, e.g. the treatment of neuroblastoma. Synthetic retinoids, specific for particular retinoic acid (RA) receptors, are tested as new therapy strategies. In the present study, application of recombinant retinoic acid (RA) lowers retinoblastoma (RB) cell viability and induces apoptosis in RB cell lines. Combined treatment of RA and bone morphogenetic protein 4 (BMP-4) increases the pro-apoptotic effect of RA in the RB cells lines WERI-Rb1, Y-79, RB355, RBL-30 and RBL-15, indicating an additive effect. We could show that in WERI-Rb1 cells RA/BMP-4 mediated cell death is at least partially caspase-dependent, whereby RA and BMP-4 additively increased (i) Apaf-1 mRNA levels, (ii) caspase-9 cleavage activity and (iii) the number of activated, cleaved caspase-3 positive cells. Compared to single application of RA and BMP-4, combined RA/BMP-4 treatment significantly augments mRNA levels of the retinoic acid receptors (RARs) RARα and RARß and the retinoic X receptor (RXR) RXRγ suggesting an interaction in the induction of these RA receptor subtypes in WERI-Rb1 cells. Agonist studies revealed that both, RARs and RXRs are involved in RA/BMP-4 mediated apoptosis in WERI-Rb1 retinoblastoma cells. Employing specific RAR subtype antagonists and a RXRß and RXRγ knockdown, we proved that RA/BMP-4 apoptosis signaling in WERI-Rb1 cells requires the RA receptor subtypes RARα, RARß, RXRß and RXRγ. Deciphering signaling mechanisms underlying apoptosis induction of RA and BMP-4 in WERI-Rb1 cells, our study provides useful starting-points for future retinoid-based therapy strategies in retinoblastoma. PMID:26173116

  5. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  6. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    PubMed Central

    Herhaus, Lina; Al-Salihi, Mazin A.; Dingwell, Kevin S.; Cummins, Timothy D.; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  7. The controversy surrounding bone morphogenetic proteins in the spine: a review of current research.

    PubMed

    Hustedt, Joshua W; Blizzard, Daniel J

    2014-12-01

    Bone morphogenetic proteins have been in use in spinal surgery since 2002. These proteins are members of the TGF-beta superfamily and guide mesenchymal stem cells to differentiate into osteoblasts to form bone in targeted tissues. Since the first commercial BMP became available in 2002, a host of research has supported BMPs and they have been rapidly incorporated in spinal surgeries in the United States. However, recent controversy has arisen surrounding the ethical conduct of the research supporting the use of BMPs. Yale University Open Data Access (YODA) recently teamed up with Medtronic to offer a meta-analysis of the effectiveness of BMPs in spinal surgery. This review focuses on the history of BMPs and examines the YODA research to guide spine surgeons in their use of BMP in spinal surgery. PMID:25506287

  8. Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

    PubMed

    Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

    1989-07-01

    A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions. PMID:2545399

  9. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Sung, Nak-Yun; Choi, Jong-il

    2015-06-01

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants

  10. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

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

    PubMed Central

    2016-01-01

    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. PMID:27433166

  12. Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

    PubMed

    Lombardo, Barbara; Rocco, Teresa; Esposito, Maria T; Cantilena, Bruno; Gargiulo, Sara; Greco, Adelaide; Montanaro, Donatella; Brunetti, Arturo; Pastore, Lucio

    2013-06-01

    Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. PMID:23317056

  13. Patterns of bone morphogenetic protein-2 expression in smooth muscle tumors of the uterine corpus and other uterine tissues.

    PubMed

    Fadare, Oluwole; Renshaw, Idris L; Liang, Sharon X

    2011-07-01

    Bone morphogenetic proteins (BMPs) are extracellular, multifunctional growth factors that constitute the largest subset of the transforming growth factor β superfamily. BMP2 is involved in cardiovascular embryogenesis, in addition to a variety of other postnatal functions, such as neovascularization, osteoinduction, tumor signaling, and in the uterus, stromal decidualization at the implantation site. Estrogen receptor signaling is common in smooth muscle tumors of the uterus, and preclinical models suggest significant interactions between BMP2 and estrogen receptor-mediated signaling. The purpose of this study is to define the patterns of BMP2 expression, as assessed by immunohistochemistry, in smooth muscle tumors and other tissues of the uterine corpus, and to establish whether BMP2 expression has any prognostic significance in uterine leiomyosarcomas. BMP2 was positive (cytoplasmic pattern, typically focal) in 24% of leiomyosarcomas and 20.7% of leiomyomata, but was either infrequently expressed or not expressed in all other tissues evaluated, including normal myometrium and endometrium, endometrial stromal tumors, typical adenomyoma, adenomyosis, and serosal endometriosis. The endothelial cells of small, thin-walled vessels were frequently, but not invariably immunoreactive for BMP2. There was no significant difference between BMP2⁺ and BMP⁻ leiomyosarcomas regarding average tumor size, average patient age, microvessel density, and proportions with high tumor grade, advanced stage and frequency of death from disease on follow-up. Two (29%) of 7 BMP2⁺ leiomyosarcomas were estrogen receptor+, compared with 5 (50%) of 10 BMP2⁻ leiomyosarcomas, a statistically insignificant difference (P=0.62). It is concluded that BMP2 is only expressed in a minority of smooth muscle tumors of the uterine corpus, and lacks prognostic significance in leiomyosarcomas. BMP2 is rarely expressed in the other nonendothelial tissues of the human uterine corpus that were

  14. Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish.

    PubMed

    Thorimbert, Valentine; König, Désirée; Marro, Jan; Ruggiero, Florence; Jaźwińska, Anna

    2015-10-01

    Zebrafish fin regeneration involves initial formation of the wound epidermis and the blastema, followed by tissue morphogenesis. The mechanisms coordinating differentiation of distinct tissues of the regenerate are poorly understood. Here, we applied pharmacologic and transgenic approaches to address the role of bone morphogenetic protein (BMP) signaling during fin restoration. To map the BMP transcriptional activity, we analyzed the expression of the evolutionarily conserved direct phospho-Smad1 target gene, id1, and its homologs id2a and id3. This analysis revealed the BMP activity in the distal blastema, wound epidermis, osteoblasts, and blood vessels of the regenerate. Blocking the BMP function with a selective chemical inhibitor of BMP type I receptors, DMH1, suppressed id1 and id3 expression and arrested regeneration after blastema formation. We identified several previously uncharacterized functions of BMP during fin regeneration. Specifically, BMP signaling is required for remodeling of plexus into structured blood vessels in the rapidly growing regenerate. It organizes the wound epithelium by triggering wnt5b expression and promoting Collagen XIV-A deposition into the basement membrane. BMP represents the first known signaling that induces actinotrichia formation in the regenerate. Our data reveal a multifaceted role of BMP for coordinated morphogenesis of distinct tissues during regeneration of a complex vertebrate appendage. PMID:26148971

  15. Control of phenotypic plasticity of smooth muscle cells by bone morphogenetic protein signaling through the myocardin-related transcription factors.

    PubMed

    Lagna, Giorgio; Ku, Manching M; Nguyen, Peter H; Neuman, Nicole A; Davis, Brandi N; Hata, Akiko

    2007-12-21

    Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFbeta and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle alpha-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases. PMID:17947237

  16. The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway ▿

    PubMed Central

    Chan, Mun Chun; Weisman, Alexandra S.; Kang, Hara; Nguyen, Peter H.; Hickman, Tyler; Mecker, Samantha V.; Hill, Nicholas S.; Lagna, Giorgio; Hata, Akiko

    2011-01-01

    Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ∼60% of familial PAH (FPAH) and ∼30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calcium-calcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH. PMID:21135135

  17. Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli.

    PubMed

    Retnoningrum, Debbie S; Pramesti, H T; Santika, P Y; Valerius, O; Asjarie, S; Suciati, T

    2012-08-01

    Codons in the open reading frame (ORF) encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38 bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39 g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC-MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system. PMID:22691543

  18. Lovastatin stimulates human vascular smooth muscle cell expression of bone morphogenetic protein-2, a potent inhibitor of low-density lipoprotein-stimulated cell growth.

    PubMed

    Emmanuele, Luca; Ortmann, Jana; Doerflinger, Tim; Traupe, Tobias; Barton, Matthias

    2003-02-28

    Bone morphogenetic proteins (BMPs) stimulate ectopic bone formation in skeletal muscle. Here we show that human vascular smooth muscle cells (VSMC) abundantly express mRNA encoding for BMP receptor type II, BMP-2, and BMP-7 proteins. Treatment with the 3-hydroxy-3-methylglutaryl coenzyme A inhibitor lovastatin (34 microM) increased BMP-2 gene transcription >14-fold as measured by real-time PCR analysis (P<0.05 vs. solvent control). Moreover, VSMC proliferation stimulated with native low-density lipoprotein (100 microg of protein/mL) was prevented by either human recombinant BMP-2 or BMP-7 at concentrations of 100 ng/mL (P<0.05). Both BMPs also inhibited basal cell proliferation (P<0.05). Induction of BMPs and subsequent inhibition of VSMC growth and/or induction of vascular bone formation could contribute to the mechanisms by which statins increase plaque stability in patients with coronary atherosclerosis. PMID:12593849

  19. Effects of antenatal application of ambroxol and glucocorticoid on lung morphometry and signal transduction of bone morphogenetic protein in the fetal rat.

    PubMed

    Chen, Xiao-Qing; Wu, Sheng-Hua; Guo, Xi-Rong; Zhou, Xiao-Yu

    2012-07-01

    Antenatal ambroxol, dexamethasone (Dex) and betamethasone (Beta) are used to prevent neonate respiratory distress syndrome. The present study aimed to investigate the role of ambroxol, Dex and Beta administered antenatally on lung morphogenesis and signal transduction of bone morphogenetic protein (BMP) in rat embryo. Fetal lungs treated with ambroxol, 1-day Beta, 3-day Dex and 3-day Beta were more mature compared to the controls as determined by light microscopy and transmission electron microscopy. Expression of BMP4 and bone morphogenetic protein receptor II (BMPR‑II) mRNA was upregulated in the 1-day-Beta-, 3-day-Dex- and 3-day-Beta-treated animals. BMP4 and BMPR-II protein were significantly increased in the 1-day-Beta-, 3-day-Dex- and 3-day-Beta-treated animals. Ambroxol, Dex and Beta promoted the morphological development of rat fetal lung; Beta was more effective than Dex. A multi-dose of glucocorticoids exhited a more beneficial effect than a single dose. The effects of Beta and Dex may be mediated by regulation of BMP signal transduction in rat fetal lung. PMID:22552703

  20. Recombinant Human Bone Morphogenetic Protein-2 in Development and Progression of Oral Squamous Cell Carcinoma.

    PubMed

    Zaid, Khaled Waleed; Chantiri, Mansour; Bassit, Ghassan

    2016-01-01

    Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β superfamily, regulate many cellular activities including cell migration, differentiation, adhesion, proliferation and apoptosis. Use of recombinant human bone morphogenic protein?2 (rhBMP?2) in oral and maxillofacial surgery has seen a tremendous increase. Due to its role in many cellular pathways, the influence of this protein on carcinogenesis in different organs has been intensively studied over the past decade. BMPs also have been detected to have a role in the development and progression of many tumors, particularly disease-specific bone metastasis. In oral squamous cell carcinoma - the tumor type accounting for more than 90% of head and neck malignancies- aberrations of both BMP expression and associated signaling pathways have a certain relation with the development and progression of the disease by regulating a range of biological functions in the altered cells. In the current review, we discuss the influence of BMPs -especially rhBMP-2- in the development and progression of oral squamous cell carcinoma. PMID:27039814

  1. Transient brown adipocyte-like cells derive from peripheral nerve progenitors in response to bone morphogenetic protein 2.

    PubMed

    Salisbury, Elizabeth A; Lazard, Zawaunyka W; Ubogu, Eroboghene E; Davis, Alan R; Olmsted-Davis, Elizabeth A

    2012-12-01

    Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling. PMID:23283549

  2. LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads.

    PubMed

    Sangadala, Sreedhara; Boden, Scott D; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

    2006-06-23

    Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway. PMID:16611643

  3. Anti-Müllerian hormone regulation by the bone morphogenetic proteins in the sheep ovary: deciphering a direct regulatory pathway.

    PubMed

    Estienne, Anthony; Pierre, Alice; di Clemente, Nathalie; Picard, Jean-Yves; Jarrier, Peggy; Mansanet, Camille; Monniaux, Danielle; Fabre, Stéphane

    2015-01-01

    In the ovary, anti-Müllerian hormone (AMH) is produced by the granulosa cells of growing follicles and can modulate the recruitment of primordial follicles and the FSH-dependent development of follicles. However, the regulation of its production remains poorly understood. Recently, a stimulating effect of the bone morphogenetic proteins (BMPs) on AMH production by granulosa cells has been shown in vitro, but the molecular mechanisms implicated in this regulation and its physiological importance in ovarian function have not yet been established. In the hyperprolific Booroola ewes carrying the FecB(B) partial loss-of-function mutation in the fecundity gene encoding the FecB/BMP receptor, type 1B, the granulosa cells of antral follicles expressed and secreted low AMH amounts, resulting in low AMH concentrations in blood, despite high numbers of AMH-secreting follicles in ovaries. The presence of the FecB(B) mutation impaired the granulosa cell response to the stimulating action of BMP4 on AMH production, indicating a crucial role of the BMP receptor, type 1B in AMH regulation. In ovine granulosa cells, BMP4 enhanced the transcriptional activity of the human AMH promoter, and this action depended on the presence of SMAD1, acting on a promoter sequence located between -423 and -202 bp upstream of the AMH transcription start site. SMAD1 and SF1 acted in concert to mediate BMP4 action on the AMH promoter. Among the 2 SF1 binding sites present on the AMH promoter, the most proximal site, located at -92 bp upstream of the AMH transcription start site, was found to be critical for ensuring the response of the AMH promoter to BMP4. In conclusion, AMH could mediate the actions of BMPs in regulating follicular development and contributing to the determination of ovulation numbers. A molecular model of regulation of the AMH promoter transactivation by BMP signaling is proposed. PMID:25322464

  4. Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

    PubMed Central

    Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

    2015-01-01

    Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

  5. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo.

    PubMed

    Teng, Fu-Yuan; Chen, Wen-Cheng; Wang, Yin-Lai; Hung, Chun-Cheng; Tseng, Chun-Chieh

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use. PMID:26977141

  6. Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro.

    PubMed

    Boden, S D; McCuaig, K; Hair, G; Racine, M; Titus, L; Wozney, J M; Nanes, M S

    1996-08-01

    Bone morphogenetic proteins (BMPs) induce cartilage and bone differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive proteins may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger bone nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to 10-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation. PMID:8754767

  7. Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

    PubMed

    Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

    2015-07-15

    Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. PMID:25952563

  8. Use of bone morphogenetic proteins in mesenchymal stem cell stimulation of cartilage and bone repair

    PubMed Central

    Scarfì, Sonia

    2016-01-01

    The extracellular matrix-associated bone morphogenetic proteins (BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell (MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted cell-type lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair. PMID:26839636

  9. Bone Morphogenetic Protein 2 Signaling Negatively Modulates Lymphatic Development in Vertebrate Embryos

    PubMed Central

    Dunworth, William P.; Cardona-Costa, Jose; Bozkulak, Esra Cagavi; Kim, Jun-Dae; Meadows, Stryder; Fischer, Johanna C.; Wang, Yeqi; Cleaver, Ondine; Qyang, Yibing; Ober, Elke A.; Jin, Suk-Won

    2014-01-01

    Rationale The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. Objective Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. Methods and Results BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell–derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. Conclusions Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development. PMID:24122719

  10. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo

    PubMed Central

    Teng, Fu-Yuan; Chen, Wen-Cheng; Wang, Yin-Lai; Hung, Chun-Cheng; Tseng, Chun-Chieh

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use. PMID:26977141

  11. Induction of a dopaminergic phenotype in cultured striatal neurons by bone morphogenetic proteins.

    PubMed

    Stull, N D; Jung, J W; Iacovitti, L

    2001-09-23

    In the present study, we examined whether the bone morphogenetic proteins (BMPs), which are important in the developmental specification of transmitter type in certain classes of neurons, might also play a role in signaling the differentiation of a dopaminergic (DA) phenotype. We found that BMP-2, -4 and -6 were each capable of inducing, in a dose and time dependent manner, moderate levels of the DA enzyme tyrosine hydroxylase (TH) in cultured neurons from the mouse embryonic striatum. In contradistinction to other TH-inducing agents, BMPs initiated de novo TH expression without the required synergy of exogenous growth factors or co-activating substances and in neurons presumably aged (E16) beyond the critical period for induction. However, the appearance of TH in induced cells was short-lived (24 h) and could not be prolonged by repeated supplementation with the BMPs. Inhibitors of the mitogen-activated protein kinase (MAPK/ERK) signaling pathway, PD98059 and apigenin, did not prevent TH induction by BMP-4, as they did other TH inducing agents, indicating that the MAPK/ERK pathway does not mediate BMPs effects on TH expression. We conclude that BMP-2, -4 and -6 can be added to the expanding inventory of agents capable of inducing TH, making them potentially important in the specification of a DA phenotype in stem/precursor cells for the treatment of Parkinson's disease. PMID:11557097

  12. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2.

    PubMed

    Zhang, Zheng; Wang, Guoxian; Li, Chen; Liu, Danping

    2013-08-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

  13. Bone graft substitutes and bone morphogenetic proteins for osteoporotic fractures: what is the evidence?

    PubMed

    Van Lieshout, Esther M M; Alt, Volker

    2016-01-01

    Despite improvements in implants and surgical techniques, osteoporotic fractures remain challenging to treat. Among other major risk factors, decreased expression of morphogenetic proteins has been identified for impaired fracture healing in osteoporosis. Bone grafts or bone graft substitutes are often used for stabilizing the implant and for providing a scaffold for ingrowth of new bone. Both synthetic and naturally occurring biomaterials are available. Products generally contain hydroxyapatite, tricalcium phosphate, dicalcium phosphate, calcium phosphate cement, calcium sulfate (plaster of Paris), or combinations of the above. Products have been used for the treatment of osteoporotic fractures of the proximal humerus, distal radius, vertebra, hip, and tibia plateau. Although there is generally consensus that screw augmentation increased the biomechanical properties and implant stability, the results of using these products for void filling are not unequivocal. In osteoporotic patients, Bone Morphogenetic Proteins (BMPs) have the potential impact to improve fracture healing by augmenting the impaired molecular and cellular mechanisms. However, the clinical evidence on the use of BMPs in patients with osteoporotic fractures is poor as there are no published clinical trials, case series or case studies. Even pre-clinical literature on in vitro and in vivo data is weak as most articles focus on the beneficial role for BMPs for restoration of the underlying pathophysiological factors of osteoporosis but do not look at the specific effects on osteoporotic fracture healing. Limited data on animal experiments suggest stimulation of fracture healing in ovariectomized rats by the use of BMPs. In conclusion, there is only limited data on the clinical relevance and optimal indications for the use of bone graft substitute materials and BMPs on the treatment of osteoporotic fractures despite the clinical benefits of these materials in other clinical indications. Given the

  14. Bone Morphogenetic Proteins in Craniofacial Surgery: Current Techniques, Clinical Experiences, and the Future of Personalized Stem Cell Therapy

    PubMed Central

    Chenard, Kristofer E.; Teven, Chad M.; He, Tong-Chuan; Reid, Russell R.

    2012-01-01

    Critical-size osseous defects cannot heal without surgical intervention and can pose a significant challenge to craniofacial reconstruction. Autologous bone grafting is the gold standard for repair but is limited by a donor site morbidity and a potentially inadequate supply of autologous bone. Alternatives to autologous bone grafting include the use of alloplastic and allogenic materials, mesenchymal stem cells, and bone morphogenetic proteins. Bone morphogenetic proteins (BMPs) are essential mediators of bone formation involved in the regulation of differentiation of osteoprogenitor cells into osteoblasts. Here we focus on the use of BMPs in experimental models of craniofacial surgery and clinical applications of BMPs in the reconstruction of the cranial vault, palate, and mandible and suggest a model for the use of BMPs in personalized stem cell therapies. PMID:23226941

  15. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    PubMed

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. PMID:26699903

  16. Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes

    PubMed Central

    Ramey, Guillemette; Deschemin, Jean-Christophe; Vaulont, Sophie

    2009-01-01

    Background The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. Design and Methods Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. Results After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum. Interestingly, addition of 30 μM holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe−/− mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. Conclusions We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin. PMID:19454495

  17. Heparin Microparticle Effects on Presentation and Bioactivity of Bone Morphogenetic Protein-2

    PubMed Central

    Hettiaratchi, Marian H.; Miller, Tobias; Temenoff, Johnna S.; Guldberg, Robert E.; McDevitt, Todd C.

    2014-01-01

    Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration. PMID:24881028

  18. Bone morphogenetic protein 7 induces cementogenic differentiation of human periodontal ligament-derived mesenchymal stem cells.

    PubMed

    Torii, D; Tsutsui, T W; Watanabe, N; Konishi, K

    2016-01-01

    Bone morphogenetic protein 7 (BMP-7) is a multifunctional differentiation factor that belongs to the transforming growth factor superfamily. BMP-7 induces gene expression of protein tyrosine phosphatase-like, member A/cementum attachment protein (PTPLA/CAP) and cementum protein 1 (CEMP1), both of which are markers of cementoblasts and cementocytes. In the previous study, we reported that BMP-7 treatment enhanced PTPLA/CAP and CEMP1 expression in both normal and immortal human periodontal ligament (PDL) cells. To elucidate the molecular mechanisms of the gene expression of these molecules, in this study, we identified a functional transcription activator binding region in the promoter region of PTPLA/CAP and CEMP1 that is responsive to BMP signals. Here, we report that some short motifs termed GC-rich Smad-binding elements (GC-SBEs) that are located in the human PTPLA/CAP promoter and CEMP1 promoter are BMP-7 responsive as analyzed with luciferase promoter assays. On the other hand, we found that transcription of Sp7/Osterix and PTPLA/CAP was up-regulated after 1 week of BMP-7 treatment on purified normal human PDL cells as a result of gene expression microarray analysis. Furthermore, transcription of Sp7/Osterix, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) was up-regulated after 2 weeks of BMP-7 treatment, whereas gene expression of osteo/odontogenic markers such as integrin-binding sialoprotein (IBSP), collagen, type I, alpha 1 (COL1A1), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP) was not up-regulated in purified normal or immortal human PDL cells as a result of qRT-PCR. The results suggest that BMP-7 mediates cementogenesis via GC-SBEs in human PDL cells and that its molecular mechanism is different from that for osteo/odontogenesis. PMID:25464857

  19. Postoperative Cyst Associated with Bone Morphogenetic Protein Use in Posterior and Transforaminal Lumbar Interbody Fusion Managed Conservatively: Report of Two Cases

    PubMed Central

    Mejía, Diana M; Drazin, Doniel; Anand, Neel

    2016-01-01

    Bone morphogenetic protein use in spinal surgery for off-label indications continues to remain popular. One area where its use has known associated radicular complications is posterior or transforaminal lumbar interbody fusion. These complications include radiculitis, cyst development, and heterotopic ossification, amongst others. Typically, cyst development has been treated surgically. We present two cases of bone morphogenetic protein-related cysts treated medically and thus, present medical treatment as an alternative treatment option. PMID:27014519

  20. Imaging symptomatic bone morphogenetic protein-2-induced heterotopic bone formation within the spinal canal: case report.

    PubMed

    Chryssikos, Timothy; Crandall, Kenneth M; Sansur, Charles A

    2016-05-01

    Heterotopic bone formation within the spinal canal is a known complication of bone morphogenetic protein-2 (BMP-2) and presents a clinical and surgical challenge. Imaging modalities are routinely used for operative planning in this setting. Here, the authors present the case of a 59-year-old woman with cauda equina syndrome following intraoperative BMP-2 administration. Plain film myelographic studies showed a region of severe stenosis that was underappreciated on CT myelography due to a heterotopic bony lesion mimicking the dorsal aspect of a circumferentially patent thecal sac. When evaluating spinal stenosis under these circumstances, it is important to carefully consider plain myelographic images in addition to postmyelography CT images as the latter may underestimate the true degree of stenosis due to the potentially similar radiographic appearances of evolving BMP-2-induced heterotopic bone and intrathecal contrast. Alternatively, comparison of sequentially acquired noncontrast CT scans with CT myelographic images may also assist in distinguishing BMP-2-induced heterotopic bony lesions from the thecal sac. Further studies are needed to elucidate the roles of the available imaging techniques in this setting and to characterize the connection between the radiographic and histological appearances of BMP-2-induced heterotopic bone. PMID:26824586

  1. Mechanical testing of recombinant human bone morphogenetic protein-7 regenerated bone in sheep mandibles.

    PubMed

    Kontaxis, A; Abu-Serriah, M; Ayoub, A F; Barbenel, J C

    2004-01-01

    A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated bone. Recombinant human bone morphogenetic protein-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the bone regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete bone regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the bone were very variable. The new bone in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, 10-20 per cent; stiffness, 6-15 per cent). The other half had better-quality bone and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated bone bridge suggesting that 35 mm is a critical-size bone defect. PMID:15648662

  2. Bone morphogenetic proteins in tumour associated angiogenesis and implication in cancer therapies.

    PubMed

    Ye, Lin; Jiang, Wen G

    2016-10-01

    Bone morphogenetic protein (BMP) belongs to transforming growth factor-β superfamily. To date, more than 20 BMPs have been identified in humans. BMPs play a critical role in embryonic and postnatal development, and also in maintaining homeostasis in different organs and tissues by regulating cell differentiation, proliferation, survival and motility. They play important roles in the development and progression of certain malignancies, including prostate cancer, breast cancer, lung cancer, etc. Recently, more evidence shows that BMPs are also involved in tumour associated angiogenesis. For example BMP can either directly regulate the functions of vascular endothelial cells or indirectly influence the angiogenesis via regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). Such crosstalk can also be reflected in the interaction with other angiogenic factors, like hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). All these factors are involved in the orchestration of the angiogenic process during tumour development and progression. Review of the relevant studies will provide a comprehensive prospective on current understanding and shed light on the corresponding therapeutic opportunity. PMID:26639195

  3. Retinoic acid signaling regulates sonic hedgehog and bone morphogenetic protein signalings during genital tubercle development.

    PubMed

    Liu, Liqing; Suzuki, Kentaro; Nakagata, Naomi; Mihara, Kenichiro; Matsumaru, Daisuke; Ogino, Yukiko; Yashiro, Kenta; Hamada, Hiroshi; Liu, Zhonghua; Evans, Sylvia M; Mendelsohn, Cathy; Yamada, Gen

    2012-02-01

    Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA-induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA-synthesizing enzyme, Raldh2, and for the RA-catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1(-/-) mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss-of-function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development. PMID:22127979

  4. Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion.

    PubMed

    Cyr-Depauw, Chanèle; Northey, Jason J; Tabariès, Sébastien; Annis, Matthew G; Dong, Zhifeng; Cory, Sean; Hallett, Michael; Rennhack, Jonathan P; Andrechek, Eran R; Siegel, Peter M

    2016-05-15

    ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling. PMID:26976638

  5. Experimental study of osteoinduction using a new material as a carrier for bone morphogenetic protein-2.

    PubMed

    Koyama, Noriaki; Okubo, Yasunori; Nakao, Kazumasa; Osawa, Kenji; Bessho, Kazuhisa

    2011-06-01

    We evaluated the usefulness of artificial collagen as a new carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) by comparing it with that of atelopeptide collagen, which is derived from porcine skin, and which we have previously shown to be useful for the induction of bone. rhBMP-2 5μg with either atelopeptide collagen 3mg or artificial collagen 3mg was implanted into the calf muscle of 10-week-old Wistar rats (n=3 in each group). Three rats were given artificial collagen alone and acted as controls (n=3). Radiographic evaluation, histological analysis, and biochemical examinations were made on day 21 after implantation. Soft radiographs (wavelength 10-0.10nm) showed opaque shadows in both groups. Histological analysis showed that new bone had formed in both experimental groups. Endochondral ossification was found at the outermost edge of the implanted collagen in the atelopeptide group. However, there was less ossification in the implanted collagen in the artificial collagen group. On biochemical examination, alkaline phosphatase activity and calcium concentrations in both experimental groups were higher than in the control group, and were higher in the atelopeptide group than in the artificial collagen group. Our results suggest that artificial collagen is useful as a carrier for rhBMP-2 designed to promote the formation of new bone. PMID:20554359

  6. Bone morphogenetic protein 4 inhibits liposaccharide-induced inflammation in the airway.

    PubMed

    Li, Zhengtu; Wang, Jian; Wang, Yan; Jiang, Hua; Xu, Xiaoming; Zhang, Chenting; Li, Defu; Xu, Chuyi; Zhang, Kedong; Qi, Yafei; Gong, Xuefang; Tang, Chun; Zhong, Nanshan; Lu, Wenju

    2014-11-01

    Bone morphogenetic protein 4 (BMP4) is a multifunctional growth factor that belongs to the TGF-β superfamily. The role of BMP4 in lung diseases is not fully understood. Here, we demonstrate that BMP4 was upregulated in lungs undergoing lipopolysaccharide (LPS)-induced inflammation, and in airway epithelial cells treated with LPS or TNF-α. BMP4 mutant (BMP4(+/-) ) mice presented with more severe lung inflammation in response to LPS or Pseudomonas aeruginosa, and lower bacterial load compared with that in BMP4(+/+) mice. Knockdown of BMP4 by siRNA increased LPS and TNF-α-induced IL-8 expression in 16HBE human airway epithelial cells and in primary human bronchial epithelial cells. Similarly, peritoneal macrophages from BMP4(+/-) mice produced greater levels of TNF-α and keratinocyte chemoattractant (KC) upon LPS treatment compared with cells from BMP4(+/+) mice. Administration of exogenous BMP4 attenuated the upregulation of TNF-α, IL-8, or KC induced by LPS and/or TNF-α in airway epithelial cells, and peritoneal macrophages. Finally, partial deficiency of BMP4 in BMP4(+/-) mice protected the animals from restrictive lung function reduction upon chronic LPS exposure. These results indicate that BMP4 plays an important anti-inflammatory role, controlling the strength and facilitating the resolution of acute lung inflammation; yet, BMP4 also contributes to lung function impairment during chronic lung inflammation. PMID:25142202

  7. Tracheal cartilage regeneration and new bone formation by slow release of bone morphogenetic protein (BMP)-2.

    PubMed

    Igai, Hitoshi; Chang, Sung Soo; Gotoh, Masashi; Yamamoto, Yasumichi; Yamamoto, Masaya; Tabata, Yasuhiko; Yokomise, Hiroyasu

    2008-01-01

    We investigated the efficiency of bone morphogenetic protein (BMP)-2 released slowly from gelatin sponge for tracheal cartilage regeneration. A 1-cm gap was made in the mid-ventral portion of each of 10 consecutive tracheal cartilages. In the control group (n = 4), the resulting gap was left untreated. In the gelatin group (n = 4), plain gelatin was implanted in the gap. In the BMP-2 group (n = 4), gelatin containing 100 microg BMP-2 was implanted. We euthanatized all dogs in each group at 1, 3, 6, and 12 months after the implantation, respectively, and then examined the implant site macro- and microscopically. In the BMP-2 group, regenerated fibrous cartilage and newly formed bone were observed at 1 and 12 months. Regenerated cartilage was observed at the ends of the host cartilage stumps, with newly formed bone in the middle portion. The gaps were filled with regenerated cartilage and newly formed bone. At 3 and 6 months, regenerated cartilage, but not newly formed bone, was evident. The regenerated cartilage was covered with perichondrium and showed continuity with the host cartilage. We succeeded in inducing cartilage regeneration and new bone formation in canine trachea by slow release of 100 microg BMP-2 from gelatin. PMID:18204324

  8. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2.

    PubMed

    Thanyaphoo, Suphannee; Kaewsrichan, Jasadee

    2016-09-01

    Silicon-substituted calcium phosphate (Si-CaP) was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP) cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2) was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future. PMID:27383886

  9. The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects.

    PubMed Central

    Takagi, K; Urist, M R

    1982-01-01

    Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP. Images Fig. 1a. Fig. 1b. Fig. 1c. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:7092346

  10. Inkjet-Based Biopatterning of Bone Morphogenetic Protein-2 to Spatially Control Calvarial Bone Formation

    PubMed Central

    Miller, Eric D.; DeCesare, Gary E.; Usas, Arvydas; Lensie, Emily L.; Bykowski, Michael R.; Huard, Johnny; Weiss, Lee E.; Losee, Joseph E.; Campbell, Phil G.

    2010-01-01

    The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix™ human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or 125I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns. PMID:20028232

  11. Immobilization of Bone Morphogenetic Protein on DOPA- or Dopamine-Treated Titanium Surfaces to Enhance Osseointegration

    PubMed Central

    Kang, Jeonghwa; Tada, Seiichi; Kitajima, Takashi; Son, Tae Il; Aigaki, Toshiro; Ito, Yoshihiro

    2013-01-01

    Titanium was treated with 3,4-dihydroxy-L-phenylalanine (DOPA) or dopamine to immobilize bone morphogenetic protein-2 (BMP2), a biomolecule. DOPA and dopamine solutions turned into suspensions, and precipitates were produced at high pH. Both treatments produced a brown surface on titanium that was thicker at high pH than low pH. Dopamine produced a thicker layer than DOPA. The hydrophobicity of the surfaces increased after treatment with dopamine independent of pH. Furthermore, there were more amino groups in the layers formed at pH 8.5 than pH 4.5 in both treatments. Dopamine treatment produced more amino groups in the layer than DOPA. BMP2 was immobilized on the treated surfaces via a coupling reaction using carbodiimide. More BMP2 was immobilized on surfaces treated at pH 8.5 than pH 4.5 in both treatments. The immobilized BMP induced specific signal transduction and alkali phosphatase, a differentiation marker. Thus, the present study demonstrates that titanium treated with DOPA or dopamine can become bioactive via the surface immobilization of BMP2, which induces specific signal transduction. PMID:24459666

  12. Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells

    PubMed Central

    ZHANG, YUNYUAN; CHEN, XIAN; QIAO, MIN; ZHANG, BING-QIANG; WANG, NING; ZHANG, ZHONGLIN; LIAO, ZHAN; ZENG, LIYI; DENG, YOULIN; DENG, FANG; ZHANG, JUNHUI; YIN, LIANGJUN; LIU, WEI; ZHANG, QIAN; YAN, ZHENGJIAN; YE, JIXING; WANG, ZHONGLIANG; ZHOU, LAN; LUU, HUE H.; HAYDON, REX C.; HE, TONG-CHUAN; ZHANG, HONGYU

    2014-01-01

    Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells. PMID:24993644

  13. [MORPHOLOGICAL CHARACTERISTICS OF OSSEOINTEGRATION AFTERAPPLICATION OF TITANIUM IMPLANTS WITH BIOACTIVE COATING AND RECOMBINANT BONE MORPHOGENETIC PROTEIN].

    PubMed

    Gaifullin, N M; Karyagina, A S; Gromov, A V; Terpilovskiy, A A; Malanin, D A; Demeshchenko, M V; Novochadov, V V

    2016-01-01

    Experiments were carried out on 22 albino male Wistar rats to study the morphological peculiarities of osseointegration of titanium grafts with bioactive surface stimulated additionally with bone plastic material "Gamalant-paste-FORTE Plus" containing recombinant human bone morphogenetic protein-2 (rhBMP-2). In 9 rats the implants were placed into femoral bones after local treatment of bone canal with rhBMP-2-containing material. Another 9 animals were implanted but received no treatment, 4 rats formed the group of intact control. Zone of osseointegration was studied 4, 8 and 12 weeks after graft placement using histological and morphometric methods as well as immune histochemistry to demonstrate osteonectin, CD68, MMP-9, and TIMP-1. The study showed that preliminary treatment of bone canal with rhBMP-2-containing material preceding implant placement was accompanied by an additional osteoinductive effect. More intense and outrunning bone formation in the area of osseointegration was observed, together with remodeling and compaction of the contiguous cancellous bone, thus providing the necessary balance between MMP-9 and TIMP-1 with a high level of each factor expression. PMID:27487669

  14. Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity.

    PubMed

    Khan, Shaukat A; Nelson, Matthew S; Pan, Chendong; Gaffney, Patrick M; Gupta, Pankaj

    2008-06-01

    Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses. PMID:18385288

  15. Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair

    PubMed Central

    Yu, Yan Yiu; Lieu, Shirley; Lu, Chuanyong; Colnot, Céline

    2010-01-01

    Bone repair depends on the coordinated action of numerous growth factors and cytokines to stimulate new skeletal tissue formation. Among all the growth factors involved in bone repair, Bone Morphogenetic Proteins (BMPs) are the only molecules now used therapeutically to enhance healing. Although BMPs are known as strong bone inducers, their role in initiating skeletal repair is not entirely elucidated. The aim of this study was to define the role of BMP2 during the early stages of bone regeneration and more specifically in regulating the fate of skeletal progenitors. During healing of non-stabilized fractures via endochondral ossification, exogenous BMP2 increased the deposition and resorption of cartilage and bone, which was correlated with a stimulation of osteoclastogenesis but not angiogenesis in the early phase of repair. During healing of stabilized fractures, which normally occurs via intramembranous ossification, exogenous BMP2 induced cartilage formation suggesting a role in regulating cell fate decisions. Specifically, the periosteum was found to be a target of exogenous BMP2 as shown by activation of the BMP pathway in this tissue. Using cell lineage analyses, we further show that BMP2 can direct cell differentiation towards the chondrogenic lineage within the periosteum but not the endosteum, indicating that skeletal progenitors within periosteum and endosteum respond differently to BMP signals. In conclusion, BMP2 plays an important role in the early stages of repair by recruiting local sources of skeletal progenitors within periosteum and endosteum and by determining their differentiation towards the chondrogenic and osteogenic lineages. PMID:20348041

  16. Bone morphogenetic protein signalling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration

    PubMed Central

    Lewis, Christopher J.; Mardaryev, Andrei N.; Poterlowicz, Krzysztof; Sharova, Tatyana Y.; Aziz, Ahmar; Sharpe, David T.; Botchkareva, Natalia V.; Sharov, Andrey A.

    2013-01-01

    Bone morphogenetic protein (BMP) signalling plays a key role in the control of skin development and postnatal remodelling by regulating keratinocyte proliferation, differentiation and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and qRT-PCR analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a, in the epidermis of K14-caSmad1 mice versus wild-type controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared to wild-type controls. Finally, siRNA-mediated silencing of Bmpr-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17 and Myo5a compared to controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds. PMID:24126843

  17. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

    SciTech Connect

    Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

    2008-10-17

    Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation.

  18. Osteoinduction by combining bone morphogenetic protein (BMP)-2 with a bioactive novel nanocomposite

    PubMed Central

    Sharma, A.; Meyer, F.; Hyvonen, M.; Best, S. M.; Cameron, R. E.; Rushton, N.

    2012-01-01

    Objectives There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. Methods BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). Results Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. Conclusions Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine. PMID:23610684

  19. Stimulation of bone healing by sustained bone morphogenetic protein 2 (BMP-2) delivery.

    PubMed

    Faßbender, Mirja; Minkwitz, Susann; Strobel, Catrin; Schmidmaier, Gerhard; Wildemann, Britt

    2014-01-01

    The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (µCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p=0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p=0.006) with reduced cartilage at Day 84 (p=0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p=0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms. PMID:24830556

  20. Stimulation of Bone Healing by Sustained Bone Morphogenetic Protein 2 (BMP-2) Delivery

    PubMed Central

    Faßbender, Mirja; Minkwitz, Susann; Strobel, Catrin; Schmidmaier, Gerhard; Wildemann, Britt

    2014-01-01

    The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (μCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p = 0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p = 0.006) with reduced cartilage at Day 84 (p = 0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p = 0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms. PMID:24830556

  1. Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

    PubMed

    Boden, S D; Hair, G; Titus, L; Racine, M; McCuaig, K; Wozney, J M; Nanes, M S

    1997-07-01

    Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation. PMID:9202223

  2. Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease.

    PubMed

    Li, Fei-Feng; Deng, Xia; Zhou, Jing; Yan, Peng; Zhao, Er-Ying; Liu, Shu-Lin

    2016-08-01

    Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and -4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2‑5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP‑2 and ‑4 inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and ‑4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non-CHD control individuals. Statistical analyses were performed using the χ2 test, implemented using SPSS software (version 13.0). The Hardy-Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P‑value=0.560; rs235768, P‑value=0.972; rs17563, P‑value=0.787). In addition, no correlation was found between the patients with CHD and the non‑CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population. PMID:27357418

  3. Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease

    PubMed Central

    Li, Fei Feng; Deng, Xia; Zhou, Jing; Yan, Peng; Zhao, Er Ying; Liu, Shu Lin

    2016-01-01

    Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and -4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2-5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP-2 and 4- inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and -4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non CHD control individuals. Statistical analyses were performed using the χ2 test, implemented using SPSS software (version 13.0). The Hardy Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P value=0.560; rs235768, P value=0.972; rs17563, P value=0.787). In addition, no correlation was found between the patients with CHD and the non-CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population. PMID:27357418

  4. Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

    PubMed

    Fenwick, Mark A; Mora, Jocelyn M; Mansour, Yosef T; Baithun, Christina; Franks, Stephen; Hardy, Kate

    2013-09-01

    Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth. PMID:23782946

  5. Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

    PubMed

    Zhao, Yong-Gang; Meng, Fan-Xing; Li, Bing-Wei; Sheng, You-Ming; Liu, Ming-Ming; Wang, Bing; Li, Hong-Wei; Xiu, Rui-Juan

    2016-02-01

    Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification. PMID:26797522

  6. Bone morphogenetic protein-2-encapsulated grafted-poly-lactic acid-polycaprolactone nanoparticles promote bone repair.

    PubMed

    Xu, Xiaojun; Yang, Jun; Ding, Lifeng; Li, Jianjun

    2015-01-01

    The aim of this study is to test the efficacy of a novel tissue-engineered bone in repairing bone defects, using poly-lactic-acid-polycaprolactone (PLA-PCL) scaffolding seeded with PEG-bone morphogenetic protein-2 (BMP-2)-transfected rBMSCs (rabbit bone marrow stromal cells). The rBMSCs were transfected with PEG/BMP-2 or liposome/BMP-2, and then implanted into a PLA-PCL tissue-engineered bone. The protein level of BMP-2 was assessed by Western blot analysis and immunohistochemistry. ELISA was used to measure the amount of BMP-2 secreted in the culture media. The mRNA level of BMP-2 and osteocalcin was assayed quantitatively by real-time PCR. The middle portion of the bilateral radius in New Zealand rabbits was excised and implanted with tissue-engineered bone, and the modified areas were monitored by X-ray, hematoxylin-eosin staining, and immunohistochemistry staining of BMP-2. PEG-BMP-2 nanoparticles (NPs) and BMP-2-loaded PEG-PLA-PCL tissue-engineered bones were successfully constructed. The novel PEG-PLA-PCL NPs/DNA complex was a superior option for transfecting BMP-2 in rBMSCs compared to normal liposomes Moreover, the mRNA level of osteocalcin and alkaline phosphatase activity was also elevated upon transfection of BMP-2-encapsulated NPs. In vivo implants with BMP-2-carried tissue-engineered bone exhibited dramatic augmentation of BMP-2 and effective bone formation in the rabbit ectopic model. The PEG-PLA-PCL NPs/BMP-2 complex had an advantageous effect on bone repair, which provided an important theoretic basis for potential clinical treatments. PMID:25158862

  7. Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation.

    PubMed

    Atalayin, Cigdem; Tezel, Huseyin; Dagci, Taner; Yavasoglu, Nefise Ulku Karabay; Oktem, Gulperi

    2016-01-01

    The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies. PMID:26981753

  8. Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

    PubMed Central

    Schwarting, Tim; Lechler, Philipp; Struewer, Johannes; Ambrock, Marius; Frangen, Thomas Manfred; Ruchholtz, Steffen; Ziring, Ewgeni; Frink, Michael

    2015-01-01

    Introduction Successful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration. Materials and Methods To study the biological effects of BMP-7 on the process of tendon-bone-integration, two independent in vitro models were used. The first model involved the mono- and coculture of bovine tendon specimens and primary bovine osteoblasts with and without BMP-7 exposure. The second model comprised the mono- and coculture of primary bovine osteoblasts and fibroblasts. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), lactate and osteocalcin (OCN) were analyzed by ELISA. Histological analysis and electron microscopy of the tendon specimens were performed. Results In both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures. Discussion This study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts. Conclusion Our findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the

  9. Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

    PubMed Central

    Shinohara, Masahiko; Mao, Maria; Keeley, Theresa M.; El–Zaatari, Mohamad; Lee, Hyuk–Joon; Eaton, Kathryn A.; Samuelson, Linda C.; Merchant, Juanita L.; Goldenring, James R.; Todisco, Andrea

    2010-01-01

    BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and griffonia simplicifolialectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis. PMID:20826155

  10. The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein-induced bone formation.

    PubMed

    Morinobu, Mikihiko; Nakamoto, Tetsuya; Hino, Kazunori; Tsuji, Kunikazu; Shen, Zhong-Jian; Nakashima, Kazuhisa; Nifuji, Akira; Yamamoto, Haruyasu; Hirai, Hisamaru; Noda, Masaki

    2005-03-21

    Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts. PMID:15781586

  11. The application of bone morphogenetic proteins to periodontal and peri-implant tissue regeneration: A literature review

    PubMed Central

    Sasikumar, Karuppanan P.; Elavarasu, Sugumari; Gadagi, Jayaprakash S.

    2012-01-01

    Progress in understanding the role of bone morphogenetic proteins (BMPs) in craniofacial and tooth development and the demonstration of stem cells in periodontal ligament have set the stage for periodontal regenerative therapy and tissue engineering. Furthermore, recent approval by the Food and Drug Administration of recombinant human BMPs for accelerating bone fusion in slow-healing fractures indicates that this protein family may prove useful in designing regenerative treatments in periodontics. In the near term, these advances are likely to be applied to periodontal surgery; ultimately, they may facilitate approaches to regenerating whole lost periodontal structures. PMID:23066304

  12. TNF-Alpha Represses Transcription of Human Bone Morphogenetic Protein-4 in Lung Epithelial Cells

    PubMed Central

    Zhu, Nian-Ling; Li, Changgong; Huang, Hao Hao; Sebald, Matthew; Londhe, Vedang A.; Heisterkamp, Nora; Warburton, David; Bellusci, Saverio; Minoo, Parviz

    2007-01-01

    Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NFkB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NFkB. Repressor effect of NFkB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease. PMID:17350185

  13. Bone Morphogenetic Protein 6 Polymorphisms Are Associated with Radiographic Progression in Ankylosing Spondylitis

    PubMed Central

    Kim, Tae-Hwan; Shim, Seung-Cheol; Lee, Seunghun; Joo, Kyung Bin; Kim, Jong Heon; Min, Hye Joon; Rahman, Proton; Inman, Robert D.

    2014-01-01

    Background and Object Nearly 25 genetic loci associated with susceptibility to ankylosing spondylitis (AS) have been identified by several large studies. However, there have been limited studies to identify the genes associated with radiographic severity of the disease. Thus we investigated which genes involved in bone formation pathways might be associated with radiographic severity in AS. Methods A total of 417 Korean AS patients were classified into two groups based on the radiographic severity as defined by the modified Stoke’ Ankylosing Spondylitis Spinal Score (mSASSS) system. Severe AS was defined by the presence of syndesmophytes and/or fusion in the lumbar or cervical spine (n = 195). Mild AS was defined by the absence of any syndesmophyte or fusion (n = 170). A total of 251 single nucleotide polymorphisms (SNPs) within 52 genes related to bone formation were selected and genotyped. Odds ratios (OR) and 95% confidence interval (95% CI) were analysed by multivariate logistic regression controlling for age at onset of symptoms, sex, disease duration, and smoking status as covariates. Results We identified new loci of bone morphogenetic protein 6 (BMP6) associated with radiographic severity in patients with AS that passed false discovery rate threshold. Two SNPs in BMP6 were significantly associated with radiologic severity [rs270378 (OR 1.97, p = 6.74×10−4) and rs1235192 [OR 1.92, p = 1.17×10−3]) adjusted by covariates. Conclusion This is the first study to demonstrate that BMP6 is associated with radiographic severity in AS, supporting the role wingless-type like/BMP pathway on radiographic progression in AS. PMID:25121767

  14. Evaluation of human recombinant bone morphogenetic protein-2-loaded tricalcium phosphate implants in rabbits' bone defects.

    PubMed

    Laffargue, P; Hildebrand, H F; Rtaimate, M; Frayssinet, P; Amoureux, J P; Marchandise, X

    1999-08-01

    Porous beta-tricalcium phosphate (betaTCP) has osteoconductive properties. The adsorption of human recombinant bone morphogenetic protein-2 (rhBMP-2) onto TCP could realize an osteoinductive bone substitute. We evaluated it on an animal model using dual-energy X-ray absorptiometry (DEXA) and solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. BetaTCP cylinders loaded with rhBMP-2 were implanted into rabbits' femoral condyle bone defects, and betaTCP alone as control into the contralateral femur. We studied two different doses of rhBMP-2 (10 and 40 microg) on two groups of four animals. Evaluation consisted in radiography, histology, and histomorphometry, DEXA, and NMR spectroscopy using an original method of quantification. With both doses of rhBMP-2, we observed on radiographs an increase of trabecular bone around implants. Histology showed resorption of the ceramic, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous betaTCP by new bone formed. Histomorphometry showed that the osteoid surface (OS/BS) was greatest with the high dose of rhBMP-2. The difference was slight between the low dose of rhBMP-2 and control. DEXA showed a dose-dependent increase of bone mineral density of rhBMP-2-loaded betaTCP vs. control. NMR spectroscopy confirmed that the amount of new bone formed in betaTCP was greater when betaTCP carried rhBMP-2, and increased with the dose of rhBMP-2 used. We showed that betaTCP was a good matrix for rhBMP-2, which gave it osteoinductive properties in an orthotopic site, in a dose-dependent manner. Thus, such composite biomaterial seems to be of great interest in reconstructive bone surgery. Further studies are needed in clinical practice to determine optimal doses. PMID:10458276

  15. Beyond osteogenesis: an in vitro comparison of the potentials of six bone morphogenetic proteins.

    PubMed

    Rivera, Jessica C; Strohbach, Cassandra A; Wenke, Joseph C; Rathbone, Christopher R

    2013-01-01

    Bone morphogenetic proteins (BMPs) other than the clinically available BMP-2 and BMP-7 may be useful for improving fracture healing through both increasing osteogenesis and creating a favorable healing environment by altering cytokine release by endogenous cells. Given the spectrum of potential applications for BMPs, the objective of this study was to evaluate various BMPs under a variety of conditions to provide further insight into their therapeutic capabilities. The alkaline phosphatase (ALP) activity of both C2C12 and human adipose-derived stem cells (hASCs) was measured after exposure of increasing doses of recombinant human BMP-2, -4, -5, -6, -7, or -9 for 3 and 7 days. BMPs-2, -4, -5, -6, -7, and -9 were compared in terms of their ability to affect the release of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (b-FGF) from human bone marrow stromal cells (hBMSCs). Gene expression of ALP, osteocalcin, SDF-1, VEGF, and b-FGF following shRNA-mediated knockdown of BMP-2 and BMP-6 in hBMSCs or human osteoblasts under osteogenic differentiation conditions was also evaluated. Collectively, BMPs-6 and -9 produced the greatest osteogenic differentiation of C2C12 and hASCs as determined by ALP. The hBMSC secretion of SDF-1 was most affected by BMP-5, VEGF by BMP-4, and b-FGF by BMP-2. The knockdown of BMP-2 in BMSCs had no effect on any of the genes measured whereas BMP-6 knockdown in hBMSCs caused a significant increase in VEGF gene expression. BMP-2 and BMP-6 knockdown in human osteoblasts caused significant increases in VEGF gene expression and trends toward decreases in osteocalcin expression. These findings support efforts to study other BMPs as potential bone graft supplements, and to consider combined BMP delivery for promotion of multiple aspects of fracture healing. PMID:24101902

  16. Is Heparin Effective for the Controlled Delivery of High-Dose Bone Morphogenetic Protein-2?

    PubMed

    Kim, Ri Youn; Lee, Beomseok; Park, Si-Nae; Ko, Jae-Hyung; Kim, In Sook; Hwang, Soon Jung

    2016-05-01

    Sustained release of bone morphogenetic protein (BMP)-2 by heparin-contained biomaterials is advantageous for bone tissue regeneration using low-dose BMP-2. However, its effect with high-dose BMP-2 is still unclear and should be clarified considering the clinical use of a high dose of BMP-2 in spine and oral surgery. This study aimed to evaluate the efficacy of a heparin-conjugated collagen sponge (HCS) with high-dose BMP-2 delivery by investigating in vivo initial osteogenic regulation and bone healing over 12 weeks in comparison with that of an absorbable collagen sponge (ACS). The in vitro BMP-2 release profile in the HCS exhibited a lower burst followed by a sustained release of BMP-2, whereas that of the ACS showed an initial burst phase only. As a result of a lower burst, the HCS-BMP group showed higher expression of bone-forming/resorbing markers and enhanced activation of osteoclasts than the ACS-BMP group within the scaffold of defect after 7 days, which is presumed to be because of retention of relatively higher amounts of BMP-2. However, the surrounding calvariae were less resorbed in the HCS-BMP group, compared with the aggressive resorptive response in the ACS-BMP group. Microcomputed tomography and histology revealed that HCS-BMP guided more effective bone regeneration of central defect over time inducing minor ossification at the defect exterior, whereas ACS-BMP exhibited excessive ossification at the defect exterior. These results showed that HCS-mediated BMP-2 delivery at a high dose has advantages over ACS, including less early resorption of surrounding bone tissue and higher efficacy in compact bone regeneration over a longer period, highlighting a clinical feasibility of this technology. PMID:27098389

  17. Salicylic Acid-Based Polymers for Guided Bone Regeneration Using Bone Morphogenetic Protein-2

    PubMed Central

    Subramanian, Sangeeta; Mitchell, Ashley; Yu, Weiling; Snyder, Sabrina; Uhrich, Kathryn

    2015-01-01

    Bone morphogenetic protein-2 (BMP-2) is used clinically to promote spinal fusion, treat complex tibia fractures, and to promote bone formation in craniomaxillofacial surgery. Excessive bone formation at sites where BMP-2 has been applied is an established complication and one that could be corrected by guided tissue regeneration methods. In this study, anti-inflammatory polymers containing salicylic acid [salicylic acid-based poly(anhydride-ester), SAPAE] were electrospun with polycaprolactone (PCL) to create thin flexible matrices for use as guided bone regeneration membranes. SAPAE polymers hydrolyze to release salicylic acid, which is a nonsteroidal anti-inflammatory drug. PCL was used to enhance the mechanical integrity of the matrices. Two different SAPAE-containing membranes were produced and compared: fast-degrading (FD-SAPAE) and slow-degrading (SD-SAPAE) membranes that release salicylic acid at a faster and slower rate, respectively. Rat femur defects were treated with BMP-2 and wrapped with FD-SAPAE, SD-SAPAE, or PCL membrane or were left unwrapped. The effects of different membranes on bone formation within and outside of the femur defects were measured by histomorphometry and microcomputed tomography. Bone formation within the defect was not affected by membrane wrapping at BMP-2 doses of 12 μg or more. In contrast, the FD-SAPAE membrane significantly reduced bone formation outside the defect compared with all other treatments. The rapid release of salicylic acid from the FD-SAPAE membrane suggests that localized salicylic acid treatment during the first few days of BMP-2 treatment can limit ectopic bone formation. The data support development of SAPAE polymer membranes for guided bone regeneration applications as well as barriers to excessive bone formation. PMID:25813520

  18. Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

    PubMed

    González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

    2012-01-01

    Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea. PMID:22451003

  19. Hydrogel Delivery of Mesenchymal Stem Cell–Expressing Bone Morphogenetic Protein-2 Enhances Bone Defect Repair

    PubMed Central

    Hsiao, Hui-Yi; Yang, Shu-Rui; Brey, Eric M.; Chu, I-Ming

    2016-01-01

    Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration. PMID:27622106

  20. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  1. High Doses of Bone Morphogenetic Protein 2 Induce Structurally Abnormal Bone and Inflammation In Vivo

    PubMed Central

    Zara, Janette N.; Siu, Ronald K.; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M.; Ting, Kang

    2011-01-01

    The major Food and Drug Association–approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL. PMID:21247344

  2. Mode of heparin attachment to nanocrystalline hydroxyapatite affects its interaction with bone morphogenetic protein-2.

    PubMed

    Goonasekera, Chandhi S; Jack, Kevin S; Bhakta, Gajadhar; Rai, Bina; Luong-Van, Emma; Nurcombe, Victor; Cool, Simon M; Cooper-White, Justin J; Grøndahl, Lisbeth

    2015-01-01

    Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active. PMID:26474791

  3. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    PubMed Central

    Sun, Han; Yang, Hui-Lin

    2015-01-01

    Objective: The purpose of this study was to review the current status of calcium phosphate (CaP) scaffolds combined with bone morphogenetic proteins (BMPs) or mesenchymal stem cells (MSCs) in the field of bone tissue engineering (BTE). Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions. PMID:25881610

  4. Demineralized dentin matrix combined with recombinant human bone morphogenetic protein-2 in rabbit calvarial defects

    PubMed Central

    2016-01-01

    Objectives The aim of this study was to compare the osteogenic effects of demineralized dentin matrix (DDM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbit calvarial defects with DDM and anorganic bovine bone (ABB) combined with rhBMP-2. Materials and Methods Four round defects with 8-mm diameters were created in each rabbit calvaria. Each defect was treated with one of the following: 1) DDM, 2) ABB/rhBMP-2, or 3) DDM/rhBMP-2. The rhBMP-2 was combined with DDM and ABB according to a stepwise dry and dip lyophilizing protocol. Histological and microcomputed tomography (µCT) analyses were performed to measure the amount of bone formation and bone volume after 2- and 8-week healing intervals. Results Upon histological observation at two weeks, the DDM and ABB/rhBMP-2 groups showed osteoconductive bone formation, while the DDM/rhBMP-2 group showed osteoconductive and osteoinductive bone formation. New bone formation was higher in DDM/rhBMP-2, DDM and ABB decreasing order. The amounts of bone formation were very similar at two weeks; however, at eight weeks, the DDM/rhBMP-2 group showed a two-fold greater amount of bone formation compared to the DDM and ABB/rhBMP-2 groups. The µCT analysis showed markedly increased bone volume in the DDM/rhBMP-2 group at eight weeks compared with that of the DDM group. Notably, there was a slight decrease in bone volume in the ABB/rhBMP-2 group at eight weeks. There were no significant differences among the DDM, ABB/rhBMP-2, and DDM/rhBMP-2 groups at two or eight weeks. Conclusion Within the limitations of this study, DDM appears to be a suitable carrier for rhBMP-2 in orthotopic sites. PMID:27162749

  5. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

    PubMed

    Zara, Janette N; Siu, Ronald K; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M; Ting, Kang; Soo, Chia

    2011-05-01

    The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL. PMID:21247344

  6. Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype*

    PubMed Central

    Midgley, Adam C.; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O.; Meran, Soma

    2015-01-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  7. Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

    PubMed

    Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

    2015-01-01

    Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment. PMID:26400336

  8. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    PubMed Central

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  9. Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype.

    PubMed

    Midgley, Adam C; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O; Meran, Soma

    2015-05-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na(+)/H(+) exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  10. Constitutively Activated ALK2 and Increased SMAD1/5 Cooperatively Induce Bone Morphogenetic Protein Signaling in Fibrodysplasia Ossificans Progressiva*S⃞

    PubMed Central

    Fukuda, Toru; Kohda, Masakazu; Kanomata, Kazuhiro; Nojima, Junya; Nakamura, Atsushi; Kamizono, Jyunji; Noguchi, Yasuo; Iwakiri, Kiyofumi; Kondo, Takeo; Kurose, Junichi; Endo, Ken-ichi; Awakura, Takeshi; Fukushi, Junichi; Nakashima, Yasuharu; Chiyonobu, Tomohiro; Kawara, Akira; Nishida, Yoshihiro; Wada, Ikuo; Akita, Masumi; Komori, Tetsuo; Nakayama, Konosuke; Nanba, Akira; Maruki, Yuichi; Yoda, Tetsuya; Tomoda, Hiroshi; Yu, Paul B.; Shore, Eileen M.; Kaplan, Frederick S.; Miyazono, Kohei; Matsuoka, Masaru; Ikebuchi, Kenji; Ohtake, Akira; Oda, Hiromi; Jimi, Eijiro; Owan, Ichiro; Okazaki, Yasushi; Katagiri, Takenobu

    2009-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP. PMID:18684712

  11. Sclerostin inhibition of Wnt-3a-induced C3H10T1/2 cell differentiation is indirect and mediated by bone morphogenetic proteins.

    PubMed

    Winkler, David G; Sutherland, May S Kung; Ojala, Ethan; Turcott, Eileen; Geoghegan, James C; Shpektor, Diana; Skonier, John E; Yu, Changpu; Latham, John A

    2005-01-28

    High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation. PMID:15545262

  12. Recombinant Human Bone Morphogenetic Protein-2 in Posterolateral Spinal Fusion: What's the Right Dose?

    PubMed Central

    Jones, Clifford Barry; Sietsema, Debra Lynn

    2016-01-01

    Study Design Single center retrospective cohort analysis. Purpose The goal was to evaluate the influence of varying amount of recombinant human bone morphogenetic protein 2 (rhBMP-2) per level on fusion rates and complications in posterolateral spinal fusions. Overview of Literature rhBMP-2 has been utilized for lumbar posterolateral fusions for many years. Initial rhBMP-2 recommendations were 20 mg/level of fusion. Dose and concentration per level in current studies vary from 4.2 to 40 mg and 1.5 to 2.0 mg/mL, respectively. Variable fusion and complication rates have been reported. Methods Patients (n=1,610) undergoing instrumented lumbar spinal fusion (2003–2009) with utilization of rhBMP-2 were retrospectively evaluated. Patient demographics, body mass index (BMI), comorbidities, number of levels, associated interbody fusion, and types of bone void filler were analyzed. Fusions rates and nonunions were subdivided into number of levels and amount of rhBMP-2 used per level. Results Patients (n=559) were evaluated with 58.5% females having an average age of 63 years, BMI of 31 kg/m2. Number of levels fused ranged from 1 to 8. rhBMP-2 averaged 7.3 mg/level (range, 1.5–24 mg/level) based upon length of collagen sponge in relation to length of fusion levels. Patients with non-union formation had lower rhBMP-2 dose per level (p=0.016). A significant difference in non-union rate was found between patients undergoing fusion with <6 mg/level compared to those with >6 mg/level (9.1% vs. 2.4%, χ2=0.012). No significant differences were noted between 6–11.9 mg/level and ≥12 mg/level. No threshold was found for seroma formation or bone overgrowth. Conclusions Previous recommendation of 20 mg/level of rhBMP-2 is more than what is required for predictable fusion rates of 98%. No dose related increase of infection, seroma formation, and bone overgrowth has been found. In order to provide variable dosing and cost reduction, industry generated rhBMP-2 kit size should be

  13. Outcomes for Single-Level Lumbar Fusion: The Role of Bone Morphogenetic Protein

    PubMed Central

    Cahill, Kevin S.; Chi, John H.; Groff, Michael W.; McGuire, Kevin; Afendulis, Christopher C.; Claus, Elizabeth B.

    2011-01-01

    Study Design Retrospective analysis of a population-based insurance claims dataset. Objective To determine the risk of repeat fusion and total costs associated with bone morphogenetic protein (BMP) use in single-level lumbar fusion for degenerative spinal disease. Summary of Background Data The use of BMP has been proposed to reduce overall costs of spinal fusion through prevention of repeat fusion procedures. Although radiographic fusion rates associated with BMP use have been examined in clinical trials, little data exists regarding outcomes associated with BMP use in the general population. Methods Using the MarketScan© claims dataset, 15,862 patients that underwent single-level lumbar fusion from 2003 to 2007 for degenerative disease were identified. Propensity scores were used to match 2,372 patients that underwent fusion with BMP to patients that underwent fusion without BMP. Logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards models were used to examine risk of repeat fusion, length of stay, and 30-day readmission by BMP use. Cost comparisons were evaluated with linear regression models using logarithmic transformed data. Results At one year from surgery, BMP was associated with a 1.1% absolute decrease in the risk of repeat fusion (2.3% with BMP vs 3.4% without BMP, p=.03) and an odds ratio for repeat fusion of 0.66 (95% confidence interval 0.47-0.94) after multivariate adjustment. BMP was also associated with a decreased hazards for long-term repeat fusion (adjusted hazards ratio =0.74, 95% confidence interval 0.58-0.93). Cost analysis indicated that BMP was associated with initial increased costs for the surgical procedure (13.9% adjusted increase, 95% confidence interval 9.9%-17.9%) as well as total one year costs (10.1% adjusted increase, 95% confidence interval 6.2%-14.0%). Conclusions At one year, BMP use was associated with a decreased risk of repeat fusion but also increased healthcare costs. PMID:21311404

  14. Pulsed Electromagnetic Fields Enhance Bone Morphogenetic Protein-2 Dependent-Bone Regeneration.

    PubMed

    Yang, Hoon Joo; Kim, Ri Youn; Hwang, Soon Jung

    2015-10-01

    The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) for the purpose of promoting bone regeneration is emerging; however, the high dose of rhBMP-2 required in humans is accompanied by several limitations, including bone resorption and swelling. To reduce the dose of rhBMP-2 required, the applicability of pulsed electromagnetic fields (PEMF) was evaluated using a rat calvarial defect model. After creating an 8-mm-diameter calvarial bone defect, a collagen sponge soaked in different concentrations (0, 2.5, 5, 10 μg) of rhBMP-2 was implanted at the defect area. One week after surgery, PEMF was applied for 8 h/day over 5 days in an experimental group of animals (n = 28) using a width of 12 μs, a pulse frequency of 60 Hz, and a magnetic intensity of 10 G. Animals were sacrificed 4 weeks after surgery and assessed by microcomputed tomography and histological and immunohistochemical analyses. In the absence of application of PEMF, bone volume, bone mineral density, trabecular thickness, trabecular number, and trabecular separation, all showed statistically significant differences, depending on the concentration of rhBMP-2 utilized (p < 0.001). PEMF accelerated bone regeneration in the groups that received 0, 2.5, and 5 μg rhBMP-2 (p < 0.05). In contrast, administration of 10 μg rhBMP-2 resulted in no additive effect on bone regeneration in combination with PEMF. Groups receiving no rhBMP-2 showed distinct bone regeneration in the central zone of the bone defect when treated with PEMF, whereas they failed to bridge the defect space without PEMF. Among the groups without PEMF, soft tissue infiltrations from the outer surface on the skin side were common. Among groups with PEMF, the groups receiving 5 and 10 μg rhBMP-2 displayed denser bone with significantly reduced dead spaces. The application of PEMF did not result in an accelerated effect on bone regeneration in groups treated with 10 μg rhBMP-2. Therefore, our data

  15. Sustained release of bone morphogenetic protein 2 via coacervate improves the osteogenic potential of muscle-derived stem cells.

    PubMed

    Li, Hongshuai; Johnson, Noah Ray; Usas, Arvydas; Lu, Aiping; Poddar, Minakshi; Wang, Yadong; Huard, Johnny

    2013-09-01

    Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle by a modified preplate technique exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. MDSCs retrovirally transduced to express bone morphogenetic proteins (BMPs) can differentiate into osteocytes and chondrocytes and enhance bone and articular cartilage repair in vivo, a feature that is not observed with nontransduced MDSCs. These results emphasize that MDSCs require prolonged exposure to BMPs to undergo osteogenic and chondrogenic differentiation. A sustained BMP protein delivery approach provides a viable and potentially more clinically translatable alternative to genetic manipulation of the cells. A unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD), was used to bind, protect, and sustain the release of bone morphogenetic protein-2 (BMP2) in a temporally and spatially controlled manner. Prolonged exposure to BMP2 released by the PEAD:heparin delivery system promoted the differentiation of MDSCs to an osteogenic lineage in vitro and induced the formation of viable bone at an ectopic site in vivo. This new strategy represents an alternative approach for bone repair mediated by MDSCs while bypassing the need for gene therapy. PMID:23884640

  16. C23 protein meditates bone morphogenetic protein-2-mediated EMT via up-regulation of Erk1/2 and Akt in gastric cancer.

    PubMed

    Yang, Yonggang; Yang, Chunyan; Zhang, Jianping

    2015-03-01

    In our previous study, the epithelial-to-mesenchymal transition (EMT) has been identified to be involved in gastric cancer progression. Notably, nuclear protein C23 and bone morphogenetic protein-2 (BMP2) have been linked into EMT. However, the specific mechanisms underlying BMP2 pathway-mediated EMT are not still unraveled. In this study, we adopted immunohistochemistry and immunoblotting to determine the expression of C23 and BMP2 receptor II (BMPR-II) in 90 gastric cancer samples and cell lines. Subsequently, relevant cell lines were selected to be treated with si-C23 or si-BMPRII and the detection of in vitro assay. Our results revealed that both C23 and BMPRII were aberrantly and constitutively expressed in gastric cancer specimens and cell lines, whose expression was positively associated with metastasis, stage and differentiation, and portended poor survival outcome of gastric cancer patients. In vitro assay validated the increased expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2 in BMP2-stimulated MGC803 cells, which was in a dose-dependent manner. By contrast, si-C23 treatment attenuated the BMP2-stimulated expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2. Also, the treatment of either si-C23 or si-BMPRII decreased the ability of migration and invasion of MGC803 cells. In conclusion, C23 mediates BMP2-induced EMT progression via the up-regulation of Erk1/2 and Akt signaling pathway in gastric cancer, which indicated both C23 and BMPRII pathway could be recommended as prospective targets or biomarkers to antagonize the progression of gastric cancer. PMID:25698539

  17. C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation*

    PubMed Central

    Zhao, Yulan; Xiao, Mu; Sun, Baoguo; Zhang, Zhengmao; Shen, Tao; Duan, Xueyan; Yu, Paul Borchyung; Feng, Xin-Hua; Lin, Xia

    2014-01-01

    The bone morphogenetic protein (BMP) signaling pathway regulates a wide range of cellular responses in metazoans. A key step in the canonical BMP signaling is the phosphorylation and activation of transcription factors Smad1, Smad5, and Smad8 (collectively Smad1/5/8) by the type I BMP receptors. We previously identified PPM1A as a phosphatase toward dephosphorylation of all receptor-regulated Smads (R-Smads), including Smad1/5/8. Here we report another nuclear phosphatase named SCP4/CTDSPL2, belonging to the FCP/SCP family, as a novel Smad phosphatase in the nucleus. SCP4 physically interacts with and specifically dephosphorylates Smad1/5/8, and as a result attenuates BMP-induced transcriptional responses. Knockdown of SCP4 in multipotent mesenchymal C2C12 cells leads to increased expression of BMP target genes and consequently promotes BMP-induced osteogenic differentiation. Collectively, our results demonstrate that SCP4, as a Smad phosphatase, plays a critical role in BMP-induced signaling and cellular functions. PMID:25100727

  18. Bone morphogenetic protein 2 decreases TRPC expression, store-operated Ca2+ entry, and basal [Ca2+]i in rat distal pulmonary arterial smooth muscle cells

    PubMed Central

    Zhang, Yi; Yang, Kai; Xu, Lei; Lai, Ning; Tian, Lichun; Jiang, Qian; Duan, Xin; Chen, Minsheng

    2013-01-01

    Recent studies indicate that multiple bone morphogenetic protein (BMP) family ligands and receptors are involved in the development of pulmonary arterial hypertension, yet the underlying mechanisms are incompletely understood. Although BMP2 and BMP4 share high homology in amino acid sequence, they appear to exert divergent effects on chronic hypoxic pulmonary hypertension (CHPH). While BMP4 promotes vascular remodeling, BMP2 prevents CHPH. We previously demonstrated that BMP4 upregulates the expression of canonical transient receptor potential channel (TRPC) proteins and, thereby, enhances store-operated Ca2+ entry (SOCE) and elevates intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs). In this study, we investigated the effects of BMP2 on these variables in rat distal PASMCs. We found that treatment with BMP2 (50 ng/ml, 60 h) inhibited TRPC1, TRPC4, and TRPC6 mRNA and protein expression. Moreover, BMP2 treatment led to reduced SOCE and decreased basal [Ca2+]i in PASMCs. These alterations were associated with decreased PASMC proliferation and migration. Conversely, knockdown of BMP2 with specific small interference RNA resulted in increased cellular levels of TRPC1, TRPC4, and TRPC6 mRNA and protein, enhanced SOCE, elevated basal [Ca2+]i, and increased proliferation and migration of PASMCs. Together, these results indicate that BMP2 participates in regulating Ca2+ signaling in PASMCs by inhibiting TRPC1, TRPC4, and TRPC6 expression, thus leading to reduced SOCE and basal [Ca2+]i and inhibition of cell proliferation and migration. PMID:23447035

  19. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility - A Review.

    PubMed

    de Castro, Fernanda Cavallari; Cruz, Maria Helena Coelho; Leal, Claudia Lima Verde

    2016-08-01

    Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review. PMID:26954112

  20. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility — A Review

    PubMed Central

    de Castro, Fernanda Cavallari; Cruz, Maria Helena Coelho; Leal, Claudia Lima Verde

    2016-01-01

    Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review. PMID:26954112

  1. Bone response to 3D periodic hydroxyapatite scaffolds with and without tailored microporosity to deliver bone morphogenetic protein 2.

    PubMed

    Dellinger, Jennifer G; Eurell, Jo Ann C; Stewart, Matthew; Jamison, Russell D

    2006-02-01

    Three types of model hydroxyapatite (HA) scaffolds were implanted in the metacarpal and metatarsal bones of goats. Scaffolds, consisting of a latticed pattern of rods, were fabricated with a solid freeform fabrication (SFF) technique. All scaffolds contained macropores; some were also fabricated with micropores (5.2 +/- 2.0 microm). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was added to some microporous scaffolds. rhBMP-2 caused increased percent filled with bone tissue compared to microporous scaffolds without rhBMP-2. Lamellar bone in the scaffolds was aligned perpendicular to the long axis of the bone near the junctions of the rods that make up the scaffold but was more random away from the junctions of rods. Microporous scaffolds stained beneath areas of contact with new bone. This staining might indicate either extracellular matrix (ECM) in the rods, byproducts of ECM production, or reaction of cellular products with the scaffold. PMID:16270335

  2. Negative feedback in the bone morphogenetic protein 4 (BMP4) synexpression group governs its dynamic signaling range and canalizes development

    PubMed Central

    Paulsen, Malte; Legewie, Stefan; Eils, Roland; Karaulanov, Emil; Niehrs, Christof

    2011-01-01

    What makes embryogenesis a robust and canalized process is an important question in developmental biology. A bone morphogenetic protein (BMP) morphogen gradient plays a key role in embryonic development, and we are beginning to understand how the self-regulating properties of its signaling circuitry ensure robust embryonic patterning. An unexplored question is why the BMP signaling circuit is organized as a modular synexpression group, with a prevalence of feedback inhibitors. Here, we provide evidence from direct experimentation and mathematical modeling that the synexpressed feedback inhibitors BAMBI, SMAD6, and SMAD7 (i) expand the dynamic BMP signaling range essential for proper embryonic patterning and (ii) reduce interindividual phenotypic and molecular variability in Xenopus embryos. Thereby, negative feedback linearizes signaling responses and confers robust patterning, thus promoting canalized development. The presence of negative feedback inhibitors in other growth factor synexpression groups suggests that these properties may constitute a general principle. PMID:21633009

  3. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    PubMed

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  4. The Use of Bone Morphogenetic Protein in Pediatric Cervical Spine Fusion Surgery: Case Reports and Review of the Literature.

    PubMed

    Molinari, Robert W; Molinari, Christine

    2016-02-01

    Study Design Case report. Objective There is a paucity of literature describing the use of bone graft substitutes to achieve fusion in the pediatric cervical spine. The outcomes and complications involving the off-label use of bone morphogenetic protein (BMP)-2 in the pediatric cervical spine are not clearly defined. The purpose of this article is to report successful fusion without complications in two pediatric patients who had instrumented occipitocervical fusion using low-dose BMP-2. Methods A retrospective review of the medical records was performed, and the patients were followed for 5 years. Two patients under 10 years of age with upper cervical instability were treated with occipitocervical instrumented fusion using rigid occipitocervical fixation techniques along with conventionally available low-dose BMP-2. A Medline and PubMed literature search was conducted using the terms "bone morphogenetic protein," "BMP," "rh-BMP2," "bone graft substitutes," and "pediatric cervical spine." Results Solid occipitocervical fusion was achieved in both pediatric patients. There were no reported perioperative or follow-up complications. At 5-year follow-up, radiographs in both patients showed successful occipital cervical fusion without evidence of instrumentation failure or changes in the occipitocervical alignment. To date, there are few published reports on this topic. Complications and the appropriate dosage application in the pediatric posterior cervical spine remain unknown. Conclusions We describe two pediatric patients with upper cervical instability who achieved successful occipital cervical fusion without complication using off-label BMP-2. This report underscores the potential for BMP-2 to achieve successful arthrodesis of the posterior occipitocervical junction in pediatric patients. Use should be judicious as complications and long-term outcomes of pediatric BMP-2 use remain undefined in the existing literature. PMID:26835215

  5. Effectiveness and safety of recombinant human bone morphogenetic protein-2 for adults with lumbar spine pseudarthrosis following spinal fusion surgery

    PubMed Central

    Balaji, V.; Kaila, R.; Wilson, L.

    2016-01-01

    Objectives We performed a systematic review of the literature to determine the safety and efficacy of bone morphogenetic protein (BMP) compared with bone graft when used specifically for revision spinal fusion surgery secondary to pseudarthrosis. Methods The MEDLINE, EMBASE and Cochrane Library databases were searched using defined search terms. The primary outcome measure was spinal fusion, assessed as success or failure in accordance with radiograph, MRI or CT scan review at 24-month follow-up. The secondary outcome measure was time to fusion. Results A total of six studies (three prospective and three retrospective) reporting on the use of BMP2 met the inclusion criteria (203 patients). Of these, four provided a comparison of BMP2 and bone graft whereas the other two solely investigated the use of BMP2. The primary outcome was seen in 92.3% (108/117) of patients following surgery with BMP2. Although none of the studies showed superiority of BMP2 to bone graft for fusion, its use was associated with a statistically quicker time to achieving fusion. BMP2 did not appear to increase the risk of complication. Conclusion The use of BMP2 is both safe and effective within the revision setting, ideally in cases where bone graft is unavailable or undesirable. Further research is required to define its optimum role. Cite this article: Mr P. Bodalia. Effectiveness and safety of recombinant human bone morphogenetic protein-2 for adults with lumbar spine pseudarthrosis following spinal fusion surgery: A systematic review. Bone Joint Res 2016;5:145–152. DOI: 10.1302/2046-3758.54.2000418. PMID:27121215

  6. Expression analysis of bone morphogenetic protein 4 between fat and lean birds in adipose tissue and serum.

    PubMed

    Cheng, B H; Leng, L; Wu, M Q; Zhang, Q; Zhang, X Y; Xu, S S; Cao, Z P; Li, Y M; Luan, P; Li, H

    2016-07-01

    The objectives of the present study were to characterize the tissue expression of chicken (Gallus gallus) bone morphogenetic protein 4 (BMP4) and compare differences in its expression in abdominal fat tissue and serum between fat and lean birds and to determine a potential relationship between the expression of BMP4 and abdominal fat tissue growth and development. The results showed that chicken BMP4 messenger RNA (mRNA) and protein were expressed in various tissues, and the expression levels of BMP4 transcript and protein were relatively higher in adipose tissues. In addition, the mRNA and protein expression levels of BMP4 in abdominal fat tissue of fat males were lower than those of lean males at 1, 2, 5, and 7 wk of age (P < 0.05). Furthermore, the serum BMP4 content of fat males was lower than that of lean males at 7 wk of age (P < 0.05). BMP4 mRNA expression levels were significantly higher in preadipocytes than those in mature adipocytes (P < 0.05), and the expression level decreased during differentiation in vitro (P < 0.05). These results suggested that chicken BMP4 might affect abdominal fat deposition through differences in its expression level. The results of this study will provide basic molecular information for studying the role of BMP4 in the regulation of adipogenesis in avian species. PMID:26945137

  7. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

    PubMed Central

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  8. Bone morphogenetic protein-induced cell differentiation involves Atg7 and Wnt16 sequentially in human stem cell-derived osteoblastic cells.

    PubMed

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Matsumoto, Toru; Nakata, Kazuhiko

    2016-09-10

    We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like cells with bone morphogenetic protein (BMP)-2. To explore the early signaling cascade for osteoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling during BMP-2-mediated human osteoblastic differentiation. In a screening experiment, BMP-2 increased the mRNA and protein levels of Atg7, Wnt16, and Lrp5/Fzd2 (a Wnt receptor), but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, Atg5, Atg12, Wnt3a, or Wnt5, together with the amounts of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg7 and Wnt16 individually suppressed the BMP-2-induced increase in osteoblastic differentiation. The osteoblastic phenotype, involving osteocalcin (BGLAP), osteopontin (SPP1), and osterix (SP7) expression, decreased when autophagy was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade of BMP-2→Atg7→Wnt16→Lrp5/Fzd2→matrix metalloproteinase-13→osteoblastic differentiation. This cascade results in a potent increase in osteoblastic cell differentiation, indicating the unique involvement of Atg7, autophagy, and Wnt16 signaling in BMP-2-induced differentiation of α7(+)hSMSCs into osteoblast-like cells at a relatively early stage. PMID:27397580

  9. Bone morphogenetic protein-2-induced signaling and osteogenesis is regulated by cell shape, RhoA/ROCK, and cytoskeletal tension.

    PubMed

    Wang, Yang-Kao; Yu, Xiang; Cohen, Daniel M; Wozniak, Michele A; Yang, Michael T; Gao, Lin; Eyckmans, Jeroen; Chen, Christopher S

    2012-05-01

    Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation. PMID:21967638

  10. Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    PubMed Central

    Chen, Fenfang; Lin, Xia; Xu, Pinglong; Zhang, Zhengmao; Chen, Yanzhen; Wang, Chao; Han, Jiahuai; Zhao, Bin; Xiao, Mu

    2015-01-01

    Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation. PMID:25755279

  11. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    PubMed

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation. PMID:26660942

  12. Soft Tissue Swelling Associated with the Use of Recombinant Human Bone Morphogenetic Protein-2 in Long Bone Non-unions

    PubMed Central

    Young, Andrew; Mirarchi, Adam

    2015-01-01

    Introduction: This report describes two cases of long bone non-union associated with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and is the first of its kind. The first case describes a 25-year-old male who sustained a left diaphyseal femoral shaft fracture initially treated with operative fixation using an intramedullary nail, which subsequently loosened distally and was treated with exchange nailing and rhBMP-2 application. This patient developed acute local soft tissue inflammation post-operatively. The second case describes a 61-year-old female who sustained a right diaphyseal humeral shaft fracture that was initially treated with intramedullary nail fixation with subsequent distal interlock screw loosening. She underwent nail removal, and compression plating with rhBMP-2 placement, and postoperatively developed severe acute local tissue swelling centered over the rhBMP-2 sponge. Surgeons should be aware that rhBMP-2 may cause local acute tissue swelling and recombinant bone morphogenic proteins such as rhBMP-2 may have a role in the management for atrophic fracture non-unions. The authors recommend careful consideration prior to rhBMP-2 use in long bone non-unions. PMID:27299059

  13. Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

    PubMed

    Geng, Yan; Dong, Yingying; Yu, Mingyan; Zhang, Long; Yan, Xiaohua; Sun, Jingxia; Qiao, Long; Geng, Huixia; Nakajima, Masahiro; Furuichi, Tatsuya; Ikegawa, Shiro; Gao, Xiang; Chen, Ye-Guang; Jiang, Dianhua; Ning, Wen

    2011-04-26

    Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-β/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. PMID:21482757

  14. Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

    PubMed

    Mandal, Chandi C; Das, Falguni; Ganapathy, Suthakar; Harris, Stephen E; Choudhury, Goutam Ghosh; Ghosh-Choudhury, Nandini

    2016-01-15

    Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through

  15. Linkage between stature and a region on chromosome 20 and analysis of a candidate gene, bone morphogenetic protein 2

    SciTech Connect

    Thompson, D.B.; Ossowski, V.; Janssen, R.C.; Knowler, W.C.; Bogardus, C.

    1995-12-04

    Sib-pair linkage analysis of the quantitative trait, stature, in over 500 Pima Indians indicates that a genetic determinant of governing stature is located on chromosome 20. Analysis of 10 short tandem repeat polymorphisms localized this linkage to a 3. cM region that includes D20S98 and D20S66. Using all possible sib-pair combinations, linkage was detected to both stature (P = 0.0001) and to leg length (P = 0.001), but not to sitting height. Single-strand conformational polymorphism analysis of exon 3 of the bone morphogenetic protein 2 (BMP2) gene, a candidate gene in this region, in genomic DNA of 20 of the tallest and 20 of the shortest individuals did not show any consistent differences associated with leg length or height. Sequence analysis of the region encoding the mature protein revealed a single nucleotide substitution, a T to G transversion, not detected by single-strand conformational polymorphism (SSCP) analysis. This transversion results in a conservative amino acid substitution of glycine for valine at codon 80 of BMP2. The frequency of this allele was 0.23 in the sample. No significant differences in height were noted in persons carrying either allele. This indicates that this structural alteration in the mature BMP2 protein does not contribute to the differences in stature observed in the Pima Indians, nor is this structural change in the mature protein likely to be responsible for the linkage observed with stature on chromosome 20. 33 refs., 2 figs., 2 tabs.

  16. The Use of Platelet Rich Plasma, Bone Morphogenetic Protein-2 and Different Scaffolds in Oral and Maxillofacial Surgery - Literature Review in Comparison with Own Clinical Experience

    PubMed Central

    Jopp, Stefan; Osadnik, Magdalena

    2011-01-01

    ABSTRACT Objectives The purpose of this article was to review and critically assess the use of platelet rich plasma, recombinant human bone morphogenetic protein-2 and different scaffolds (i.e. tricalciumphosphate, polycaprolactone, demineralized bone matrix and anorganic bovine bone mineral) in oral and maxillofacial surgery comparing the relevant literature and own clinical experience. Material and Methods A literature review was conducted using MEDLINE, MEDPILOT and COCHRANE DATABASE OF SYSTEMATIC REVIEWS. It concentrated on manuscripts and overviews published in the last five years (2006-2010). The key terms employed were platelet rich plasma, bone morphogenetic proteins and their combinations with the above mentioned scaffolds. The results of clinical studies and animal trials were especially emphasized. The statements from the literature were compared with authors’ own clinical data. Results New publications and overviews demonstrate the advantages of platelet rich plasma in bone regeneration. The results from the literature review were discussed and compared with the publications detailing authors' own experiences. Conclusions A favourable outcome concerning newly grown bone was achieved combining platelet rich plasma in addition to optimal matrices with or without recombinant human bone morphogenetic protein-2, depending on the clinical case. As a consequence, the paradigm shift from transplantation of autogenous bone to bone tissue engineering appears promising. PMID:24421984

  17. The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model.

    PubMed

    Ampuja, M; Alarmo, E L; Owens, P; Havunen, R; Gorska, A E; Moses, H L; Kallioniemi, A

    2016-06-01

    Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings. PMID:26970275

  18. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    NASA Astrophysics Data System (ADS)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  19. Delayed myelination in an intrauterine growth retardation model is mediated by oxidative stress upregulating bone morphogenetic protein 4.

    PubMed

    Reid, Mary V; Murray, Kaitlin A; Marsh, Eric D; Golden, Jeffrey A; Simmons, Rebecca A; Grinspan, Judith B

    2012-07-01

    Intrauterine growth retardation (IUGR) is associated with neurological deficits including cerebral palsy and cognitive and behavioral disabilities. The pathogenesis involves oxidative stress that leads to periventricular white matter injury with a paucity of mature oligodendrocytes and hypomyelination. The molecular mechanisms underlying this damage remain poorly understood. We used a rat model of IUGR created by bilateral ligation of the uterine artery at embryonic Day 19 that results in fetal growth retardation and oxidative stress in the developing brain. The IUGR rat pups showed significant delays in oligodendrocyte differentiation and myelination that resolved by 8 weeks. Bone morphogenetic protein 4 (BMP4), which inhibits oligodendrocyte maturation, was elevated in IUGR brains at postnatal time points and returned to near normal by adulthood. Despite the apparent recovery, behavioral deficiencies were found in 8-week-old female animals, suggesting that the early transient myelination defects have permanent effects. In support of these in vivo data, oligodendrocyte precursor cells cultured from postnatal IUGR rats retained increased BMP4 expression and impaired differentiation that was reversed with the BMP inhibitor noggin. Oxidants in oligodendrocyte cultures increased BMP expression, which decreased differentiation; however, abrogating BMP signaling with noggin in vitro and in BMP-deficient mice prevented these effects. Together, these findings suggest that IUGR results in delayed myelination through the generation of oxidative stress that leads to BMP4 upregulation. PMID:22710965

  20. Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

    PubMed Central

    Chiang, Chih-Wei; Chen, Wei-Chuan; Liu, Hsia-Wei; Wang, I-Chun; Chen, Chih-Hwa

    2015-01-01

    Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering. PMID:26426006

  1. Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells.

    PubMed

    Csiszar, Anna; Labinskyy, Nazar; Jo, Hanjoong; Ballabh, Praveen; Ungvari, Zoltan

    2008-08-01

    There is increasing evidence that TGF-beta family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased O(2)(*-) and H(2)O(2) generation, induced NF-kappaB activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased O(2)(*-) production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation. PMID:18539760

  2. Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla

    PubMed Central

    Wang, Jinhua; Zhang, Hongmei; Zhang, Wenwen; Huang, Enyi; Wang, Ning; Wu, Ningning; Wen, Sheng; Chen, Xian; Liao, Zhan; Deng, Fang; Yin, Liangjun; Zhang, Junhui; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Zhang, Zhonglin; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.

    2014-01-01

    Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration. PMID:24517722

  3. Genetic analysis of the bone morphogenetic protein-related gene, gbb, identifies multiple requirements during Drosophila development.

    PubMed Central

    Wharton, K A; Cook, J M; Torres-Schumann, S; de Castro, K; Borod, E; Phillips, D A

    1999-01-01

    We have isolated mutations in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-beta signaling molecule (formerly referred to as 60A) with highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5-8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, we show that gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, we show a requirement for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independently of dpp. PMID:10353905

  4. Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling

    PubMed Central

    Kobielak, Krzysztof; Stokes, Nicole; de la Cruz, June; Polak, Lisa; Fuchs, Elaine

    2007-01-01

    During the hair cycle, follicle stem cells (SCs) residing in a specialized niche called the “bulge” undergo bouts of quiescence and activation to cyclically regenerate new hairs. Developmental studies have long implicated the canonical bone morphogenetic protein (BMP) pathway in hair follicle (HF) determination and differentiation, but how BMP signaling functions in the hair follicle SC niche remains unknown. Here, we use loss and gain of function studies to manipulate BMP signaling in the SC niche. We show that when the Bmpr1a gene is conditionally ablated, otherwise quiescent SCs are activated to proliferate, causing an expansion of the niche and loss of slow-cycling cells. Surprisingly, follicle SCs are not lost, however, but rather, they generate long-lived, tumor-like branches that express Sox4, Lhx2, and Sonic Hedgehog but fail to terminally differentiate to make hair. A key component of BMPR1A-deficient SCs is their elevated levels of both Lef1 and β-catenin, which form a bipartite transcription complex required for initiation of the hair cycle. Although β-catenin can be stabilized by Wnt signaling, we show that BMPR1A deficiency enhances β-catenin stabilization in the niche through a pathway involving PTEN inhibition and PI3K/AKT activation. Conversely, sustained BMP signaling in the SC niche blocks activation and promotes premature hair follicle differentiation. Together, these studies reveal the importance of balancing BMP signaling in the SC niche. PMID:17553962

  5. [Comparison between gene therapy and gradual release carrier for bone morphogenetic protein-2 in repairing bone defects].

    PubMed

    Li, Jianjun; Bai, Lunhao; Cui, Shaoqian; Wang, Huan; Xu, Xinxiang

    2007-06-01

    To compare the effects between gene therapy and gradual release carrier for bone morphogenetic protein-2 (BMP-2) in repairing bone defects, bone defects for 15 mm were created.on the bilateral radius in rabbits and treated with four kinds of implantations, ie, composite of transgeneic MSCs and PLA/PCL (Group A), composite of MSCs and gradual release carrier for BMP-2 (Group B), composite of MSCs and PLA/PCL (Group C), and PLA/PCL alone (Group D). After 4, 8, and 12 weeks of the operations, X-ray, histological examination, biomechanics analysis, and bone density measurement were conducted. Results showed that both osteoblasts and mesenchymal cells displayed strongly positive expression of BMP-2 in Group A after 4 weeks of the operation, the speed and quality of bone formation in Group A were much better than those in Group B. After 12 weeks of the operations, bone defects were completely repaired in Group A. BMP-2 gene therapy is really a good method to repair segmental bone defects. PMID:17713285

  6. Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors

    PubMed Central

    Calpe, Silvia; Correia, Ana C. P.; Sancho-Serra, Maria del Carmen; Krishnadath, Kausilia K.

    2016-01-01

    ABSTRACT Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications. PMID:26967714

  7. Protein kinase signalling pathways involved in the up-regulation of the rat alpha1(I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells.

    PubMed Central

    Palcy, S; Goltzman, D

    1999-01-01

    Transforming growth factor beta (TGFbeta) family members are known for their important role in bone physiology. TGFbeta(1) and, to a smaller extent, bone morphogenetic protein 2 (BMP-2) have been reported to regulate the gene expression of different osteoblast markers in vitro. However, little is known about the molecular mechanisms involved in these actions. Here we report that BMP-2, like TGFbeta(1), up-regulated alpha1(I) collagen mRNA expression in ROS 17/2.8 osteoblastic cells. This was mediated through an increase in the transcriptional rate of the gene rather than through the stabilization of alpha1(I) collagen mRNA, and required new protein synthesis. In addition, TGFbeta(1)- and BMP-2-induced increases in alpha1(I) collagen mRNA levels were both dependent on protein kinase C and protein tyrosine kinase activities. Furthermore, the mitogen-activated protein kinase (MAPK) [MAPK/extracellular signal-regulated protein kinase kinase 1/extracellular signal-regulated protein kinase (MEK-1/ERK)] pathway participated in the up-regulation of alpha1(I) collagen gene expression by TGFbeta(1) and BMP-2. In response to either TGFbeta(1) or BMP-2, the stimulation of alpha1(I) collagen mRNA levels was paralleled by an early increase in extracellular signal-regulated kinase protein activity. Moreover, the effects of both TGFbeta(1) and BMP-2 on alpha1(I) collagen gene expression were markedly decreased in transfected ROS 17/2.8 cells expressing a dominant-negative MEK-1. Our findings therefore show that TGFbeta(1) and BMP-2, which signal through discrete cell-surface receptors, are able to trigger analogous, if not identical, protein-phosphorylation-transducing cascades leading to comparable actions on the transcription of the alpha1(I) collagen gene in osteoblastic cells. PMID:10493907

  8. The factor VII-activating protease (FSAP) enhances the activity of bone morphogenetic protein-2 (BMP-2).

    PubMed

    Roedel, Elfie Kathrin; Schwarz, Elisabeth; Kanse, Sandip Madhav

    2013-03-01

    Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg(282)↓Gln(283)), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg(289)↓Lys(290)). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg(289)↓Lys(290)). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases. PMID:23341458

  9. The Chordin Morphogenetic Pathway.

    PubMed

    De Robertis, Edward M; Moriyama, Yuki

    2016-01-01

    The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments. PMID:26970622

  10. Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog.

    PubMed

    Manning, Alyssa J; Peters, Kimberly A; Peifer, Mark; Rogers, Stephen L

    2013-11-12

    Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells and used RNA interference-based screening to identify Mist, a Drosophila G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes. PMID:24222713

  11. Bone Morphogenetic Protein Signaling and Olig1/2 Interact to Regulate the Differentiation and Maturation of Adult Oligodendrocyte Precursor Cells

    PubMed Central

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R.; Cao, Qilin

    2009-01-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. PMID:17872503

  12. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

    PubMed

    Kato, Satoshi; Sangadala, Sreedhara; Tomita, Katsuro; Titus, Louisa; Boden, Scott D

    2011-03-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. PMID:21110071

  13. Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

    PubMed

    Auclair, Sylvain; Rossetti, Raffaella; Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  14. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits

    PubMed Central

    Li, Wenju; Wei, Haifeng; Xia, Chunmei; Zhu, Xiaomeng; Hou, Guozhu; Xu, Feng; Song, Xinghua; Zhan, Yulin

    2015-01-01

    Purpose: Transferring the bone morphogenetic protein 2 (BMP-2) genes into the tissues or cells can improve the bone healing of the fracture has been widely accepted. We evaluated the efficiency of using gene gun to transfer the BMP-2 gene thereby affected the healing of a fractured bone. Methods: The vector coding for BMP-2 was constructed by a non-replicating encephalo-myocarditis virus (ECMV)-based vector. The segmental bone defect (1.5 cm) model was created by a wire-saw at the middle part of the radius bone of the New Zealand white rabbits. Then either BMP-2 gene or control vector without BMP-2 gene was injected into the tissues around the fracture site. Healing of the defects was monitored radiographically for 9 weeks, bone consolidation was determined by the Lane-Sandhu score pre- and post-operatively, which can evaluated bone formation, bone connect and bone plasticity. Results: The radiographic score and bone consolidation rates were significantly higher in animals injected with BMP-2 gene group as compared with control vector-injected animals (P<0.05). The control group still showed no radiological signs of stable healing. Western-blot and RT-PCR showed BMP-2 expression was significant increase in the tissues around the site of osseous lesions in comparison with the control vector-injected animals (P<0.05). Conclusions: Our results suggested that BMP-2 gene transferred by gene gun could increase the expression of BMP-2 protein and improved the bone callus formation therefore shortened the time of bone defect healing. PMID:26884910

  15. Transcriptional regulation of gilthead seabream bone morphogenetic protein (BMP) 2 gene by bone- and cartilage-related transcription factors.

    PubMed

    Marques, Cátia L; Cancela, M Leonor; Laizé, Vincent

    2016-01-15

    Bone morphogenetic protein (BMP) 2 belongs to the transforming growth factor β (TGFβ) superfamily of cytokines and growth factors. While it plays important roles in embryo morphogenesis and organogenesis, BMP2 is also critical to bone and cartilage formation. Protein structure and function have been remarkably conserved throughout evolution and BMP2 transcription has been proposed to be tightly regulated, although few data is available. In this work we report the cloning and functional analysis of gilthead seabream BMP2 promoter. As in other vertebrates, seabream BMP2 gene has a 5′ non-coding exon, a feature already present in DPP gene, the fruit fly ortholog of vertebrate BMP2 gene, and maintained throughout evolution. In silico analysis of seabream BMP2 promoter revealed several binding sites for bone and cartilage related transcription factors (TFs) and their functionality was evaluated using promoter-luciferase constructions and TF-expressing vectors. Runt-related transcription factor 3 (RUNX3) was shown to negatively regulate BMP2 transcription and combination with the core binding factor β (CBFβ) further reduced transcriptional activity of the promoter. Although to a lesser extent, myocyte enhancer factor 2C (MEF2C) had also a negative effect on the regulation of BMP2 gene transcription, when associated with SRY (sex determining region Y)-box 9 (SOX9b). Finally, v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) was able to slightly enhance BMP2 transcription. Data reported here provides new insights toward the better understanding of the transcriptional regulation of BMP2 gene in a bone and cartilage context. PMID:26456102

  16. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  17. The Role of TGF-β2 and Bone Morphogenetic Proteins in the Trabecular Meshwork and Glaucoma

    PubMed Central

    Sharma, Tasneem; Clark, Abbot F.

    2014-01-01

    Abstract Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary risk factor associated with POAG. Increased aqueous humor (AH) outflow resistance through the trabecular meshwork (TM) results in elevated IOP in POAG patients. Resistance to AH outflow is associated with increased accumulation of extracellular matrix (ECM) proteins in the TM. In addition, levels of transforming growth factor-beta2 (TGF-β2) are elevated in the AH and TM tissue of POAG patients. Elevated levels of TGF-β2 in other tissues have been associated with fibrosis and increased tissue stiffness. However, locally produced effectors that maintain homeostatic relationships must also be present. Bone morphogenetic proteins (BMPs) serve this purpose in the TM as they inhibit TGF-β2-induced ECM changes in TM cells. This review article first describes the TGF-β superfamily of growth factors including BMPs and their canonical and noncanonical signaling pathways. The article then addresses the role of TGF-β2 in the pathophysiology of POAG as related to the ECM and ECM crosslinking enzymes. This is followed by a discussion of potential homeostatic control mechanisms of TGF-β2 signaling in the TM including the inhibitory role of BMP-4 and BMP-7. We then describe the relationship of TGF-β2 and BMPs in TM fibrosis including the role of antagonists. Lastly, in future directions, we identify potential future studies that explore new and unique cellular interactions within the TM for potential therapeutic interventions. PMID:24517218

  18. Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis*

    PubMed Central

    Wei, Zhenquan; Salmon, Richard M.; Upton, Paul D.; Morrell, Nicholas W.; Li, Wei

    2014-01-01

    BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes. PMID:25237187

  19. Alterations in bone morphogenetic protein 15, growth differentiation factor 9, and gene expression in granulosa cells in preovulatory follicles of dairy cows given porcine LH.

    PubMed

    Behrouzi, Amir; Colazo, Marcos Germán; Ambrose, Divakar Justus

    2016-04-15

    In a previous work, using porcine LH (pLH) in lieu of GnRH for synchronizing ovulation in dairy cows improved pregnancy rates without increasing plasma progesterone concentrations after ovulation. The LH profile is known to remain elevated above basal concentrations (≥1 ng/mL) for up to 20 hours in pLH-treated cows compared to less than 6 hours in GnRH-treated cows. Because LH triggers a cascade of signaling networks in the preovulatory follicle to promote final maturation and support oocyte competence, we hypothesized that dissimilar LH profiles will differentially regulate the intrafollicular factors and expression of downstream genes associated with improved oocyte competence. Specific objectives were to determine differences in the abundance of oocyte-secreted factors in the preovulatory follicular fluid and target genes in granulosa cells associated with oocyte competence, in response to exogenous porcine LH or GnRH-induced endogenous bovine LH exposure, in dairy cows. Follicular contents were aspirated by a transvaginal ultrasound-guided procedure from the preovulatory follicle of cyclic, nonlactating Holstein cows 21 ± 1 hour after administration of either pLH (25-mg) or GnRH (100-μg). Mature forms of bone morphogenetic protein 15, growth differentiation factor 9, and transforming growth factorβ1 were approximately 2-fold more abundant in pLH-treated cows which were exposed to an extended, low LH profile, than in GnRH-treated cows that had a short, high LH profile. The relative abundance of messenger RNA for cyclooxygenase-2, LH receptor, and progesterone receptor in granulosa cells, was about two-, eight-, and two-fold higher, respectively, in cows subjected to pLH than GnRH treatment. We infer that the improved pregnancy rate after pLH-induced ovulation reported previously, occurred through greater activation of intrafollicular transforming growth factor-β1 superfamily members, as these proteins promote cumulus expansion and oocyte competence

  20. Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-β and bone morphogenetic protein signaling.

    PubMed

    Sawada, Shunsuke; Chosa, Naoyuki; Takizawa, Naoki; Yokota, Jun; Igarashi, Yasuyuki; Tomoda, Koichi; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

    2016-03-01

    Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG‑2, ‑3, and ‑5 from the bone marrow of green fluorescent protein (GFP)‑transgenic mice. These cell lines clearly expressed mouse MSC markers Sca‑1 and CD44, and SG‑2 and ‑5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)‑β superfamily. By contrast, SG‑3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG‑3 cells, which underwent osteogenesis in response to BMP, while TGF‑β receptor II was most highly expressed in SG‑3 and ‑5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF‑β1 in SG‑2 cells but not in SG‑3 or ‑5 cells. Furthermore, TGF‑β1 clearly induced the expression of Smad‑interacting transcription factor CCAAT/enhancer binding protein‑β in SG‑2 but not in SG‑3 or ‑5 cells. These results demonstrated the establishment of TGF‑β‑responsive SG‑2 MSCs, BMP‑responsive SG‑3 MSCs and TGF‑β/BMP‑unresponsive SG‑5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF‑β superfamily affects the proliferation and differentiation status of MSCs in vivo. PMID:26781600

  1. A comprehensive assessment of the risk of bone morphogenetic protein use in spinal fusion surgery and postoperative cancer diagnosis.

    PubMed

    Cahill, Kevin S; McCormick, Paul C; Levi, Allan D

    2015-07-01

    The risk of postoperative cancer following the use of recombinant human bone morphogenetic protein (BMP)-2 in spinal fusion is one potential complication that has received significant interest. Until recently, there has been little clinical evidence to support the assertion of potential cancer induction after BMP use in spinal surgery. This report aims to summarize the findings from clinical data available to date from the Yale University Open Data Access (YODA) project as well as more recently published large database studies regarding the association of BMP use in spinal fusion and the risk of postoperative cancer. A detailed review was based on online databases, primary studies, FDA reports, and bibliographies of key articles for studies that assessed the efficacy and safety of BMP in spinal fusion. In an analysis of the YODA project, one meta-analysis detected a statistically significant increase in cancer occurrence at 24 months but not at 48 months, and the other meta-analysis did not detect a significant increase in postoperative cancer occurrence. Analysis of 3 large health care data sets (Medicare, MarketScan, and PearlDiver) revealed that none were able to detect a significant increase in risk of malignant cancers when BMP was used compared with controls. The potential risk of postoperative cancer formation following the use of BMP in spinal fusion must be interpreted on an individual basis for each patient by the surgeon. There is no conclusive evidence that application of the common formulations of BMP during spinal surgery results in the formation of cancer locally or at a distant site. PMID:25860517

  2. A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects.

    PubMed

    Diab, Tamim; Pritchard, Eleanor M; Uhrig, Brent A; Boerckel, Joel D; Kaplan, David L; Guldberg, Robert E

    2012-07-01

    The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects. PMID:22658161

  3. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. PMID:26500262

  4. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    PubMed

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  5. Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2.

    PubMed

    Xu, Xian; Jha, Amit K; Duncan, Randall L; Jia, Xinqiao

    2011-08-01

    We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. To this end heparin (HP)-decorated, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase the amount of HP integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized HP retained its ability to bind bone morphogenetic protein-2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared with pure HA particles the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per mg HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as shown by up-regulation of the mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration. PMID:21550426

  6. Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2

    PubMed Central

    Xu, Xian; Jha, Amit K.; Duncan, Randall L.; Jia, Xinqiao

    2011-01-01

    We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. Towards this end, heparin (HP)-decorated, hyaluronic acid (HA)-based, hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase, the amount of heparin integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized heparin retained its ability to bind bone morphogenetic protein 2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared to the pure HA particles, the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero-order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per milligram HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as evidenced by the up-regulation of mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration. PMID:21550426

  7. Recombinant human bone morphogenetic protein-2 suspended in fibrin glue enhances bone formation during distraction osteogenesis in rabbits

    PubMed Central

    Li, Yunfeng; Li, Rui; Hu, Jing; Song, Donghui; Jiang, Xiaowen

    2016-01-01

    Introduction Bone morphogenetic protein-2 (BMP-2) has high potential for bone formation, but its in vivo effects are unpredictable due to the short life time. This study was designed to evaluate the effects of recombinant human (rh) BMP-2 suspended in fibrin on bone formation during distraction osteogenesis (DO) in rabbits. Material and methods The in vitro release kinetics of rhBMP-2 suspended in fibrin was tested using an enzyme-linked immunosorbent assay. Unilateral tibial lengthening for 10 mm was achieved in 48 rabbits. At the completion of osteodistraction, vehicle, fibrin, rhBMP-2 or rhBMP-2 suspended in fibrin (rhBMP-2 + fibrin) was injected into the center of the lengthened gap, with 12 animals in each group. Eight weeks later, the distracted callus was examined by histology, micro-CT and biomechanical testing. Radiographs of the distracted tibiae were taken at both 4 and 8 weeks after drug treatment. Results It was found that fibrin prolonged the life span of rhBMP-2 in vitro with sustained release during 17 days. The rhBMP-2 + fibrin treated animals showed the best results in bone mineral density, bone volume fraction, cortical bone thickness by micro-CT evaluation and mechanical properties by the three-point bending test when compared to the other groups (p < 0.05). In histological images, rhBMP-2 + fibrin treatment showed increased callus formation and better gap bridging compared to the other groups. Conclusions The results of this study suggest that fibrin holds promise to be a good carrier of rhBMP-2, and rhBMP-2 suspended in fibrin showed a stronger promoting effect on bone formation during DO in rabbits. PMID:27279839

  8. Modulating Hydrogel Crosslink Density and Degradation to Control Bone Morphogenetic Protein Delivery and In Vivo Bone Formation

    PubMed Central

    Holloway, Julianne L.; Ma, Henry; Rai, Reena; Burdick, Jason A.

    2014-01-01

    Bone morphogenetic proteins (BMPs) show promise in therapies for improving bone formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on bone repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1–5 wt%) or maleimide functionalization (10–40 %f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of bone repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new bone tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, bone volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation. PMID:24905414

  9. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion

    PubMed Central

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  10. Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels.

    PubMed

    Bosman, Erika A; Lawson, Kirstie A; Debruyn, Joke; Beek, Lisette; Francis, Annick; Schoonjans, Luc; Huylebroeck, Danny; Zwijsen, An

    2006-09-01

    Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion. PMID:16887830