Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). We have analyzed the molecular mechanism by which Tsg regulates BMP signaling. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, as they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay revealed that Tsg has two distinct and sequential activities on BMP signaling. First, Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. Second, after cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated.

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

Development ◽

10.1242/dev.128.22.4463 ◽

2001 ◽

Vol 128 (22) ◽

pp. 4463-4474 ◽

Cited By ~ 8

Author(s):

Sandrine Pizette ◽

Cory Abate-Shen ◽

Lee Niswander

Keyword(s):

Apical Ectodermal Ridge ◽

Bmp Signaling ◽

Signal Loss ◽

Dorsoventral Patterning ◽

Morphogenetic Protein ◽

Vertebrate Limb ◽

Common Signal ◽

Necessary And Sufficient ◽

Bmp Antagonist ◽

Dorsal Pattern

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

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BMP Activity Is Required for Tooth Development from the Lamina to Bud Stage

Journal of Dental Research ◽

10.1177/0022034512448660 ◽

2012 ◽

Vol 91 (7) ◽

pp. 690-695 ◽

Cited By ~ 40

Author(s):

Y. Wang ◽

L. Li ◽

Y. Zheng ◽

G. Yuan ◽

G. Yang ◽

...

Keyword(s):

Cell Proliferation ◽

Molecular Marker ◽

Tooth Development ◽

Tooth Germ ◽

Bmp Signaling ◽

Expression Of Genes ◽

Dental Epithelium ◽

Initiation Stage ◽

Transgenic Embryos ◽

Bmp Antagonist

Several Bmp genes are expressed in the developing mouse tooth germ from the initiation to the late-differentiation stages, and play pivotal roles in multiple steps of tooth development. In this study, we investigated the requirement of BMP activity in early tooth development by transgenic overexpression of the extracellular BMP antagonist Noggin. We show that overexpression of Noggin in the dental epithelium at the tooth initiation stage arrests tooth development at the lamina/early-bud stage. This phenotype is coupled with a significantly reduced level of cell proliferation rate and a down-regulation of Cyclin-D1 expression, specifically in the dental epithelium. Despite unaltered expression of genes known to be implicated in early tooth development in the dental mesenchyme and dental epithelium of transgenic embryos, the expression of Pitx2, a molecular marker for the dental epithelium, became down-regulated, suggesting the loss of odontogenic fate in the transgenic dental epithelium. Our results reveal a novel role for BMP signaling in the progression of tooth development from the lamina stage to the bud stage by regulating cell proliferation and by maintaining odontogenic fate of the dental epithelium.

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BMP-binding modules in chordin: a model for signalling regulation in the extracellular space

Development ◽

10.1242/dev.127.4.821 ◽

2000 ◽

Vol 127 (4) ◽

pp. 821-830 ◽

Cited By ~ 10

Author(s):

J. Larrain ◽

D. Bachiller ◽

B. Lu ◽

E. Agius ◽

S. Piccolo ◽

...

Keyword(s):

Dissociation Constants ◽

Extracellular Proteins ◽

Full Length ◽

Dorsoventral Patterning ◽

C Elegans ◽

Growth Factor Signalling ◽

Bmp Antagonist ◽

Nanomolar Range ◽

Shed Light ◽

Novel Protein

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

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Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development

Development ◽

10.1242/dev.126.18.4005 ◽

1999 ◽

Vol 126 (18) ◽

pp. 4005-4015 ◽

Cited By ~ 14

Author(s):

M. Weaver ◽

J.M. Yingling ◽

N.R. Dunn ◽

S. Bellusci ◽

B.L. Hogan

Keyword(s):

Cell Types ◽

Surfactant Protein ◽

Bmp Signaling ◽

Dominant Negative ◽

Clara Cell ◽

Mouse Lung ◽

Marker Genes ◽

Distal Marker ◽

Morphogenetic Protein

In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693–1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.

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Suppression of GATA factor activity causes axis duplication in Xenopus

Development ◽

10.1242/dev.125.23.4595 ◽

1998 ◽

Vol 125 (23) ◽

pp. 4595-4605 ◽

Cited By ~ 4

Author(s):

T.G. Sykes ◽

A.R. Rodaway ◽

M.E. Walmsley ◽

R.K. Patient

Keyword(s):

Cell Fate ◽

Homeobox Gene ◽

Dominant Negative ◽

Gata Factor ◽

Dorsoventral Axis ◽

Morphogenetic Protein ◽

Head Formation ◽

Bmp Signalling ◽

Bmp Antagonist

In Xenopus, the dorsoventral axis is patterned by the interplay between active signalling in ventral territories, and secreted antagonists from Spemann's organiser. Two signals are important in ventral cells, bone morphogenetic protein-4 (BMP-4) and Wnt-8. BMP-4 plays a conserved role in patterning the vertebrate dorsoventral axis, whilst the precise role of Wnt-8 and its relationship with BMP-4, are still unclear. Here we have investigated the role played by the GATA family of transcription factors, which are expressed in ventral mesendoderm during gastrulation and are required for the differentiation of blood and endodermal tissues. Injection ventrally of a dominant-interfering GATA factor (called G2en) induced the formation of secondary axes that phenocopy those induced by the dominant-negative BMP receptor. However, unlike inhibiting BMP signalling, inhibiting GATA activity in the ectoderm does not lead to neuralisation. In addition, analysis of gene expression in G2en injected embryos reveals that at least one known target gene for BMP-4, the homeobox gene Vent-2, is unaffected. In contrast, the expression of Wnt-8 and the homeobox gene Vent-1 is suppressed by G2en, whilst the organiser-secreted BMP antagonist chordin becomes ectopically expressed. These data therefore suggest that GATA activity is essential for ventral cell fate and that subsets of ventralising and dorsalising genes require GATA activity for their expression and suppression, respectively. Finally, using G2en, we show that suppression of Wnt-8 expression, in conjunction with blocked BMP signalling, does not lead to head formation, suggesting that the head-suppressing Wnt signal may not be Wnt-8.

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Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

Journal of the American Society of Nephrology ◽

10.1681/asn.2008070768 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1020-1031 ◽

Cited By ~ 24

Author(s):

Barry W. Larman ◽

Michele J. Karolak ◽

Derek C. Adams ◽

Leif Oxburgh

Keyword(s):

Kidney Injury ◽

Bmp Signaling ◽

Twisted Gastrulation

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Hemojuvelin Acts as a Bone Morphogenetic Protein Co-Receptor To Regulate Hepcidin Expression.

Blood ◽

10.1182/blood.v106.11.511.511 ◽

2005 ◽

Vol 106 (11) ◽

pp. 511-511 ◽

Cited By ~ 4

Author(s):

Franklin W. Huang ◽

Jodie L. Babitt ◽

Diedra M. Wrighting ◽

Tarek A. Samad ◽

Yin Xia ◽

...

Keyword(s):

Signal Transduction ◽

Bone Morphogenetic Protein ◽

Signal Transduction Pathway ◽

Bmp Signaling ◽

Dominant Negative ◽

Transduction Pathway ◽

Reporter Construct ◽

Hepcidin Expression ◽

Morphogenetic Protein ◽

Juvenile Hemochromatosis

Abstract Juvenile hemochromatosis is a severe iron overload disorder resulting from mutations in the hemojuvelin (HJV) gene. To understand its pathogenesis, we developed Hjv−/− mice. Similar to human patients, Hjv−/− animals accumulate excess iron in the liver, pancreas and heart early in life. Tissue macrophages are iron-depleted. Hjv−/− mice express very low levels of hepcidin mRNA and, likely as a consequence, have elevated expression of the iron transporter ferroportin in enterocytes and macrophages. These results suggested that Hjv plays a role in regulating hepcidin expression. Two known Hjv homologs, Rgma and Rgmb, have previously been shown to act as bone morphogenetic protein (BMP) co-receptors. We hypothesized that Hjv regulates hepcidin expression through a BMP signal transduction pathway. We found that Hjv binds radiolabeled BMP, supporting the contention that it is a BMP co-receptor. Transfection of HepG2 cells with Hjv cDNA activated a BMP-responsive reporter construct and augmented its response to exogenous BMP. Both an anti-BMP neutralizing antibody and the natural BMP antagonist Noggin blocked this response, as did co-expressed dominant negative BMP receptor proteins. When cells were transfected with a construct carrying an Hjv mutation known to cause human disease, BMP reporter activation was significantly reduced in the presence and absence of exogenous BMP. Treatment with BMP stimulated hepcidin production in hepatoma cells and activated a reporter construct containing a fragment of the hepcidin promoter. To extend these results, we studied tissues from Hjv−/− mice. BMP signals are transduced through phosphorylation of Smad proteins. We found that Smads 1, 5 and 8 were hypophosphorylated in Hjv−/− liver, consistent with impaired BMP signaling. BMP treatment of wild type and Hjv−/− primary hepatocytes induced hepcidin expression, but induction was blunted in cells from Hjv−/− animals. Taken together, these data suggest that the normal hepatic function of Hjv is to serve as a BMP co-receptor, modulating a signal transduction pathway that culminates in hepcidin expression. [Note - Jodie L. Babitt is the first author of this abstract, but it will be presented by Franklin W. Huang, the second author]

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Twisted Gastrulation (TWSG1) Is Expressed at Elevated Levels in Thalassemia and Regulates Bone Morphogenic Protein Signaling.

Blood ◽

10.1182/blood.v110.11.1785.1785 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1785-1785

Author(s):

Toshihiko Tanno ◽

Prashanth Porayette ◽

Ajoy Bhupatiraju ◽

Pamela Staker ◽

Y. Terry Lee ◽

...

Keyword(s):

Mouse Model ◽

Quantitative Pcr ◽

Hepatoma Cell ◽

Bone Morphogenic Protein ◽

Bmp Signaling ◽

Hepatoma Cell Line ◽

Wild Type ◽

Expression Levels ◽

Hepcidin Expression ◽

Twisted Gastrulation

Abstract Iron overload and bony abnormalities cause considerable morbidity among patients with thalassemia syndromes. One possible explanation for this phenomenon is that proteins normally secreted into the marrow microenvironment during erythropoiesis are over-expressed in thalassemia patients due to expanded and ineffective erythropoiesis. We previously discovered that GDF15 is produced at very high levels in thalassemia patients and inhibits hepcidin expression. Transcriptome screens of erythroblasts were utilized here to identify twisted gastrulation (TWSG1) as a second candidate protein for further study. Quantitative PCR using the β-thalassemia murine model (Hbbth3/+ β-thalassemia intermedia mouse model, n=13; Hbbth3/th3 β-thalassemia major mouse model, n=5) revealed that splenic expression levels of Tsg (murine TWSG1) were significantly higher in thalassemia mice (Hbbth3/+, 2.2E02 ± 2.7E01 copies/ng RNA, p<0.01; Hbbth3/th3, 5.3E02 ± 6.8E01 copies/ng RNA, p<0.01) than among wild type mice (4.7E01 ± 2.4E01 copies/ng RNA, n=7). Bone marrow expression of Tsg was elevated (Hbbth3/+, 1.1E02 ± 3.2E01 copies/ng RNA, p=0.17; Hbbth3/th3, 1.3E02 ± 2.2E01 copies/ng RNA, p<0.05) compared with wild type mice (5.3E01 ± 2.5E01 copies/ng RNA). Tsg expression levels in the murine liver were also significantly higher (Hbbth3/+, 2.8E02 ± 4.6E01 copies/ng RNA, p<0.05; Hbbth3/th3, 3.9E02 ± 4.9E01 copies/ng RNA, p<0.01) than in wild type mice (1.5E02 ± 4.0E01 copies/ng RNA). These results suggest that expression of Tsg is up-regulated in the murine β-thalassemia model. By comparison, murine Tsg expression was up-regulated to a greater extent than GDF15 in the thalassemia mice. In addition to murine studies, human studies of TWSG1 were performed. Quantitative PCR using cultured human CD34+ cells demonstrated the highest-level expression of TWSG1 at the early stages of erythroblast differentiation (9.3E02 ± 1.4E02 copies/ng RNA). Preliminary ELISA analyses demonstrated statistically significant elevations in TWSG1 levels in serum from thalassemia patients (n=18, 463 ± 41 ng/ml) when compared to serum from healthy volunteers (n=10, 310 ± 45 ng/ml, p<0.05), but the relative increase in TWSG1 in humans was far less than previously reported for GDF15. To determine whether TWSG1 regulates hepcidin expression, assays were performed using a human hepatoma cell line (HuH-7). Unlike GDF15, TWSG1 did not directly affect hepcidin expression as measured by quantitative PCR in dosed assays (1–1,000 ng/ml TWSG1). However, TWSG1 was found to suppress hepcidin through an indirect mechanism involving bone morphogenic protein (BMP). BMPs regulate several tissue-specific processes including bone remodeling and induction of hepcidin expression in liver cells. In dosed-titrations, ≥100 ng/ml of TWSG1 resulted in a 50% reduction (p<0.05) in the BMP2 augmentation of hepcidin expression. These novel data suggest that TWSG1 is expressed at elevated levels in thalassemia and has the potential to affect BMP signaling processes including the regulation of hepcidin.

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Circulating Plasma Levels of Bone Morphogenic Protein Antagonist Gremlin-1 Are Increased in Patients with Pulmonary Arterial Hypertension

Blood ◽

10.1182/blood.v120.21.5189.5189 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5189-5189

Author(s):

Jasmin Wellbrock ◽

Jan K. Hennigs ◽

Björn Schulz ◽

Gabi Vohwinkel ◽

Hans Jörg Baumann ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Pulmonary Artery ◽

Arterial Hypertension ◽

Plasma Levels ◽

Research Funding ◽

Bmp Signaling ◽

Pulmonary Arterial ◽

Bmp Antagonist ◽

Minute Walk Distance ◽

Gremlin 1

Abstract Abstract 5189 Background: Pulmonary arterial hypertension (PAH) is a severe and life-threatening disease. It is characterized by excessive growth of pulmonary artery endothelial and smooth muscle cells leading to a profound pulmonary artery remodeling and consequently increased pulmonary artery pressure and vascular resistance. Most patients with the heritable form of PAH harbor a mutation in the bone morphogenic protein (BMP) receptor 2 (BMPR2) resulting in dysregulated BMP signaling. In addition, aberrant BMP signaling was also observed in the idiopathic form of PAH although the underlying molecular mechanisms have not been elucidated. Recently, it was shown that BMP antagonist Gremlin-1 was elevated in pulmonary vessels of mice during development of hypoxic pulmonary hypertension (Cahill et al, Circulation. 2012;125(7):920–30). Methods and Results: The aim of this prospective study was to investigate the plasma levels of Gremlin-1 in PAH patients (Dana point classification group I) and to correlate Gremlin-1 levels to clinical and hemodynamic parameters. Thirty subjects were included in the study (19 patients with PAH treated at the PH clinics of the University Medical Center Hamburg-Eppendorf, Germany and 11 healthy volunteers) after giving informed consent. The mean Gremlin-1 plasma level was 2. 6-fold increased with 333 ± 160 ng/ml, in patients with pulmonary arterial hypertension compared to those of healthy control subjects with a mean Gremlin-1 plasma level of 118 ± 115 ng/ml (p=0. 001 in t-test). Gremlin-1 plasma levels of PAH patients were correlated to demographic, clinical and hemodynamic parameters including age, sex, 6-minute walk distance, systemic and pulmonary blood pressure & vascular resistance, lung function testing, NT-proBNP (N terminal pro-brain natriuretic peptide) and NYHA/WHO functional classification. A positive correlation between Gremlin-1 plasma levels and NT-proBNP plasma levels was observed (Spearman Rho 0. 809 with p<0. 001). Furthermore, a negative correlation was observed between the Gremlin-1 levels and the 6-minute walk distance (Spearman Rho −0. 522 with p=0. 032). Conclusion: The plasma levels of BMP antagonist Gremlin-1 are significantly elevated in patients with pulmonary arterial hypertension and may serve as new serological marker. Gremlin-1 might mirror the state of BMP dysregulation and represent a potential follow up marker under a future targeted therapy. Furthermore, since Gremlin-1 was shown to induce proliferative effects on both endothelial as well as smooth muscle cells, it might also contribute directly to the aberrant vessel growth observed in PAH. Gremlin-1 plasma levels of patients with pulmonary hypertension (n=19) were analyzed in an enzyme-linked immunosorbent assay. Compared to healthy subjects (n=11), mean plasma levels of Gremlin-1 were 2. 6-fold increased in PH patients (t-test p=0. 001). Box plots show the median (center horizontal line), the 25th to the 75th percentile (box) and the range (whiskers).** indicates p<0. 01. Disclosures: Hennigs: Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Actelion: Research Funding; GlaxoSmithKline: Honoraria; Novartis: Honoraria. Fiedler:Pfizer Inc. : Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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