Bone Morphogenetic Protein Co-receptor Research Articles

The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt-mTOR signaling

Myofiber size regulation is critical in health, disease, and aging. MuSK (muscle-specific kinase) is a BMP (bone morphogenetic protein) co-receptor that promotes and shapes BMP signaling. MuSK is expressed at all neuromuscular junctions and is also present extrasynaptically in the mouse soleus, whose predominantly oxidative fiber composition is akin to that of human muscle. To investigate the role of the MuSK-BMP pathway in vivo, we generated mice lacking the BMP-binding MuSK Ig3 domain. These ∆Ig3-MuSK mice are viable and fertile with innervation levels comparable to wild type. In 3-month-old mice, myofibers are smaller in the slow soleus, but not in the fast tibialis anterior (TA). Transcriptomic analysis revealed soleus-selective decreases in RNA metabolism and protein synthesis pathways as well as dysregulation of IGF1-Akt-mTOR pathway components. Biochemical analysis showed that Akt-mTOR signaling is reduced in soleus but not TA. We propose that the MuSK-BMP pathway acts extrasynaptically to maintain myofiber size in slow muscle by promoting protein synthetic pathways including IGF1-Akt-mTOR signaling. These results reveal a novel mechanism for regulating myofiber size in slow muscle and introduce the MuSK-BMP pathway as a target for promoting muscle growth and combatting atrophy.

Anti-USAG-1 therapy for tooth regeneration through enhanced BMP signaling.

Uterine sensitization-associated gene-1 (USAG-1) deficiency leads to enhanced bone morphogenetic protein (BMP) signaling, leading to supernumerary teeth formation. Furthermore, antibodies interfering with binding of USAG-1 to BMP, but not lipoprotein receptor-related protein 5/6 (LRP5/6), accelerate tooth development. Since USAG-1 inhibits Wnt and BMP signals, the essential factors for tooth development, via direct binding to BMP and Wnt coreceptor LRP5/6, we hypothesized that USAG-1 plays key regulatory roles in suppressing tooth development. However, the involvement of USAG-1 in various types of congenital tooth agenesis remains unknown. Here, we show that blocking USAG-1 function through USAG-1 knockout or anti-USAG-1 antibody administration relieves congenital tooth agenesis caused by various genetic abnormalities in mice. Our results demonstrate that USAG-1 controls the number of teeth by inhibiting development of potential tooth germs in wild-type or mutant mice missing teeth. Anti-USAG-1 antibody administration is, therefore, a promising approach for tooth regeneration therapy.

Antiviral activity of bone morphogenetic proteins and activins.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Neurexin, Neuroligin and Wishful Thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions.

Trans-synaptic interactions involving Neurexins and Neuroligins are thought to promote adhesive interactions for precise alignment of the pre- and postsynaptic compartments and organize synaptic macromolecular complexes across species. In Drosophila, while Neurexin (Dnrx) and Neuroligins (Dnlg) are emerging as central organizing molecules at synapses, very little is known of the spectrum of proteins that might be recruited to the Dnrx/Dnlg trans-synaptic interface for organization and growth of the synapses. Using full length and truncated forms of Dnrx and Dnlg1 together with cell biological analyses and genetic interactions, we report novel functions of Dnrx and Dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. We show that Dnrx and Dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of Dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the Bone Morphogenetic Protein (BMP) receptor Wishful thinking (Wit), and Dnrx and Dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of Dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. Our findings also demonstrate that loss of Dnrx and Dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the Neuromuscular Junction (NMJ) synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together our findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development.

MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells.

Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway's output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca(2+) signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling.

Repulsive guidance molecule b inhibits renal cyst development through the bone morphogenetic protein signaling pathway

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disease that still lacks effective therapy. Repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins (BMPs) and a ligand for neogenin, is expressed in renal tubular epithelial cells. Previous studies showed that RGMb plays negative roles in several types of tumors and prevents the immune system from over activation. The present study was designed to explore the effects of RGMb in ADPKD development. We found that expression of RGMb in kidney was less in PKD mice than wild-type mice. With stimulation of 8-bromo-cAMP, RGMb-null embryonic kidneys had greater cyst index, though their ureteric bud branched less than wild-type mice at E13.5. Postnatal RGMb-null kidneys showed interstitial hyperplasia and decreased tubular structures, especially in the boundary area of renal cortex and medulla. RGMb overexpression dramatically inhibited cyst development and promoted tubulogenesis in MDCK cells grown in 3D collagen gels. Biochemical analysis showed increased p-Smad1/5/8 and decreased p-ERK in RGMb-overexpressing MDCK cells, suggesting modulated BMP signaling. Specific inhibition of p-Smad1/5/8 by LDN193189 reversed the suppression of RGMb on MDCK cyst model. These results reveal RGMb as a novel regulator for ADPKD by promoting renal tubule branching and regulating BMP signaling pathway. Elevating RGMb and enhancing p-Smad1/5/8 are promising new strategies to treat ADPKD.

Reversibility of Iron Loading in Transferrin-Deficient Mice

An abundant serum metal-binding protein, transferrin is a critical factor in mammalian iron homeostasis. As transferrin is essential for iron delivery for erythropoiesis, transferrin-deficient patients and mice develop anemia but also a paradoxical systemic iron overload. Iron overload results from deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Using transferrin-deficient mice, we have previously shown that transferrin deficiency leads to hepcidin deficiency via two mechanisms. First, the anemia and/or hypoxia that results from inadequate iron delivery to bone marrow actively suppresses hepcidin expression by the liver. Second, transferrin directly stimulates hepcidin expression by the liver independently of transferrin's role in erythropoiesis; this second mechanism is dependent upon hemojuvelin, a bone morphogenetic protein coreceptor essential for hepcidin expression that is mutated in juvenile hemochromatosis. Here we present data examining the reversibility of iron loading in transferrin deficiency. Measurement of tissue metal levels in adult transferrin-deficient mice demonstrated progressive and systemic iron loading yet minimal to moderate changes in levels of other metals. Treatment of adult transferrin-deficient mice with exogenous transferrin normalized red cell parameters and hepcidin levels and reduced iron load in most organs. To further investigate the reduced tissue iron burden in transferrin-treated mice, we employed iron-loaded cell lines to demonstrate that cellular iron efflux can be stimulated by transferrin and that this efflux is specific to iron but not manganese or zinc. Current studies focus on two goals. First, we are determining if the reduction in tissue iron load represents solely a redistribution of iron stores to the erythron or additionally excretion of iron via urine and/or feces by measuring whole body iron levels in mice before and after transferrin treatment. Second, we are identifying transporters required for mobilization of iron stores using expression profiling of known and putative iron transporters in mouse tissues and validating the role of transporters of interest using our cell culture model. Results of these studies and their implications will be presented. Disclosures No relevant conflicts of interest to declare.

Expression of repulsive guidance molecule b (RGMb) in the uterus and ovary during the estrous cycle in rats

Repulsive guidance molecule b (RGMb; a.k.a. Dragon), initially identified in the embryonic dorsal root ganglion, is the first member of the RGM family shown to enhance bone morphogenetic protein (BMP) signaling by acting as a BMP co-receptor. BMP signaling has been demonstrated to play an important role in the reproductive organs. Our previous study found that RGMb was expressed in the reproductive axis, but whether RGMb expression in reproductive organs changes across the estrous cycle remains unknown. Here, we show in the rat that RGMb mRNA expression in the uterus was significantly higher during metesterus and diestrus than during proestrus and estrus. Western blotting indicated that RGMb protein was significantly lower during estrus compared with the other three stages. Immunohistochemistry revealed that RGMb protein was mainly localized to the uterine luminal and glandular epithelial cells of the endometrium. RGMb mRNA and protein in the ovary remained unchanged during the estrous cycle. RGMb protein was expressed in the oocytes of all follicles. Weak staining for RGMb protein was also found in corpora lutea. RGMb was not detected in granulosa cells and stromal cells. Taken together, RGMb expression in the uterus and ovary across the estrus cycle demonstrate that RGMb may be involved in the regulation of uterine function, follicular development as well as luteal activity.

Bone morphogenetic proteins as regulators of iron metabolism.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-β) superfamily of signaling molecules. In addition to protean roles in embryonic development, germ-line specification, and cellular differentiation, a central role in iron homeostasis has recently been demonstrated for certain BMPs. Specifically, BMP6 serves to relate hepatic iron stores to the hepatocellular expression of the iron-regulatory hormone hepcidin. This regulation occurs via cellular SMAD-signaling molecules and is strongly modulated by the BMP coreceptor hemojuvelin (HJV). Mutations in certain genes influencing signaling to hepcidin via the BMP/SMAD pathway are associated with human disorders of iron metabolism, such as hereditary hemochromatosis and iron-refractory iron-deficiency anemia. Evidence suggests that signals in addition to iron stores influence hepcidin expression via the BMP/SMAD pathway. This review summarizes the details of BMP/SMAD signaling, with a particular focus on its role in iron homeostasis and iron-related diseases.

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance (IRC5P.465)

Abstract We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb) which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, while lung dendritic cells express PD-L2. Blockade of the RGMb:PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2 deficient mice showed PD-L2 expression on non-T cells was critical for respiratory tolerance but expression on T cells was not required. Since PD-L2 binds to both PD-1, which inhibits anti-tumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer and other immune-mediated disorders. Understanding this pathway may give insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance.

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb), which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, whereas lung dendritic cells express PD-L2. Blockade of the RGMb-PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2-deficient mice showed that PD-L2 expression on non-T cells was critical for respiratory tolerance, but expression on T cells was not required. Because PD-L2 binds to both PD-1, which inhibits antitumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer, and other immune-mediated disorders. Understanding this pathway may provide insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.

Matriptase-2, a Regulatory Protease of Iron Homeostasis: Possible Substrates, Cleavage Sites and Inhibitors

Matriptase-2 is a cell surface serine protease with a modular structure. The exploration of its function in iron homeostasis was of significance for the understanding of the regulation of hepcidin expression, the master protein in iron control. Mutations in matriptase- 2 cause iron-refractory iron deficiency anemia (IRIDA), an iron deficiency disorder where the level of hepcidin is inappropriately high. Matriptase-2 controls hepcidin expression through the suppression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic homologue protein (SMAD) signaling, probably by cleaving the BMP co-receptor hemojuvelin. Since prospective studies revealed that genetic inactivation of matriptase-2 reduces iron loading in different mouse models, matriptase-2 becomes highly attractive as a novel target for the design of low-molecular weight inhibitors. The first synthetic peptidomimetic matriptase-2 inhibitors have been reported. A computational model of the active site of matriptase-2 based on the X-ray crystal structure of the close homologue matriptase was generated and mutational studies were performed in order to identify critical amino acids specifying the preferred recognition site of matriptase-2. So far, the only known putative natural substrates of matriptase-2 are hemojuvelin and matriptase-2 itself, as this protease undergoes complex auto-processing during zymogen activation. Cleavage sites within both natural substrates were identified.

Neogenin Interacts with Matriptase-2 to Facilitate Hemojuvelin Cleavage

Hemojuvelin (HJV) and matriptase-2 (MT2) are co-expressed in hepatocytes, and both are essential for systemic iron homeostasis. HJV is a glycosylphosphatidylinositol-linked membrane protein that acts as a co-receptor for bone morphogenetic proteins to induce hepcidin expression. MT2 regulates the levels of membrane-bound HJV in hepatocytes by binding to and cleaving HJV into an inactive soluble form that is released from cells. HJV also interacts with neogenin, a ubiquitously expressed transmembrane protein with multiple functions. In this study, we showed that neogenin interacted with MT2 as well as with HJV and facilitated the cleavage of HJV by MT2. In contrast, neogenin was not cleaved by MT2, indicating some degree of specificity by MT2. Down-regulation of neogenin with siRNA increased the amount of MT2 and HJV on the plasma membrane, suggesting a lack of neogenin involvement in their trafficking to the cell surface. The increase in MT2 and HJV upon neogenin knockdown was likely due to the inhibition of cell surface MT2 and HJV internalization. Analysis of the Asn-linked oligosaccharides showed that MT2 cleavage of cell surface HJV was coupled to a transition from high mannose oligosaccharides to complex oligosaccharides on HJV. These results suggest that neogenin forms a ternary complex with both MT2 and HJV at the plasma membrane. The complex facilitates HJV cleavage by MT2, and release of the cleaved HJV from the cell occurs after a retrograde trafficking through the TGN/Golgi compartments.

Hepcidina: su interacción con la hemojuvelina y su aporte en el diagnóstico de las enfermedades relacionadas con el metabolismo del hierro

La hepcidina es una hormona peptídica, producida en el hígado y considerada un regulador del metabolismo del hierro. Su regulación está dirigida a la absorción intestinal del hierro y a la función de la ferroportina dentro de la célula, a fin de mantener un balance entre el consumo y las reservas de hierro. Esta hormona, a su vez, está regulada por procesos inflamatorios, estados de anemia ferropénica, actividad hematopoyética y algunas vías de señalización como SMAD, STAT y JAK. En estas vías interviene una proteína de membrana llamada hemojuvelina, la cual actúa como un correceptor de la proteína morfogenética ósea, cuya función es inducir el gen de transcripción de la hepcidina mediante la vía SMAD1/5/8-SMAD4. Debido a la asociación de la hemojuvelina con la expresión de la hepcidina y la relación de esta última con procesos inflamatorios y regulación del metabolismo del hierro, se han propuesto algunas técnicas para la determinación de ambas proteínas como Elisa, Dotblot, inmunoensayos y SELDI-TOF MS.

The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation

The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP-SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes. We explore protein-protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells. Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120-139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS. Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV.

Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia

AbstractInappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by β-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbbth3/+ mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)–son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown “erythroid regulator.” The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbbth3/+ mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbbth3/+ mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbbth3/+ male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves β-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition.

Tumour necrosis factor alpha downregulates human hemojuvelin expression via a novel response element within its promoter

BackgroundIron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation.MethodsTo study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression.ResultsHere we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional.ConclusionsFrom these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.

The BMP Coreceptor RGMb Promotes While the Endogenous BMP Antagonist Noggin Reduces Neurite Outgrowth and Peripheral Nerve Regeneration by Modulating BMP Signaling

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

High Levels of Soluble Serum Hemojuvelin in Patients with Congenital Dyserythropoietic Anemia Type I,

Abstract 3179Ineffective erythropoiesis in congenital dyserythropoietic anemia type I (CDA I) is characterized by increased iron absorption mediated by down-regulation of hepcidin. It has been suggested that growth differentiation factor 15 (GDF15) contributes to the inappropriate suppression of hepcidin. Hemojuvelin (HJV), an important regulator of hepcidin production, is highly expressed in skeletal muscle and the liver. Mutations in the gene encoding HJV lead to severe hepcidin deficiency and juvenile hemochromatosis. Membrane-bound HJV (m-HJV) acts as a co-receptor for bone morphogenetic proteins (BMPs). HJV also exists in a soluble form (s-HJV) which competitively down-regulates hepcidin by interfering with BMP signaling. At least two proteases, furin and matriptase-2, cleave m-HJV into s-HJV. Recently, Brasse-Lagnel et al. (Heamtologica 95:2031–7, 2010) reported a new assay for measuring both the furin- and the matriptase-2-cleaved forms of s-HJV. The aim of the present study was to evaluate the possible role of s-HJV in the development of iron overload in CDA I. The study group included eight (8) Israeli Bedouins with CDA I who were homozygous for the founder mutation, c.3124C>T. All were young adults of mean age 27 years. One patient had undergone splenectomy and is currently transfusion-dependent; 3 female patients required occasional blood transfusion during intercurrent infection, pregnancy, and delivery. Nine healthy volunteers served as the comparative group. Laboratory data, including levels of serum hepcidin, ferritin, erythropoietin, soluble transferrin receptor (sTfR), and GDF 15, were previously collected in both groups (Tamary et al. Blood 112:5241–4, 2008). Serum levels of s-HJV were measured in stored frozen samples using a competitive ELISA assay based on an anti-HJV polyclonal antibody (Brasse-Lagnel et al. Heamtologica 95:2031–7, 2010). Compared to the healthy volunteers, the patient group was characterized by significantly higher levels of s-HJV (1.99±0.98mg/L vs 0.36±0.25mg/L; p=0.001), significantly lower level of hemoglobin, and significantly higher levels of serum ferritin, erythropoietin, GDF 15, and sTfR. Serum hepcidin levels were similar in the CDA I patients and the healthy volunteers, but the hepcidin-to-ferritin ratio was significantly lower in the patient group, suggesting that serum hepcidin concentrations were inappropriately low. Levels of s-HJV negatively correlated with hemoglobin levels (R= −0.69 p=0.002) and positively correlated with the iron loading parameter, serum ferritin (R=0.73 p=0.000), and parameters of erythropoiesis, such as serum erythropoietin (R=0.63 p= 0.006) and sTfR (R=0.66 p= 0.008). Additionally, GDF 15 level was positively correlated with s-HJV level (R= 0.64 p=0.006), whereas the hepcidin-to-ferritin ratio (R=−0.797 p=0.000) was negatively correlated with s-HJV level. In summary, our study suggests that s-HJV is overexpressed in patients with CDA I and probably contributes to the down-regulation of hepcidin and the iron-loading pathology. To our knowledge, this is the first study to analyze s-HJV levels in a disease characterized by ineffective erythropoiesis. The main limitation of our study is the small number of patients. Larger studies including patients with CDA and thalassemia are indicated. The source of serum s-HJV and the trigger for its overexpression in CDA I still need to be established. Disclosures:No relevant conflicts of interest to declare.

Repulsive guidance molecules, novel bone morphogenetic protein co-receptors, are key regulators of the growth and aggressiveness of prostate cancer cells

Repulsive guidance molecule (RGM) family members RGMA, RGMB and RGMC are GPI-linked membrane proteins recently identified as co-receptor of bone morphogenetic proteins (BMPs). BMPs are a group of proteins enriched in bone and play important roles in prostate cancer. The current study aimed to investigate roles played by RGMs in prostate cancer. Expression of RGMs was examined in prostate cancer cell lines and prostate cancer tissues using RT-PCR and immunohistochemical staining. Knockdown of each RGM in prostate cancer cells was performed using the respective anti-RGMA, RGMB and RGMC transgenes. A variety of in vitro function tests were employed to analyze the influence on cancer cell functions by RGM knockdown. The implications of RGM knockdown in BMP signalling were also examined using both Western blot and real-time quantitative PCR. Knockdown of RGMA had no effect on cell growth, migration and invasion, but promoted cell-matrix adhesion. Knockdown of RGMB and RGMC increased growth and adhesion, but only RGMB knockdown increased capacities of migration and invasion in PC-3 cells. Further investigations showed an increase in Smad-3 activation and reduced levels of Smad-1 in PC-3 cells by RGMB and RGMC knockdown, and also an up-regulation of ID1, a BMP target gene particularly in exposure to BMP7. RGMs play inhibitory roles in prostate cancer by suppressing cell growth, adhesion, migration and invasion. RGMs can coordinate Smad-dependent signalling of BMPs in prostate cancer cells.