Blockade of activin type II receptors with a dual anti-ActRIIA/IIB antibody is critical to promote maximal skeletal muscle hypertrophy.

The TGF-ß family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc "trap," to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.

A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-canonical Wnt-5a/Ror2/Disheveled Pathway Activation.

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

A gradient of ROR2 protein stability and membrane localization confers brachydactyly type B or Robinow syndrome phenotypes.

Mutations in ROR2 cause dominant brachydactyly type B (BDB1) or recessive Robinow syndrome (RRS), each characterized by a distinct combination of phenotypic features. We here report a novel nonsense mutation in ROR2 (c.1324C>T; p.R441X) causing intracellular protein truncation in a patient exhibiting features of RRS in conjunction with severe recessive brachydactyly. The mutation is located at the same position as a previously described frame shift mutation causing dominant BDB1. To investigate the apparent discrepancy in phenotypic outcome, we analysed ROR2 protein stability and distribution in stably transfected cell lines expressing exact copies of several human RRS and BDB1 intracellular mutations. RRS mutant proteins were less abundant and retained intracellularly, although BDB1 mutants were stable and predominantly located at the cell membrane. The p.R441X mutation showed an intermediate pattern with membrane localization but also high endoplasmic reticulum retention. Furthermore, we observed a correlation between the severity of BDB1, the location of the mutation, and the amount of membrane-associated ROR2. Membrane protein fraction quantification revealed a gradient of distribution and stability correlating with the clinical phenotypes. This gradual model was confirmed by crossing mouse models for RRS and BDB1, yielding double heterozygous animals that exhibited an intermediate phenotype. We propose a model in which the RRS versus the BDB1 phenotype is determined by the relative degree of protein retention/degradation and the amount of mutant protein reaching the plasma membrane.

Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation.

Wnt signalling plays important roles in patterning and outgrowth of the vertebrate limb. Different mutations in Wnt genes, their antagonists or (co-)receptors result in patterning and outgrowth defects as well as chondrocyte and bone phenotypes in mouse and human. Understanding Wnt activity during mouse limb development and chondrogenesis requires a temporal and spatial overview of Wnt signalling key factor expression. Here we present a comparative expression analysis of all 19 Wnt genes and their major secreted antagonists of the Dickkopf (Dkk), Wisp and the secreted frizzled related protein (Sfrp) families during mouse limb development. Our study reveals new domains of expression for Wnt2, Wnt2b, Wnt5b, Wnt6, Wnt7b, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16, in the limb. We also identified novel expression domains for the Wnt antagonists Sfrp1, Sfrp3, Sfrp5, Wisp1 as well as Dkk2 and Dkk3. We provide a full expression pattern for Wif1 in limb development, for which no limb expression had been documented so far.