Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Endoglin (CD105), a transmembrane protein of the transforming growth factor ␤ superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. Endoglin (CD105) is a homodimeric glycosylated cell-surface protein of 180 kDa previously identified as a co-receptor belonging to the TGF␤ superfamily (1). Several lines of evidence support an important role of endoglin in cardiovascular development and vascular remodeling (2). Loss-of-function mutations in endoglin are implicated in the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1), 3 which is a bleeding disorder characterized by arteriovenous malformations in the brain, lungs, and liver and is attributed to haploinsufficiency (3-5). Homozygous endoglin knock-out mice die during early gestation due to the lack of development of normal mature blood vessels (6). Adult endoglin heterozygous mice (7-8) or mice with a conditional mutation in the endoglin gene (9) exhibited similar angiogenic abnormalities and were used as animal models for HHT1. Mutations in the endoglin gene found in numerous HHT1 patients are localized most exclusively in the extracellular domain (4). The specific role of endoglin in the vascular dysplasia observed in HHT patients is not known, but it is likely to be related to the role of TGF␤ family signaling in angiogenesis (2, 10). Interestingly, another form of HHT, known as HHT2, which is also characterized by the presence of telangiectases as well as arteriovenous malformations in brain, lungs and liver, results from the loss of TGF␤ type I receptor ALK1 (11), which suggests an interrelatedness between endoglin and ALK1 and possibly involvement of the same ligand(s) in the mechanism of action of both molecules.A soluble form of endoglin has been observed in the serum of patients with different types of solid malignancies (12) and of pregnant women suffering from preeclampsia, a disease leading to vascular permeability (13), hypertension, and placental abruption (14). This soluble form, which reportedly results from partial shedding of the membrane-bound form of endoglin by the matrix metalloproteinase 14 (MT1-MMP) (15), a phenomenon also observed for the other type III receptor betaglycan (16), has been proposed to act as a scavenger or trap for circulating TGF␤ family liga...

Background: Activin receptor-like kinase 1 (ALK1) is an important regulator of normal blood vessel formation and pathological tumor angiogenesis. Results: Crystal structure of ALK1-BMP9-ACTRIIB signaling complex and kinetic and thermodynamic properties of receptorligand interactions are described. Conclusions: ALK1 achieves high specificity for BMP9/10 through unusual receptor positioning and unique receptor-ligand interface. Significance: Structural and mechanistic insights into ALK1 signaling provide a framework for novel anti-angiogenic therapies.

Adrenodoxin is an iron‐sulfur protein that belongs to the broad family of the [2Fe‐2S]‐type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH‐dependent adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate‐type ferredoxin, the truncated adrenodoxin Adx(4‐108), that offers the unique opportunity for better understanding of the structure‐function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe‐2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein‐protein recognition and kinetics of adrenodoxin dependent reactions. Proteins 2000;40:590–612. © 2000 Wiley‐Liss, Inc.

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