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.

In B. subtilis, the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) regulate the initiation of sporulation. Soj is a negative regulator of sporulation gene expression, and Spo0J antagonizes Soj function. Using fusions of Soj to green fluorescent protein, we found that Soj localized near the cell poles and upon entry into stationary phase oscillated from pole to pole. In the absence of Spo0J, Soj was associated predominantly with DNA. By in vivo cross-linking and immunoprecipitation, we found that Soj physically associates with developmentally regulated promoters, and this association increased in the absence of Spo0J. These results show that Soj switches localization and function depending on the chromosome partitioning protein Spo0J. We further show that mutations in the Soj ATPase domain disrupt localization and function and render Soj insensitive to regulation by Spo0J.

The phosphorylated form of the response regulator Spo0A (Spo0AϳP) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0AϳP-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed mediumdependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.In response to starvation and high cell density, Bacillus subtilis can initiate a developmental pathway leading to the formation of dormant endospores (3, 13, 46). The transcription factor Spo0A is a critical regulator of the shift from exponential growth to sporulation. Spo0A is activated by phosphorylation {reviewed in references 3 and 13), and sporulation begins with accumulation of a threshold amount of phosphorylated Spo0A (Spo0AϳP) (5).Spo0A is a response regulator, but unlike most members of this protein family, Spo0A does not obtain phosphate directly from a histidine kinase. Spo0A phosphorylation is controlled by a phosphotransfer pathway, known as the phosphorelay, composed of Spo0F, Spo0B, and at least four histidine kinases, KinA, KinB, KinC, and KinD (1,2,20,21,24,27,36,47). The kinases donate phosphate to Spo0F, a response regulator with no output domain (2, 36). The phosphate from Spo0FϳP is transferred to Spo0B and finally from Spo0BϳP to Spo0A (2). spo0B and spo0F null mutants do not sporulate, indicating that phosphorelay-independent phosphorylation of Spo0A is normally insufficient to activate sporulation.A range of intra-and extracellular signals control sporulation by affecting the flow of phosphate through the phosphorelay. KinA, KinB, KinC, and KinD all have different roles in Spo0A activation and probably respond to distinct stimuli (7,28,47). KinA is the most important kinase for sporulation in rich sporulation media (28,47). KinB is the most important kinase when cells are grown in glucose minimal medium (28). KinC and KinD contribute very little to sporulation under any condition but appear to regulate Spo0A during vegetative growth (20,21,24,27).Spo0AϳP levels are also regulated by phosphatases. Spo0E is a Spo0AϳP-specific phosphatase (33). Several phosphatases indirectly decrease Spo0AϳP levels by dephosphorylating Spo0FϳP. At least three members of the Rap (response regulator asparty...

Human genetics, biomarker, and animal studies implicate loss of function in bone morphogenetic protein (BMP) signaling and maladaptive transforming growth factor–β (TGFβ) signaling as drivers of pulmonary arterial hypertension (PAH). Although sharing common receptors and effectors with BMP/TGFβ, the function of activin and growth and differentiation factor (GDF) ligands in PAH are less well defined. Increased expression of GDF8, GDF11, and activin A was detected in lung lesions from humans with PAH and experimental rodent models of pulmonary hypertension (PH). ACTRIIA-Fc, a potent GDF8/11 and activin ligand trap, was used to test the roles of these ligands in animal and cellular models of PH. By blocking GDF8/11- and activin-mediated SMAD2/3 activation in vascular cells, ACTRIIA-Fc attenuated proliferation of pulmonary arterial smooth muscle cells and pulmonary microvascular endothelial cells. In several experimental models of PH, prophylactic administration of ACTRIIA-Fc markedly improved hemodynamics, right ventricular (RV) hypertrophy, RV function, and arteriolar remodeling. When administered after the establishment of hemodynamically severe PH in a vasculoproliferative model, ACTRIIA-Fc was more effective than vasodilator in attenuating PH and arteriolar remodeling. Potent antiremodeling effects of ACTRIIA-Fc were associated with inhibition of SMAD2/3 activation and downstream transcriptional activity, inhibition of proliferation, and enhancement of apoptosis in the vascular wall. ACTRIIA-Fc reveals an unexpectedly prominent role of GDF8, GDF11, and activin as drivers of pulmonary vascular disease and represents a therapeutic strategy for restoring the balance between SMAD1/5/9 and SMAD2/3 signaling in PAH.

We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinbardfii in response to phosphorus deprivation. Phosphorus-deprived cells increase extracellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the alkaline phosphatase activity. lhis phosphatase has optimal activity at pH 9.5 and a K,,, of 120 to 262 PM for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. l h e information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells.Phosphorus is often a limiting nutrient in both terrestrial and aquatic ecosystems. Most organisms acquire phosphorus through active transport of Pi from the surrounding medium. However, in many soils more than 50% of the soluble phosphate is esterified to organic compounds, rendering it impossible for most organisms to absorb directly (Halstead and McKercher, 1975). Esterified Pi can be released from organic phosphates by the action of extracellular phosphatases, which catalyze the hydrolysis of the