Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1(+) osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH-NIAMS)Harvard Sch Dent Med, Dept Dev Biol, 188 Longwood Ave, Boston, MA 02115 USASaitama Med Univ, Res Ctr Genom Med, Div Pathophysiol, 1397-1 Yamane, Hidaka, Saitama 3501241, JapanUniv Fed Sao Paulo, Inst Ciencia & Tecnol, Rua Talim 330, BR-12231280 Sao Jose Dos Campos, SP, BrazilUniversidade Federal de São Paulo, Rua Talim, 330, São José dos Campos, São Paulo, CEP 12231-280, BrazilNIH-NIAMS: R01 AR055904Web of Scienc

Monapinone Coupling Enzyme Produces Non-Natural Heterodimers

The monapinone coupling enzyme (MCE), a fungal multicopper oxidase, catalyzes the regioselective C–C coupling between tricyclic monapinone A (the primary substrate) and other monapinones (secondary substrates) to produce atropisomeric biaryl homo- or heterodimers. In this study, mono-, bi- and tricyclic compounds were tested to determine whether they worked as secondary substrates for MCE. Among 14 cyclic compounds, MCE utilized semivioxanthin, YWA1, 1,3-naphthalenediol and flaviolin as secondary substrates to produce non-natural heterodimers. The atropisomeric biaryl heterodimers produced by MCE from monapinone A and semivioxanthin were isolated, and their structures were elucidated by NMR and MS. These findings indicate that MCE recognizes bi- and tricyclic compounds with a 1,3-dihydroxy or 1-hydroxy-3-methoxy benzene ring as a secondary substrate

Circulating level of microRNA-126 may be a potential biomarker for recovery from smoking-related vascular damage in middle-aged habitual smokers

Cigarette smoking promotes vascular endothelial damage and accelerates progression of atherosclerosis. The purpose of this study was to examine whether the circulating level of vascular endothelium-enriched microRNA-126 (miR-126), which is highlighted as a regulator of gene expression, would serve as a novel biomarker for recovery from smoking-related vascular damage. Middle-aged male smokers (n = 30) were enrolled and instructed to stop smoking. Their clinical profiles and laboratory findings including expression of miR-126 were investigated before and after 8 weeks of smoking cessation. Serum levels of cotinine, metabolites of nicotine, were measured to confirm smoking cessation. Endothelial function for peripheral small vessels was assessed and expressed as reactive hyperemia peripheral arterial tonometry (RH-PAT) index. The expression of miR-126 in plasma was analyzed by quantitative real-time PCR. At baseline, serum cotinine levels were inversely correlated with RH-PAT index (r = − 0.48, P < 0.01) and positively correlated with levels of metabolic parameters such as non-HDL cholesterol (r = 0.53, P < 0.01) and HOMA-IR (r = 0.52, P < 0.01). The RH-PAT index was not significantly changed after 8 weeks in all subjects, because only 13 subjects could attain smoking cessation. However, changes in the RH-PAT index showed a significant correlation with those in systolic blood pressure (r = − 0.54, P < 0.01). In smokers who completely attained smoking cessation (n = 13), RH-PAT index and plasma levels of miR-126 were significantly increased (P < 0.05, respectively). Endothelial damage was improved and plasma levels of circulating miR-126 were increased after 8 weeks of smoking cessation. These findings suggested a potential use of miR-126 as a biomarker for recovery from smoking-induced vascular damage

Circulating level of microRNA-126 may be a potential biomarker for recovery from smoking-related vascular damage in middle-aged habitual smokers

Background: Cigarette smoking promotes vascular endothelial damage and accelerates progression of atherosclerosis. The purpose of this study was to examine whether the circulating level of vascular endothelium-enriched microRNA-126 (miR-126), which is highlighted as a regulator of gene expression, would serve as a novel biomarker for recovery from smoking-related vascular damage. Methods: Middle-aged male smokers (n = 30) were enrolled and instructed to stop smoking. Their clinical profiles and laboratory findings including expression of miR-126 were investigated before and after 8 weeks of smoking cessation. Serum levels of cotinine, metabolites of nicotine, were measured to confirm smoking cessation. Endothelial function for peripheral small vessels was assessed and expressed as reactive hyperemia peripheral arterial tonometry (RH-PAT) index. The expression of miR-126 in plasma was analyzed by quantitative real-time PCR. Results: At baseline, serum cotinine levels were inversely correlated with RH-PAT index (r = −0.48, P < 0.01) and positively correlated with levels of metabolic parameters such as non-HDL cholesterol (r = 0.53, P < 0.01) and HOMA-IR (r = 0.52, P < 0.01). The RH-PAT index was not significantly changed after 8 weeks in all subjects, because only 13 subjects could attain smoking cessation. However, changes in the RH-PAT index showed a significant correlation with those in systolic blood pressure (r = −0.54, P < 0.01). In smokers who completely attained smoking cessation (n = 13), RH-PAT index and plasma levels of miR-126 were significantly increased (P < 0.05, respectively). Conclusions: Endothelial damage was improved and plasma levels of circulating miR-126 were increased after 8 weeks of smoking cessation. These findings suggested a potential use of miR-126 as a biomarker for recovery from smoking-induced vascular damage

Fungal pyrrolidine-containing metabolites inhibit alkaline phosphatase activity in bone morphogenetic protein-stimulated myoblastoma cells

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. A constitutively activated mutation of a bone morphogenetic protein (BMP) receptor, ALK2, has been identified in patients with FOP. We report here that four structurally related compounds, lucilactaene, hydroxylucilactaene, NG-391 and NG-393, produced by fungal strain Fusarium sp. B88, inhibit BMP signaling in vitro. Alkaline phosphatase activity, a marker enzyme of osteoblastic differentiation, was decreased in C2C12 myoblasts stably expressing mutant ALK2 by treatment with those compounds with IC50 values of 5.7, 6.8, 6.9 and 6.1 μM, respectively. Furthermore, NG-391 and NG-393 inhibited BMP-specific luciferase reporter activity, which is directly regulated by transcription factor Smads, with IC50 values of 1.4 and 2.1 μM, respectively. These findings suggest that these fungal metabolites may provide a new direction in the development of FOP therapeutics

A blocking monoclonal antibody reveals dimerization of intracellular domains of ALK2 associated with genetic disorders

Abstract Mutations in activin receptor-like kinase 2 (ALK2) can cause the pathological osteogenic signaling seen in some patients with fibrodysplasia ossificans progressiva and other conditions such as diffuse intrinsic pontine glioma. Here, we report that intracellular domain of wild-type ALK2 readily dimerizes in response to BMP7 binding to drive osteogenic signaling. This osteogenic signaling is pathologically triggered by heterotetramers of type II receptor kinases and ALK2 mutant forms, which form intracellular domain dimers in response to activin A binding. We develop a blocking monoclonal antibody, Rm0443, that can suppress ALK2 signaling. We solve the crystal structure of the ALK2 extracellular domain complex with a Fab fragment of Rm0443 and show that Rm0443 induces dimerization of ALK2 extracellular domains in a back-to-back orientation on the cell membrane by binding the residues H64 and F63 on opposite faces of the ligand-binding site. Rm0443 could prevent heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva that carries the human R206H pathogenic mutant