Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Relative p‐SMAD2/3 (left) and p‐SMAD1/5/8 (right) response, evaluated by AlphaLISA signal, of HEK293T cells after 1‐h exposure to recombinant myostatin (Mstn), activin A, GDF11, TGFβ, and BMP2. Relative p‐SMAD2/3 response of C2C12 myoblasts (left) and myotubes (right) after 1‐h exposure to Mstn, activin A, GDF11, and TGFβ. Phosphorylation of SMAD2 and SMAD3 in differentiated C2C12 myotubes following stimulation with recombinant Mstn or GDF11 for 30 and 60 min, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. EC 50 values (in nM) for p‐SMAD2/3 and p‐SMAD1/5/8 responses of HEK293T, C2C12 myoblasts, and C2C12 myotubes to the above listed ligands, as well as p‐SMAD1/5/8 response to BMP4, BMP6, and BMP7. Data information: Values are displayed as mean ± SEM; n = 4 for all data points.

Article Snippet: To assess the phosphorylation of SMAD in response to TGFβ superfamily members in undifferentiated cells, C2C12 myoblasts (ATTC CRL‐1772; passage 14) and HEK293T cells (ATCC CRL‐11268; passage 13) were plated on 96‐well tissue culture‐treated plates (Greiner‐Bio‐One 655098) in growth medium [high glucose Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin streptomycin (PS)], grown to confluency, and were treated with various doses of the following recombinant ligands for 1 h ( n = 4): Mstn (R&D Systems #788‐G8), GDF11 (R&D Systems # 1958‐GD), TGFβ (R&D Systems #240‐B), activin A (R&D Systems # 338‐AC), BMP‐2 (R&D Systems # 355‐BM), BMP‐4 (R&D Systems # 314‐BP), BMP‐6 (R&D Systems # 507‐BP), or BMP‐7 (R&D Systems # 354‐BP).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Staining

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

doi: 10.1074/jbc.m109.002881

Figure Lengend Snippet: FIGURE 1. BMP9 selectively activates mRNA transcription in HPAECs. A, confluent serum-restricted HPAECs were treated with BMP2, BMP4, BMP6, BMP9 (10 ng/ml), or TGF1 (2 ng/ml) in M199, 0.1% FBS (0.1%) for 1, 4, or 8 h. Total RNA was extracted and cDNA prepared. The expression of Id1, Id2, IL-6, IL-8, E-selectin, L-selectin, P-selectin, and fibroblast growth factor 2 (FGF2) were determined by qPCR, with expression being normalized to -actin and expressed as the fold-change relative to 0.1% at that time point. Data are presented as the mean S.E. of three experiments. *, p 0.05; **, p 0.01; or ***, p 0.001 compared with 0.1% FBS (0.1%). B, serum-restricted HPAECs were treated with BMP9 (1 ng/ml) in M199, 0.1% FBS (0.1%) for 0.5, 1, 2, and 4 h. Immunoblotting was performed with antibodies against Id1 and Id2. All blots were reprobed for -actin to ensure equal loading. C, HPAECs were treated with BMP9 (1 ng/ml) in M199, 0.1% FBS (0.1%) for 24 h. Conditioned medium was assayed for IL-8 using a specific enzyme-linked immunosorbent assay and data are expressed as picograms of IL-8/105 cells. Data are mean S.E. (n 6) from a representative experiment from 3 repeats.

Article Snippet: Western Blotting—Cells were grown to confluence in 6-cm dishes and serum-restricted in M199, 0.1% FBS for 16 h. Cells were then treated with recombinant human BMP2, BMP4, BMP6, BMP9, or TGF 1 (R&D Systems) diluted in 0.1% for 1 h. For ALK5 inhibition experiments, cells were preincubated for 30minwith SD208 (TGF Receptor kinase inhibitor V, Calbiochem) or dimethyl sulfoxide vehicle control in 0.1%, prior to treatment with ligands.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

doi: 10.1074/jbc.m109.002881

Figure Lengend Snippet: FIGURE 2. BMP9 induction of Smad phosphorylation and mRNA tran- scription in HPAECs is concentration-dependent. A, serum-restricted HPAECs were treated with BMP9 (0.01–10 ng/ml), BMP2 (10 or 50 ng/ml), BMP4 (10 or 50 ng/ml), BMP6 (10 or 50 ng/ml), or TGF1 (2 or 5 ng/ml) in M199, 0.1% FBS (0.1%) for 1 h. Immunoblotting was performed with anti- bodies against phospho-Smad1/5, Smad1, phospho-Smad2, Smad2, phospho-Smad1/3, or Smad3. All blots were reprobed for -actin to ensure equal loading. B, serum-restricted HPAECs were treated with BMP9 (0.01–10 ng/ml) in 0.1% for 8 h. Total RNA was extracted and cDNA pre- pared. The expression of Id1, Id2, IL-8, and E-selectin were determined by qPCR, with expression being normalized to -actin and expressed as the fold-change relative to 0.1%. Data are presented as the mean S.E. of three experiments. C, serum-restricted HPAECs, HAECs, HMEC-1, and HPASMCs were treated with BMP9 (1 ng/ml) or TGF1 (5 ng/ml) in M199,

Article Snippet: Western Blotting—Cells were grown to confluence in 6-cm dishes and serum-restricted in M199, 0.1% FBS for 16 h. Cells were then treated with recombinant human BMP2, BMP4, BMP6, BMP9, or TGF 1 (R&D Systems) diluted in 0.1% for 1 h. For ALK5 inhibition experiments, cells were preincubated for 30minwith SD208 (TGF Receptor kinase inhibitor V, Calbiochem) or dimethyl sulfoxide vehicle control in 0.1%, prior to treatment with ligands.

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Expressing

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is induced by BMP6. (A) Hep3B cells were treated with 5, 25, and 50 ng/mL of human BMP6 for 16 hours and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 3 to 8 (depending of the dose) independent experiments is presented. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock. (B-C) Hep3B cells were transfected with siRNA control (5nM), siRNA TMPRSS6 (5nM), or TMPRSS6-FLAG (8 μg), and treated with 25 ng/mL of BMP6 for 48 hours. Cells were analyzed for matriptase-2 level relative to pan-cadherin protein by Western blot (B) followed by chemiluminescence quantification (C). (B) *A shorter exposure of lane 5 to better distinguish the 2 bands. (C) The mean of 3 experiments is presented, and results are reported as the mean ± SEM. (D) A total of 15 μg of protein from conditioned media of Hep3B cells transfected with siRNA control (5nM) and siRNA TMPRSS6 (5nM) and treated with BMP6 (25 ng/mL) for 48 hours were incubated with 666μM of N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide. Activity of matriptase-2 was assessed by measurement of the release of the dye p-nitroaniline during up to 20 minutes at a wavelength of 405 nm at 37°C using a spectrophotometer. The resulting activities (1 U corresponds to a release rate of 1 mmol of p-nitroaniline per minute) were measured in duplicate in 3 independent experiments. Results are reported as the mean ± SEM. (A,C-D) Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Incubation, Activity Assay, Spectrophotometry

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Regulation of TMPRSS6 mRNA expression in response to BMP6. Hep3B cells were treated with 25 ng/mL of BMP6 for several time points between 1 and 48 hours (A), and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. (B-C) Hep3B cells received 10 μg/mL of cycloheximide (B) or 60nM of LDN-193189 (C) before BMP6 (25 ng/mL) treatment and were analyzed for gene expression relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 6 (A-B) and 3 (C) independent experiments is presented. (B-C) Results are reported as the mean ± SEM for the fold change from mock (just before adding BMP6) and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is controlled by ID1 in response to BMP6. Hep3B cells transfected with 10nM of siRNA control, siRNA SMAD7 (A-B), or siRNA ID1 (C-D), and treated in the absence or presence of 25 ng/mL of BMP6 for 24 hours were analyzed for TMPRSS6 (B,D) SMAD7 (A), and ID1 (C) mRNA expression relative to RPL19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock (siRNA control alone). Significant changes are as follows: *P < .05.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is up-regulated in vivo by BMP6 and chronic iron treatment. (A) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of BMP6 750 μg/kg animal weight (BMP6, black bars) or vehicle alone (mock, gray bars; n = 3 per group) for 6 and 12 hours. (B) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of neutralizing BMP6 antibody 15 mg/kg once a day for 1 week (n = 5 per group). (C) Seven-week-old male C57BL/6 mice were killed at time zero (baseline) or after initiation of a 2% carbonyl iron diet for 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks (n = 6 per group). Tissues were analyzed for hepatic hepcidin and Tmprss6 relative to Rpl19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, In Vivo, Injection, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Schematic representation showing proposed role of TMPRSS6 regulation by the BMP6-SMAD signaling pathway and iron via ID1. We propose that, in addition to being stimulated by several signals that inhibit hepcidin, such as iron deficiency, erythropoeitic drive, and hypoxia, TMPRSS6 expression is also stimulated indirectly by the hepcidin activators BMP6 and iron. Stimulation by BMP6 and/or iron induces an increase of the BMP6-HJV-SMAD pathway activity, possibly through a mechanism involving HFE and transferrin receptor 2 (TFR2), leading to binding of SMAD complexes to BMP-responsive elements (BMP-REs) on the hepcidin promoter and up-regulation of hepcidin transcription. In parallel, BMP6-SMAD pathway directly up-regulates SMAD7 and ID1 transcription. ID1 induction leads to the up-regulation of TMPRSS6 expression. TMPRSS6 then serves as a negative feedback inhibitor of BMP-SMAD pathway activity and hepcidin expression by cleaving the BMP coreceptor HJV. Inhibitory SMAD7 can also act as a negative feedback inhibitor by blocking SMAD activation. By acting as negative feedback inhibitors, TMPRSS6 and SMAD7 are important to prevent excessive hepcidin increases in response to BMP6 and iron, thereby maintaining tight control of iron homeostasis. Abbreviations: BMPR indicates BMP receptor; TF-Fe, holotransferrin; sHJV, soluble hemojuvelin; and BMP-RE, BMP responsive element.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Activity Assay, Binding Assay, Blocking Assay, Activation Assay, Control

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: BMP6 expression is uniquely upregulated in response to loss of LKB1 in invasive HBECs and patients with LUAD. A, Western blot analysis of indicated proteins in a 3D invasive spheroid panel. B, Volcano plot depicting significant DEGs (upregulated, N = 583; downregulated, N = 401) between invasive KRAS /LKB1 (KL) vs. KRAS /TP53 (KP) 3D spheroids with respect to noninvasive control (C) HBEC spheroids. C, Heatmap depicting the mean log 2 -transformed expression levels of selected differentially upregulated genes (KL > KP and KL > K > C) between isogenic 3D spheroids. D, Graph depicting fold change in BMP6 gene expression from qRT-PCR validation in isogenic HBECs. E, Confocal images of immunofluorescence for BMP6 protein expression (red) in 3D HBECs of the indicated genotypes (DAPI labels nuclei, and all cells express cytoplasmic GFP). Scale bar, 100 μm. F, Graph generated using cBioPortal depicting the mean BMP6 mRNA expression from genetic subtypes of patients with LUAD. Each circle represents an individual patient sample. Error bars, SD. G, Western blot analysis of indicated proteins in isogenic 3D spheroids. H, Graph of pathway enrichment analysis sorted by significance. ****, P < 0.0001. RESM, RNA-seq by expectation maximization; TCGA, The Cancer Genome Atlas.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Expressing, Western Blot, Control, Transformation Assay, Gene Expression, Quantitative RT-PCR, Biomarker Discovery, Immunofluorescence, Generated, RNA Sequencing

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: LKB1 restricts BMP6 signaling using a kinase-dependent mechanism, and suppression of BMP6 signaling and ALK2 inhibition suppresses 3D proliferation and invasion in multiple KL cell lines. A, Western blot analysis of BMP6 pathway activation in stable control pLKO.1 and shLKB1 H1299 lung cancer cells. B, Western blot of BMP6-regulated Smad signaling components in LKB1-null H157 cells that express vector control, LKB1-WT, or kinase-dead LKB1 (LKB1-K78I). C, Western blot analysis of indicated proteins in control IgG (−)- or anti-BMP6 (+)–treated KL, JK43-P, and JK43-M cells. D, Representative brightfield images (top) and quantitative graphs (bottom) of control IgG-treated or anti-BMP6–treated invasive KL, JK43-P, or JK43-M spheroids. E, Graph depicting cell viability of indicated cell lines with increasing concentrations of LDN214117 (top). Table of LDN214117 IC 50 in indicated cell lines. F, Representative images (left) and quantitative graphs (right) of A549 3D spheroids assayed for Ki67. Scale bar, 70 μm. G, Representative images (left) and quantitative graphs (right) of JK43-M 3D spheroids assayed for Ki67. Scale bar, 70 μm. H, Brightfield images of 3D spheroids of the indicated cell lines either treated with vehicle control (−) or treated with the indicated concentration of LDN214117 and embedded in the invasion matrix for 72 hours. Scale bar, 100 μm. I, Quantitation of the invasive area for LDN214117-treated KL spheroids (the graph depicts the mean of three biological replicates). J, Quantitation of the invasive area for LDN214117-treated A549 spheroids (the graph depicts the mean of three biological replicates). K, Western blot of BMP6/ALK2-regulated Smad signaling in KL HBECs and A549 (LKB1-null) cells treated with increasing concentrations of LDN214117 for 24 hours. L, Western blot analysis of BMP6/ALK2-regulated Smad signaling in JK43-P and JK43-M mouse tumor cell lines (KrasG12D/Lkb1-null) treated with increasing concentrations of LDN214117 for 24 hours. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Inhibition, Western Blot, Activation Assay, Control, Plasmid Preparation, Concentration Assay, Quantitation Assay

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: Efficacy of targeting ALK2 in LKB1-mutant lung cancer in vivo. LKB1 restricts iron homeostasis pathways using a kinase-dependent mechanism. A, Mean tumor volume from syngeneic mice (JK-M cells) treated with vehicle (6 mice/group) and LDN214117 (7 mice/group). B, Graph depicting the mean tumor weight from vehicle- and LDN214117-treated mice. C, Brightfield images of tumors isolated from vehicle- and LDN214117-treated mice. D, The mean tumor volume from NSG mice with A549 lung tumor xenografts treated with either vehicle (7 mice/group), LDN214117 (7 mice/group), or LDN193189 (7 mice/group). E, Graph depicting the mean tumor weight from vehicle-, LDN214117-, and LDN193189-treated mice. F, Brightfield images of A549 tumors isolated from vehicle-, LDN214117-, and LDN193189-treated mice. G, Representative brightfield images of tumor sections treated with vehicle or LDN214117 and stained by the indicated Ab IHC or special stain. Scale bar, 200 μm or 2 mm (TUNEL). H, Western blot to assess BMP6 and hepcidin levels in tumors from vehicle- and LDN214117-treated mice. I, Western blot of indicated proteins from JK43-M cells treated with 1 uM of LDN214117 for 24, 48, and 72 hours. J, Western blot analysis of indicated proteins in vector, LKB1-WT, or LKB1-K78I add-back H157 lung cancer cells. K, Model depicting the mechanism of altered iron homeostasis signaling in LKB1-mutant tumor cells. **, P < 0.01; ****, P < 0.0001.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Mutagenesis, In Vivo, Isolation, Staining, TUNEL Assay, Western Blot, Plasmid Preparation