Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: Msx2 is a downstream target of BMP2 signaling in the uterus during decidualization. (A) The primary cultures of mouse endometrial stromal cells (MESCs) were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points, as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of Msx2. The relative levels of gene expression were determined by setting the expression level of the GFP-treated sample at 24 h to 1.0 (n = 3). Rplp0, encoding a ribosomal protein, was used to normalize the level of RNA. *P < 0.05. (B) The nucleotide positions of the SBEs on the Msx2 promoter were analyzed by ChIP. (C) Mouse stromal cells were treated with E + P or E, P, and BMP2 (E + P + BMP2) for 90 min. ChIP, using the Smad4 antibody, was performed, as described in “Materials and Methods.” Chromatin enrichment was quantified by real-time PCR using primers flanking the potential SBE in the Msx2 promoter and also a negative control region in the ORF of Msx2. Enrichments were normalized to 1% of input DNA. The experiment was repeated twice, and representative data are shown.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression, Negative Control

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: MSX1 and MSX2 mediate BMP2-induced HESC decidualization. The primary cultures of HESCs were established as described in “Materials and Methods.” The cells were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of MSX1 and MSX2. The relative levels of gene expression were determined by setting the expression level on day 0 of the GFP-treated sample at 1.0. RPLP0, encoding a ribosomal protein, was used to normalize the level of RNA. Data were collected from three independent clinical samples, which were subjected to the same experimental conditions. *P < 0.05; **P < 0.005.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: eNOS and NO‐donors stimulate bone growth. eNOS mRNA expression in MC3T3 (A) and 2T3 (B) cells treated with RSV (1–100 μM) or vehicle for 24 h ( n = 5) determined by real time PCR. Nitrite (NO 2 − ) levels (C) or total eNOS protein (D) from 2T3 or MC3T3 cells treated with RSV (1–100 μM) or vehicle ( n = 5) for 48 h, determined by colorimetric assay. ALP levels from primary osteoblasts treated with NO‐donor (NOC22, 0.1–10 μM) or vehicle for 4 days ( n = 5) normalized to total cell protein (E). Calvariae from newborn mice cultured with NO‐donor (NOC22, 0.1–1 μM) or vehicle control for 4 days ( n = 5) processed for histology and H&E staining (F). mRNA expression of osteoblast marker genes Runx2, osteocalcin (OCN) and BMP2 determined in 2T3 cells treated with NOC22 (1.0 μM) or vehicle for 24 h ( n = 5) by real time PCR (G). BMP2 protein levels assessed in conditioned media from NO‐donor‐treated osteoblasts (NOC22 or SNP, 3–200 μM, 48 h, n = 5), by ELISA (with rhBMP2 as standard) (H). The μCT analysis of dissected tibia from homozygous eNOS knockout ( −/− ) or wild‐type ( +/+ ) control mice (4 month, n = 10) (I) and trabecular bone volume (J) and BMD (K) analysis (* P < 0.05 vs. vehicle; * P < 0.01 vs. wild‐type control animals).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Colorimetric Assay, Cell Culture, Control, Staining, Marker, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: The eNOS‐SIRT1 axis is necessary for the pro‐osteogenic effects of RSV. Different effects on ALP levels (A) BMP2 gene expression (qPCR, B) and promoter activity (C) in primary osteoblasts from eNOS knockout ( −/− ) or wild‐type ( +/+ ) control mice ( n = 5) treated with RSV (5 μM) or vehicle (24 h). RSV‐induced ALP levels (5 μM) in the presence of the BMP inhibitor noggin (500 ng·mL −1 , 48 h, n = 5), normalized to total protein (D). SIRT1 gene expression in 2T3 osteoblasts following RSV (1–100 μM) treatment, quantified by real time PCR (E), along with eNOS (F) and BMP2 (G) mRNA levels transfected with SIRT1 siRNA or scramble control ( n = 5) with and without RSV treatment (5 μM). RSV‐induced ALP levels (5 μM) in the presence of SIRT1 (or scrambled control) siRNA (H) ( n = 5) (* P < 0.05 vs. vehicle treated WT/scrambled ctrl; # P < 0.001 vs. RSV‐treated WT/scrambled ctrl).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

Techniques: Gene Expression, Activity Assay, Knock-Out, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: British Journal of Pharmacology

Article Title: The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1‐bone morphogenic protein 2 longevity axis

doi: 10.1111/bph.14477

Figure Lengend Snippet: Ageing decreases bone volume and eNOS‐BMP2 expression. μCT analysis of tibia of young (3 month) or aged (12 month) mice ( n = 10) (A) determining BV/TV (B) and BMD (C). Gene expression changes (real time PCR) of eNOS (D), BMP2 (E) in ageing long bones ( n = 10). Proposed mechanism of RSV action within osteoblasts (F) (* P < 0.05).

Article Snippet: Recombinant human BMP2 (hBMP2; R&D Systems Inc.) was used as a standard.

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor (TGF)-β-activated Kinase 1 Mimics and Mediates TGF-β-induced Stimulation of Type II Collagen Synthesis in Chondrocytes Independent of Col2a1 Transcription and Smad3 Signaling

doi: 10.1074/jbc.m500646200

Figure Lengend Snippet: FIG. 4. TAK1a mediates the stimulation of type II collagen synthesis by TGF- and BMP2 and exhibits TAB1-independent kinase activity. A, chondrocytes were infected 24 h after plating with dominant-negative TAK1 adenoviral vectors, Ad-pC-hTAK1a-K63A, and Ad-pC-hTAK1a-K63W. Seventy-two hours after initiating infec- tion, cells were treated for 24 h with 5 ng/ml TGF-1 (T) or 100 ng/ml BMP2 (B) in the presence of [3H]proline. Samples from triplicate wells were pooled before SDS-PAGE. B, for immune complex kinase assays, chondrocytes were infected with Ad-pC-hTAK1a (TK), kinase-negative (KN) Ad-pC-hTAK1a-KWSA, and the tandem vector (TK,TB) pCEA3- hTAB1(pC-hTAK1a). Forty-eight hours after beginning infection, cells were treated with TGF-1 (40 ng/ml; T) or IL-1 (40 ng/ml; IL) for 10 min prior to lysis, immunoprecipitation with TAK-ct antibody, and TAK1a immune complex kinase assay using bacterially expressed GST- MKK6 as substrate. Lysates were equally divided prior to immunopre- cipitation to permit direct kinase assays in the presence of [-32P]ATP (upper panels) or kinase assay after pretreatment with cold ATP (lower panels). The right panels are 7-fold shorter exposures of the last two lanes. C, Western blots (IB) demonstrating activation of endogenous TAK1 and TAB1 and their overexpressed counterparts. Chondrocytes were treated for 10 or 30 min with TGF-1 (5 ng/ml) or BMP2 (100 ng/ml) and lysed with SDS sample buffer at the same time as cells exposed to 48 h of adenoviral expression of TAK1a or coexpressed TAK1a and TAB1 (tandem construct). TAK-ct and TAB-m primary antibodies were used for detection. Long dashes, unmodified TAK1 or TAB1 bands; short dashes, activated/phosphorylated bands. Right panel, shorter exposures of lanes 5 and 6.

Article Snippet: Subsequently, cells were fed with the same medium, treated with recombinant human TGF- 1 or BMP2 (R&D Systems) for 24 h and then labeled in fresh medium with tritiated proline for 24 h. Labeling medium was 0.3% ITS /DMEM, 62.5 g/ml -aminoproprionitrile, 25 g/ml ascorbate, 40 Ci/ml [5-3H]proline (Amersham Biosciences), and growth factors.

Techniques: Activity Assay, Infection, Dominant Negative Mutation, SDS Page, Immune Complex Kinase Assay, Plasmid Preparation, Lysis, Immunoprecipitation, Kinase Assay, Western Blot, Activation Assay, Expressing, Construct

Journal: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Microarray, Software, Microscopy

Journal: Development (Cambridge, England)

Article Title: A novel chordin-like BMP inhibitor, CHL2, expressed preferentially in chondrocytes of developing cartilage and osteoarthritic joint cartilage.

doi: 10.1242/dev.00901

Figure Lengend Snippet: Fig. 2. Direct interaction of mCHL2 with BMPs, and inhibition of BMP4 binding to BMP receptor ectodomain by mCHL2. (A) FLAG-tagged CHL2 protein. Proteins in the peak eluate from the hydroxyapatite column chromatography were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then silver stained (Sambrook et al., 1989). The mCHL2-FLAG band was excised and the NH2- terminal amino acid sequence (vertical arrow in Fig. 1B) determined. (B) Immunoprecipitation/western blot analysis of mCHL2-FLAG individually mixed with BMP2 (a), BMP4 (b), BMP5 (c), BMP6 (d), BMP7 (e), GDF5 (f), activin A (g), TGFβ1 (h), TGFβ2 (i) or TGFβ3 (j), followed by treatment with αCHL2-COOH (lanes underlined). Immunocomplexes were detected using the corresponding antibodies (upper panels). Reactions only with mCHL2-FLAG, BMP, GDF, activin or TGFβ were also performed as negative controls. Each blot was further developed with M2 to confirm the presence of precipitated mCHL2-FLAG (lower panels). The TGFβ immunocomplexes (h-j) were separated into two sets; one was loaded on a non-reducing gel to visualize TGFβ (upper panels), and the other on a reducing gel to detect CHL2 (lower panels). Lanes not underlined were directly loaded with the indicated amount (ng) of mCHL2-FLAG, BMP, GDF, activin or TGFβ (for standards). (C) Inhibition of BMP4 binding to BMPR1B ectodomain by mCHL2-FLAG. The indicated amount of mCHL2-FLAG was first mixed with or without BMP4, and then BMPR1B-Fc or IgG-Fc was added. Protein complexes containing BMPR1B-Fc or IgG-Fc were selectively precipitated with protein A and subjected to western blot analysis (lanes underlined). Upper panel: bound BMP4 visualized with anti-BMP4 antibody. Middle panel: co-precipitation of mCHL2-FLAG checked with M2. Lower panel: precipitation of BMPR1B-Fc/IgG-Fc confirmed with anti-IgG-Fc antibody. For the standards, 0.04 µg of mCHL2-FLAG and 0.04 µg of BMP4 were loaded directly.

Article Snippet: Mouse chordin (mCHD-His); human BMP2, BMP7 and TGFβ1 (and mouse monoclonal antibodies against them); mouse monoclonal anti-human TGFβ3; mouse growth and differentiation factor 5 (GDF5); and affinity-purified goat polyclonal anti-mouse GDF5 were from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Binding Assay, Column Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Immunoprecipitation, Western Blot

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Imbalance Between Bone Morphogenetic Protein 2 and Noggin Induces Abnormal Osteogenic Differentiation of Mesenchymal Stem Cells in Ankylosing Spondylitis.

doi: 10.1002/art.39433

Figure Lengend Snippet: Figure 4. Levels of bone morphogenetic protein 2 (BMP-2) and Noggin secreted by AS-MSCs compared with HD-MSCs during osteogenic dif- ferentiation. A and B, BMP-2 (A) and Noggin (B) protein levels in HD-MSCs and AS-MSCs were detected by enzyme-linked immunosorbent assay from day 0 to day 21 of induction. C, Top, Activation levels of the Smad1/5/8, p38 MAPK, JNK, and ERK-1/2 signaling pathways in HD- MSCs and AS-MSCs were determined by Western blotting. Bottom, Results of Western blotting were quantified as the mean 6 SD intensity ratio of phosphorylated to nonphosphorylated proteins. Values are the mean 6 SD of 12 samples per group. * 5 P , 0.05. See Figure 1 for other definitions.

Article Snippet: In addition, BMP-2 and Noggin were measured in the serum using a Quantikine ELISA kit for BMP-2 (R&D Systems) and an ELISA kit for human Noggin (Uscn Life Science).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Protein-Protein interactions, Western Blot

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Bone Morphogenic Protein Type 2 Receptor Mutation-Independent Mechanisms of Disrupted Bone Morphogenetic Protein Signaling in Idiopathic Pulmonary Arterial Hypertension

doi: 10.1165/rcmb.2015-0402OC

Figure Lengend Snippet: Gremlin-1 is up-regulated in human patients with pulmonary arterial hypertension (PAH). (A) Gremlin-1 mRNA expression from human control (n = 10) and PAH (n = 10) lung tissue. The target gene, Gremlin-1, was determined and compared with GAPDH in both control and PAH and calculated as fold change using 2(−ΔΔCT) . (B) PAH plasma levels of Gremlin-1 compared with control samples as determined by ELISA. Error bars represent the SDs of independent triplicate experiments. The P values were given on the basis of a Student's t test determined from the independent triplicate experiments. Ctrl, control.

Article Snippet: Names of primers for human genes are preceded by the letter h. List of Primers Used in RT-qPCR ELISA A human BMP2 ELISA DuoSet (R&D Systems, Minneapolis, MN) and a human Gremlin-1 ELISA (Wuhan USCN Business Co., Ltd) were used to measure BMP2 and Gremlin-1 levels from human PAH ( n = 29) and control ( n = 12) plasma ( ) according to the manufacturer’s recommendations.

Techniques: Expressing, Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Bone Morphogenic Protein Type 2 Receptor Mutation-Independent Mechanisms of Disrupted Bone Morphogenetic Protein Signaling in Idiopathic Pulmonary Arterial Hypertension

doi: 10.1165/rcmb.2015-0402OC

Figure Lengend Snippet: Bone morphogenic protein 2 (BMP2) mRNA and protein expression levels are not significantly different in patients with PAH (without the BMPR2 mutation) compared with control subjects. (A) BMP2 levels from human plasma were measured by ELISA. (B) mRNA fold change in expression of BMP2 in whole lung was determined as in Figure 1. (C) Western blot analysis of whole lung BMP2 protein expression in patients with PAH and control subjects. (D) Densitometry of BMP2 expression normalized to β-actin from C. Error bars represent the SDs of independent triplicate experiments. IB, immunoblot; n.s., not significant.

Article Snippet: Names of primers for human genes are preceded by the letter h. List of Primers Used in RT-qPCR ELISA A human BMP2 ELISA DuoSet (R&D Systems, Minneapolis, MN) and a human Gremlin-1 ELISA (Wuhan USCN Business Co., Ltd) were used to measure BMP2 and Gremlin-1 levels from human PAH ( n = 29) and control ( n = 12) plasma ( ) according to the manufacturer’s recommendations.

Techniques: Expressing, Mutagenesis, Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot