bmp2 6  (R&D Systems)

Journal: Blood

Article Title: Erythroferrone lowers hepcidin by sequestering BMP2/6 heterodimer from binding to the BMP type I receptor ALK3

doi: 10.1182/blood.2019002620

Figure Lengend Snippet: ERFE binds BMP2/6 heterodimer and inhibits hepcidin expression by sequestering BMP2/6 from binding to ALK3. (A) 0.5 μg (+), 2 μg (++), 6 μg (+++) BMP2, or 0.5 μg (+) BMP6 were incubated with 0.3 μL (+), 1 μL (++), or 3 μL (+++) of ERFE-FLAG at 52.9 ng/μl. (B,F-G) 0.5 μg (+) or 2 μg (++) ALK3-Fc, ALK2-Fc, Fc, BMP6, or BMP2/6 were incubated with 0.3 μL (+), 1 μL (++), or 3 μL (+++) of ERFE-FLAG at 52.9 ng/μL. Three percent of the total mixture was saved as input. Samples were immunoprecipitated (IP) with anti-FLAG M2 affinity gel (A-B,F) or protein A agarose (G) in 500 μL of NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 0.5% Nonidet P-40) supplemented with 1× protease inhibitor cocktail (MilliporeSigma P8340) at 4°C overnight. This was followed by elution with 150 μg/mL 3× FLAG peptide in Tris-buffered saline at 4°C for 30 minutes (A-B,F) or 2× Laemmli buffer containing 100 mM of β-mercaptoethanol at 95°C for 5 minutes (G) before sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analyses. Experiments were repeated 3 times, with 1 representative blot shown. (C-D) Hep3B cells were transfected with 40 nM of ALK2 or ALK3 siRNA for 30 hours, serum-starved overnight with 1% fetal bovine serum, and treated with PBS or 5 ng/mL of BMP2, BMP6, or BMP2/6 for 6 hours. (E) Hep3B cells were transfected with 200 ng of complementary DNA encoding Erfe or pCMV6-entry empty vector (CTRL) for 48 hours and treated with PBS or 5 ng/mL of BMP2, BMP6, or BMP2/6 in growth medium containing 1% fetal bovine serum for 6 hours. Panels C-E, n = 3 independent experiments. Data are presented as mean ± SEM. **P < .01, ***P < .001 relative to the respective control by the Student t test or one-way analysis of variance with Tukey’s post hoc test. (H) Proposed model depicting ERFE’s mechanism of action: BMP2/6 heterodimeric ligand is secreted by liver endothelial cells and binds to the BMP receptor complex that has been proposed to contain two BMP type II receptors (RII), two type I receptors in the form of ALK3/ALK3 homodimers or ALK2/3 heterodimers, the coreceptor hemojuvelin (HJV), and possibly other interacting proteins, including HFE, transferrin receptor 2 (TFR2), and neogenin (Neo).10,14,17 Activated type I receptors phosphorylate SMAD1/5/8 proteins, which complex with SMAD4 and translocate to the nucleus to induce hepcidin transcription. In the context of erythropoietic drive, secreted ERFE binds to BMP2/6 to prevent BMP2/6 from binding and activating the BMP receptor complex, thereby suppressing hepcidin transcription.

Article Snippet: For immunoprecipitation and pull-down assays, 0.5 to 2 μg of ALK3-Fc, ALK2-Fc, BMP2, BMP6, or BMP2/6 (R&D Systems) were mixed with 0.3 to 3 μL of ERFE-FLAG at 52.9 ng/μL concentrated from serum-free ERFE-CM using a 30 KDa Filter Unit (Ambion) in NETN buffer.

Techniques: Expressing, Binding Assay, Incubation, Immunoprecipitation, Protease Inhibitor, Saline, Polyacrylamide Gel Electrophoresis, Western Blot, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration

doi: 10.3390/ijms21082720

Figure Lengend Snippet: Glycosaminoglycan (GAG) production by human degenerated nucleus pulposus (NP) cells from 4 donors cultured on filters coated with type II collagen and treated with different bone morphogenetic proteins (BMPs, 4 nM total concentration, also with combinations of homodimers and heterodimers (H)) and compared to transforming growth factor (TGF)-β1 controls (4 and 0.4 nM, equal to 100 and 10 ng/mL). GAG and DNA content, GAG/DNA are displayed relative to 0.4 nM TGF-β1-treated NP cells, which were set at 1 (indicated by the dashed line). All the data are n = 12 (3 separate repetitions per donor per condition). ( A ) GAG content was highest in differentiation cultures with BMP4 and BMP2/6H and lowest for 4 nM TGF-β1 and BMP2. ( B ) DNA content was highest when cells were cultured with BMP2/6H, BMP4, BMP2/7H, and BMP7. ( C ) The amount of GAG corrected for DNA was highest in BMP4/7H, followed by BMP4+7, BMP6, and BMP4. ( D ) Total GAG production, including the GAGs released into the medium and contained in the neotissue, was highest in BMP4, BMP2/7H, and BMP4+7. ( E ) Incorporation efficiency was highest in the cells cultured with BMP4/7H. ( F ) Safranin O/Fast Green staining on histological sections of the NP cells cultured on transwell filters reveals that GAGs were deposited in the presence of all BMPs, although the staining was less intense for BMP2, BMP6, or BMP7. Cells in the presence of controls with 0.4 and especially 4 nM TGF-β1 produced hypercellular tissues with limited extracellular matrix (ECM). Representative images for all donors are shown. Significant differences are indicated as follows: * p ≤ 0.001, + p ≤ 0.005, # p ≤ 0.01, ^ p ≤ 0.025, $ p ≤ 0.05.

Article Snippet: BMP2, BMP4, BMP6, BMP7, heterodimers BMP2/6H, BMP2/7H, and BMP4/7H (all with BSA as the carrier, R&D Systems) were added to the culture medium at 4 nM, and combinations of BMP2+6, BMP2+7, and BMP4+7 homodimers were added at 2 nM per homodimer.

Techniques: Cell Culture, Concentration Assay, Staining, Produced

Journal: International Journal of Molecular Sciences

Article Title: Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration

doi: 10.3390/ijms21082720

Figure Lengend Snippet: Human degenerated nucleus pulposus (NP) cells were cultured on type II collagen-coated transwell inserts in the presence of 4 nM bone morphogenetic proteins (BMPs, alone, in combination, or heterodimers (H)) or 0.4 or 4 nM transforming growth factor (TGF)-β1. ( A ) Procollagen type II production (PIICP), measured by enzyme-linked immunosorbent assay (ELISA), is displayed relative to 0.4 nM TGF-β1-treated NP cells, which were set at 1 (indicated by the dashed line). The data are n = 12 (3 separate repetitions per donor per condition). Procollagen II production in week 2 was higher by the NP cells cultured with BMP7 than with BMP2, BMP4+7, and BMP2/6H. The cells cultured with BMP4, BMP6, and BMP4+7 also produced more type II procollagen than the cells cultured with BMP2/6H. Significant differences are indicated as follows: * p ≤ 0.001, + p ≤ 0.005, # p ≤ 0.01, ^ p ≤ 0.025, $ p ≤ 0.05. ( B ) Immunohistochemistry shows that type II collagen was deposited in all the neotissues, but only localized staining was seen occasionally in the presence of 4 nM TGF-β1. The negative control is a cultured construct, the positive control is an osteoarthritic cartilage. ( C ) All the constructs cultured with BMPs and TGF-β1 were positive for type I collagen visualized by immunohistochemistry. The intensity was higher for BMP2+6 and very faint for BMP2/7H. Both negative and positive controls are cultured constructs. ( D ) Type X collagen was not detectable in any of the neotissues while the growth plate was immunopositive. Both negative and positive controls are fetal vertebra.

Article Snippet: BMP2, BMP4, BMP6, BMP7, heterodimers BMP2/6H, BMP2/7H, and BMP4/7H (all with BSA as the carrier, R&D Systems) were added to the culture medium at 4 nM, and combinations of BMP2+6, BMP2+7, and BMP4+7 homodimers were added at 2 nM per homodimer.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced, Immunohistochemistry, Staining, Negative Control, Construct, Positive Control