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: The BMP type I receptor ALK3 is functionally important for hepcidin suppression by ERFE/EPO in primary hepatocytes and in mice. (A-B) Primary hepatocytes were transfected with negative control (Ctrl), Alk2, Alk3, ActrIIa, Actrllb, or Bmpr2 siRNA (80 nM) for 30 hours, followed by treatment with conditioned medium containing 50% (vol/vol) cell supernatant from control HEK293T cells (CTRL-CM) or HEK293T cells overexpressing ERFE (ERFE-CM)2 for 15 hours. Panel A, n = 4 independent experiments; panel B, n = 5 independent experiments. Hepatocyte-specific Alk2-deficient mice (C, Alk2fl/fl;Alb-Cre), hepatocyte-specific Alk3-deficient mice (D, Alk3fl/fl;Alb-Cre) and their respective controls (Alk2fl/fl or Alk3fl/fl; n = 4-8 per group) at 8 weeks of age were given 1 intraperitoneal dose of EPO (200 U per mouse) or PBS, and livers were collected after 15 hours. Relative hepcidin (Hamp) mRNA levels were determined by using quantitative reverse transcription–polymerase chain reaction. Transcripts were normalized to Rpl19 (in cells) or 18S (in mice) as internal controls, and the average of Ctrl cells treated with CTRL-CM or the respective Alk2fl/fl or Alk3fl/fl control mice was set to 1. Data are presented as box plots with minimum to maximum whiskers. *P < .05, **P < .01, ***P < .001 relative to the indicated control by the Student t test or Mann Whitney U test. ns, not significant.

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: Transfection, Negative Control, Reverse Transcription, Polymerase Chain Reaction, MANN-WHITNEY

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