Figure 2.
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.

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.

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