SUPPLEMENTARY MATERIALS BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice Elena Corradini, Paul J. Schmidt, Delphine Meynard, Cinzia Garuti, Giuliana Montosi, Shanzhuo Chen, Slobodan Vukicevic, Antonello Pietrangelo, Herbert Y. Lin, and Jodie L. Babitt
SUPPLEMENTARY MATERIALS AND METHODS Animals All animal protocols were approved by the Institutional Animal Care and Use Committee at the Massachusetts General Hospital (MGH), Children s Hospital Boston (CHB), or the University Hospital of Modena (UHM). Wildtype mice with iron deficiency anemia (WT anemic) were housed in the MGH rodent facility and were generated by placing female C57BL/6 mice on an iron deficient diet (2-6 ppm iron, TD.80396, Harlan Teklad) for 3 weeks until 8-10 weeks of age followed by phlebotomy of 0.4 ml blood by retro-orbital puncture. Mice were sacrificed 24 hours after phlebotomy. In parallel, a control group of female WT C57BL/6 mice were housed in the MGH rodent facility and maintained on a Prolab 5P75 Isopro RMH 3000 diet with 380 ppm iron until sacrifice at 8-10 weeks of age. For BMP6 time course experiments, eight-week-old male 129S6/SvEvTac WT mice housed in the MGH rodent facility and maintained on a Prolab 5P75 Isopro RMH 3000 diet with 380 ppm iron were treated with a single intraperitoneal injection of BMP6 at 500 µg/kg or an equal volume of vehicle alone. Six, 8, or 24 hours after injection, mice were sacrificed and analyzed for serum iron and transferrin saturation. Tissue iron measurement Quantitative measurement of nonheme iron was performed on liver, spleen, heart, and pancreas as previously described (Babitt et al. J Clin Invest. 2007; 117:1933-1939). Quantitative real-time RT-PCR Total RNA was isolated from mouse livers and real-time quantification of Hfe or Crp 1
relative to Rpl19 mrna were performed using two-step quantitative real-time RT-PCR as previously described (Babitt et al. Nat Genet. 2006; 38:531-539) using primers: Hfe forward 5 -CACCGCGTTCACATTCTCTAA-3, Hfe reverse 5 - CTGGCTTGAGGTTTGCTCC-3 (Primer Bank ID 31981697a1; Spandidos et al. Nucl. Acids Res. 2010 38:D792-9.); and Crp forward 5' ATG GAG AAG CTA CTC TGG TGC 3', Crp reverse 5' ACA CAC AGT AAA GGT GTT CAG TG 3'. Western blot Western blot of liver lysates for phosphorylated Smad1/5/8 relative to total Smad1 and Actin and chemiluminescence quantitation was performed as previously described (Corradini et al. Gastroenterology. 2009;137:1489-1497). For Ferroportin Western blots, membrane proteins were extracted using the ProteoJET Membrane Protein Kit (Fermentas), following the manufacturer s instructions. After dilution in 1x NuPAGE LDS Sample Buffer and Sample Reducing Agent (Invitrogen), samples were incubated for 10 minutes at 70 C and 5 minute on ice. Then, 2 µg of spleen proteins or 10 µg of liver proteins per sample were resolved by SDS- PAGE using pre-cast NuPAGE Novex 4 12% Bis-Tris gels (Invitrogen) and transferred onto PDVF membranes, using the liquid transfer method. The blots were blocked with 5% of non-fat dry milk in TBS containing 0.1% Tween-20 (Sigma) and probed overnight at 4 C with rabbit polyclonal antibodies to Ferroportin (1:1000, Alpha Diagnostic) diluted in the same blocking solution. Following washes, the blots were incubated for 1 hour with peroxidase-coupled goat anti-rabbit IgG (1:5000, Sigma). Blots were stripped and re-probed with mouse anti-actin monoclonal antibody (1:10000 Chemicon). Enzyme activity was detected by the Western Lightning Plus-ECL method (Perkin Elmer). Chemiluminescence was quantitated using IPLab Spectrum software version 3.9.5 r2 (Scanalytics), by normalizing Ferroportin to Actin. 2
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Supplementary Figure 1. Hepatic Hfe mrna is increased in Hfe Tg mice relative to WT mice. Eight-week-old female Hfe Tg mice (N = 6) versus littermate WT mice (N = 6) were analyzed for hepatic Hfe relative to Rpl19 mrna by quantitative real-time RT-PCR. Results are reported as the mean ± SD for the fold change from WT mice. Exact P values are shown for the comparison with WT mice. 4
Supplementary Table 1. Hematologic and iron parameters of Hfe Tg male versus female mice Gender n Hgb (g/dl) HCT (%) Serum iron (µg/dl) Tf Sat (%) LIC (µg/g) Male 4 10.2 ± 1.3 41.3 ± 1.9 85.7 ± 11.5 38 ± 5.3 29.3 ± 2.1 Female 5 12.0 ± 0.5 44.3 ± 1.9 98.7 ± 9.7 44 ± 3.6 31.7 ± 4.0 Hemoglobin (Hgb), serum iron, transferrin saturation (Tf Sat), and liver iron content (LIC) were measured in 8-week old Hfe Tg male versus female mice on a C57BL/6 background. Data are presented as the mean ± SD. P > 0.05 between genders for all parameters. Male Hfe Tg mice have a similar iron deficiency anemia phenotype compared with female Hfe Tg mice, although there was a trend for a more severe phenotype in the male mice. Genders were therefore matched between treatment groups in BMP6 antibody treatment experiments. 5
Supplementary Table 2. Hematologic and iron parameters of Hfe Tg, WT, and anemic WT mice Genotype/ n Hgb (g/dl) Serum iron (µg/dl) Tf Sat (%) LIC (µg/g) Treatment WT 6 15.3 ± 0.3 149 ± 16 61 ± 5 55 ± 12 Hfe Tg 6 12.7 ± 0.4 ** 82 ± 4 ** 36 ± 2 ** 40 ± 2 ** WT 6 14.4 ± 0.3 167 ± 10 60 ± 4 86 ± 9 Anemic WT 10 11.5 ± 1.0 ** 154 ± 21 53 ± 5 * 58 ± 17 ** The hemoglobin (Hgb), serum iron, transferrin saturation (Tf Sat), and liver iron content (LIC) were measured in 8-week old female Hfe Tg versus littermate WT mice, or in 8-10-week-old female control WT mice maintained on a normal diet versus WT mice maintained on an iron deficient diet for 3 weeks and phlebotomized (Anemic WT). All mice were on a C57BL/6 background. Data are presented as the mean ± SD. **P 0.002 for Hfe Tg versus WT mice or anemic WT mice versus control WT mice. * P = 0.01 for anemic WT mice versus control WT mice. P not significant from WT. Anemic WT mice had a similar reduction in hemoglobin and liver iron content relative to matched WT mice compared with the Hfe Tg mice relative to their WT littermates. Although the anemic WT mice also had slightly reduced serum Tf Sat compared with WT mice, this was considerably less pronounced than in Hfe Tg mice. 6
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Supplementary Figure 2. Hepcidin (Hamp), Id1, and Smad7 mrna are decreased in anemic WT mice relative to control WT mice. Eight- to ten-week-old female WT mice maintained on a normal diet (WT, N = 6) versus WT mice maintained on an iron deficient diet for three weeks followed by phlebotomy of 0.4 ml blood (Anemic WT, N = 10) from Supplementary Table 2 were analyzed 24-hours after phlebotomy for hepatic Hamp, Id1, Smad7, and Bmp6 mrna relative to Rpl19 mrna by quantitative real-time RT-PCR. Results are reported as the mean ± SD for the fold change from WT mice. Exact P values (or NS if not significant) are shown for the comparison with control WT mice. 8
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Supplementary Figure 3. There is a nonsignificant trend toward increased hepatic P-Smad1/5/8 in Hfe Tg mice versus WT mice. Eight-week-old Hfe Tg mice (N = 6) versus littermate WT mice (N = 6) were analyzed for hepatic P-Smad1/5/8 relative to total Smad1 and Actin by Western blot (A) followed by chemiluminescence quantitation (B-C). Results are reported as the mean ± SD. 10
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Supplementary Figure 4. Neutralizing anti-bmp6 antibody decreases hepatic Id1 mrna in Hfe Tg mice. Eight to ten-week-old Hfe Tg mice that received an intraperitoneal injection of BMP6 antibody (BMP6 Ab, N = 5) or vehicle alone (Mock, N = 5) once daily for ten days from Figure 2 were analyzed for hepatic Id1 relative to Rpl19 mrna by quantitative real-time RT-PCR. Results are reported as the mean ± SD. Exact P values are shown for the comparison of BMP6 Ab treatment versus mock treatment. 12
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Supplementary Figure 5. Dose and time curve of the effects of exogenous BMP6 administration on serum iron and transferrin saturation in mice. A) Eight to ten week-old male and female Hfe -/- mice and WT mice receiving a single intraperitoneal injection of BMP6 at 100 µg/kg or 250 µg/kg or vehicle alone (Control) as indicated (N = 6-10 per group, genders were matched between groups) from Figure 3 were analyzed for serum transferrin saturation (Tf Sat). BMP6 at 250 µg/kg caused only a nonsignificant trend toward reduced transferrin saturation in Hfe -/- mice. A higher dose of BMP6 at 500 µg/kg was therefore chosen for further experiments. B-C) Eight week-old WT mice on a 129S6/SvEvTac background received a single intraperitoneal injection of BMP6 at 500 µg/kg or an equal volume of vehicle alone (Control), and were sacrificed 6, 8, or 24 hours after injection (N = 6 per group). Serum was analyzed for iron (B) and transferrin saturation (C). Results are reported as the mean ± SD. Exact P values (or NS if not significant) are shown for the comparison between BMP6-treated versus Control mice. 14
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Supplementary Figure 6. Exogenous BMP6 administration in Hfe -/- mice causes a trend toward increased hepatic Id1 mrna but does not affect hepatic Crp mrna. Eight-to-ten week-old Hfe -/- mice treated with BMP6 at 500 µg/kg or vehicle alone (Mock) twice daily for 10 days from Figure 4 (N = 8 per group) were analyzed for A) hepatic Id1 relative to Rpl19 mrna and B) hepatic Crp relative to Rpl19 mrna by quantitative realtime RT-PCR. Results are reported as the mean ± SD. NS indicates P > 0.05 for BMP6 treated mice compared to vehicle treated mice. 16
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Supplementary Figure 7. Exogenous BMP6 does not affect liver, heart, or pancreas iron content in Hfe -/- mice. A) Eight-to-ten week-old Hfe -/- mice treated with BMP6 at 500 µg/kg or vehicle alone (Mock) twice daily for 10 days from Figure 4 (N = 8 per group) were analyzed for liver, heart, and pancreas iron content. Results are reported as the mean ± SD. All P values were non-significant for the comparison of BMP6 treated mice (black bars) with vehicle treated mice (grey bars). B) Perls Prussian blue staining of liver iron in Mock versus BMP6-treated Hfe -/- mice from panel A (original magnification x10). 18
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Supplementary Figure 8. Splenic Ferroportin protein expression is not affected by exogenous BMP6 treatment in Hfe -/- mice, and is not significantly changed in Hfe -/- mice compared with WT mice. A) Eight-to-ten week-old male Hfe -/- mice treated with BMP6 at 500 µg/kg or vehicle alone (Mock) twice daily for 10 days from Figure 4 were analyzed for splenic Ferroportin relative to Actin protein expression by Western blot followed by chemiluminescence quantitation. B) Eight-to-ten week-old male WT mice versus Hfe-/- mice on a C57BL/6 background were analyzed for splenic Ferroportin relative to Actin protein expression by Western blot followed by chemiluminescence quantitation. BMP6 treated Hfe-/- mice on a C57BL/6 background from panel A are also shown as a comparison. Results are reported as the mean ± SD. P values were nonsignificant for comparison between all groups. 20
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Supplementary Figure 9. Exogenous BMP6 does not affect liver Ferroportin expression in Hfe -/- mice. Eight-to-ten week-old Hfe -/- mice treated with BMP6 at 500 µg/kg or vehicle alone (Mock) twice daily for 10 days from Figure 4 were analyzed for liver Ferroportin relative to Actin protein expression by Western blot followed by chemiluminescence quantitation. Results are reported as the mean ± SD. The P value was non-significant for the comparison of BMP6 treated mice with vehicle treated mice. 22