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Endothelial cells produce bone morphogenetic protein 6 required for iron homeostasis in mice

Susanna Canali, Kimberly B. Zumbrennen-Bullough, Amanda B. Core, Chia-Yu Wang, Manfred Nairz, Richard Bouley, Filip K. Swirski and Jodie L. Babitt

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Generation of Bmp6 floxed (Bmp6fl/fl) and Bmp6−/−global knockout mice. (A) Schematic representation of Bmp6 gene structure and the floxed Bmp6 allele. Cre-mediated excision of exons 5 to 7 encoding the mature Bmp6 protein generates a functionally null allele. Genotyping primer positions are indicated. (B) PCR of tail genomic DNA from Bmp6 floxed and global knockout mouse colonies shows the presence of wild-type (WT) (+), floxed (fl), and cre-excised (–) alleles. (C) qRT-PCR of liver Bmp6 relative to Rpl19 mRNA in the Bmp6+/+, Bmp6+/−, and Bmp6−/− mice (n = 7 to 11 males and 5 to 7 females per group; supplemental Table 2). Results are reported as mean ± standard error of the mean (SEM) of –ΔCt = –[Ct target mRNA – Ct Rpl19], with a higher –ΔCt indicating higher mRNA expression. Fold change in mRNA expression between groups is calculated by 2–ΔΔCt, where ΔΔCt = ΔCt higher expressing group – ΔCt lower expressing group. *P < .05 relative to Bmp6+/+ mice by Student t test. nd, not detectable.

  • Figure 2.

    Validation of conditional Bmp6 knockout mice. (A-C) Immunofluorescence microscopy of liver from offspring of Rosa26tdT Cre-reporter mice mated with (A) Alb-Cre, (B) LysM-Cre, and (C) Tek-Cre mice. Cells expressing Cre recombinase are labeled red throughout the cytoplasm because of the removal of a stop codon upstream of the tdT fluorescent protein. Other NPC populations are labeled green by using CD31 (ECs), desmin (HSCs), and F4/80 (KCs) antibodies. Nuclei are labeled blue with 4′,6-diamidino-2-phenylindole (DAPI). Dotted areas are shown with increased brightness below the right panel. Scale bars represent 10 μM. One representative mouse of 4 to 7 per group is shown. (D-F,J-L) qRT-PCR of (D) the EC marker Cd146, (E) the leukocyte/KC marker Cd45, (F) the hepatocyte (Hepa) marker Tmprss6, and (J-L) Bmp6 relative to Rpl19 mRNA from isolated hepatocytes, NPCs, ECs, or KCs from Bmp6 conditional knockout and littermate control Bmp6fl/fl;Cre– mice (n = 3-5 per group). Results are reported as mean ± SEM of –ΔCt as in Figure 1. (G-I) PCR of genomic DNA for floxed and cre-excised Bmp6 alleles from isolated hepatocytes, NPCs, ECs, or KCs from Bmp6 conditional knockout and littermate control Bmp6fl/fl;Cre– mice (n = 3-5 per group). Representative gels are shown. ***P < .001 for NPCs relative to hepatocytes by Student t test or for ECs relative to hepatocytes or KCs by one-way ANOVA with Tukey post hoc test for mice of the same genotype. ++P < .01 in Bmp6fl/fl;Cre+ relative to Bmp6fl/fl;Cre– mice for the same cell type by Student t test. nd, not detectable; ns, not significant.

  • Figure 3.

    Eight-week-old Bmp6fl/fl;Tek-Cre+ mice exhibit liver Bmp6 and Hamp deficiency and serum iron overload, whereas Bmp6fl/fl;Alb-Cre+ and Bmp6fl/fl;LysM-Cre+ mice do not. Eight-week-old littermate male (left) and female (right) Bmp6+/+, Bmp6+/−, and Bmp6−/− global knockout mice (red bars) and Bmp6 conditional knockout mice in ECs (Bmp6fl/fl;Tek-Cre+ [blue bars]), macrophages (Bmp6fl/fl;LysM-Cre+ [green bars]), and hepatocytes (Bmp6fl/fl;Alb-Cre+ [purple bars]) compared with littermate controls (Bmp6fl/fl;Cre–) were analyzed for (A) total liver hepcidin (Hamp) and (D) Bmp6 relative to Rpl19 mRNA by qRT-PCR, (B) serum iron, and (C) transferrin (Tf) saturation. n = 4-11 mice per sex per group (supplemental Table 2). Results are reported as mean ± SEM of –ΔCt as in Figure 1. Bmp6 mRNA levels for Bmp6+/+ and Bmp6−/− mice, stratified by sex, were replotted from Figure 1 for comparison. *P < .05, **P < .01, ***P < .001 relative to control Bmp6+/+ mice or Bmp6fl/fl;Cre– mice by one-way ANOVA with Dunnett’s post hoc test or Student t test. nd, not detectable.

  • Figure 4.

    Liver iron is increased and spleen iron is reduced in Bmp6fl/fl;Tek-Cre+ mice, but not Bmp6fl/fl;Alb-Cre+ or Bmp6fl/fl;LysM-Cre+ mice. Eight- and 16-week-old littermate male and female Bmp6+/+, Bmp6+/−, and Bmp6−/− global knockout mice and Bmp6 conditional knockout mice in ECs (Bmp6fl/fl;Tek-Cre+), macrophages (Bmp6fl/fl;LysM-Cre+), and hepatocytes (Bmp6fl/fl;Alb-Cre+) compared with littermate controls (Bmp6fl/fl;Cre–) were analyzed for tissue iron in (A,C) liver and (B,C) spleen by (A-B) biochemical analysis or (C) Perls’ Prussian blue. n = 4-7 mice per group (supplemental Table 2), with tissues from 1 representative mouse shown in (C) (original magnification ×20; scale bar represents 100 μM). *P < .05, **P < .01, ***P < .001 relative to control Bmp6+/+ mice by one-way ANOVA with Dunnett’s post hoc test; +P < .05, ++P < .01, +++P < .001 relative to control Bmp6fl/fl;Cre– mice by Student t test.

  • Figure 5.

    Bmp6fl/fl;Tek-Cre+ mice exhibit extrahepatic iron loading, whereas Bmp6fl/fl;Alb-Cre+ and Bmp6fl/fl;LysM-Cre+ mice do not. Eight- and 16-week-old littermate male and female Bmp6+/+, Bmp6+/−, and Bmp6−/− global knockout mice and Bmp6 conditional knockout mice in ECs (Bmp6fl/fl;Tek-Cre+), macrophages (Bmp6fl/fl;LysM-Cre+), and hepatocytes (Bmp6fl/fl;Alb-Cre+) compared with littermate controls (Bmp6fl/fl;Cre–) from Figure 4 were analyzed for tissue iron in (A,C) pancreas and (B,C) heart by (A-B) biochemical analysis or (C) Perls’ Prussian blue. Diaminobenzidine (DAB)-enhanced Perls’ stain was performed on sections that appeared negative or faint by standard Perls’ Prussian blue. n = 4 to 7 mice per group (supplemental Table 2), with tissues from 1 representative mouse shown in (C) (original magnification ×20; scale bar represents 100 μM; boxed areas shown enlarged ×5 for pancreas and ×3 for heart in insets for some panels). **P < .01, ***P < .001 relative to control Bmp6+/+ mice by one-way ANOVA with Dunnett’s post hoc test; +P < .05, ++P < .01 relative to control Bmp6fl/fl;Cre– mice by Student t test.

  • Figure 6.

    Hjv is expressed in hepatocyte sinusoidal membranes adjacent to Bmp6-producing SECs. (A) Immunofluorescence microscopy of liver tissue from Rosa26tdT;Tek-Cre+ mice. Liver SECs are labeled with tdT (red). Hjv expression is visualized with anti-Hjv antibody (green). Nuclei are labeled with DAPI (blue). Scale bar represents 10 μM. Right panel shows boxed area enlarged ×3. (B) Proposed model: BMP6 is produced in liver SECs and acts in a paracrine fashion on hepatocytes to bind the co-receptor HJV, which together with BMP type I and type II receptors induces SMAD1/5/8 phosphorylation and hepcidin transcription.