Figure 1.
Fpn1Tek/Tek mice develop anemia and have iron loading in their hepatocytes, macrophages, and duodenal enterocytes.
(A) Fpn1 mRNA levels in the liver, spleen and duodenum. (B) Liver mRNA levels of genes implicated in Hamp1 regulation (n = 6–7 per group). (C) DAB iron staining in the liver (left panels; brown staining indicates iron), Perls' Prussian Blue iron staining in the liver, spleen, and duodenum (right panels; brown staining indicates iron). Iron accumulated in hepatocytes (White arrowheads), liver kupffer cells and spleen macrophages (Black arrowheads), and duodenum enterocytes (Yellow arrowheads). (D) Wright-Giemsa-stained peripheral blood smears. (E) Photographs of 13–15-day-old male Fpn1flox/flox and Fpn1Tek/Tek mice. DAB iron staining was used to detect tissue iron levels when Prussian Blue staining was not sufficiently sensitive. Images were captured on an Olympus BX61 microscope with a UPlanApo 20×/0.70 or 40×/0.85 objective, Q Imaging QICAM camera and Q Capture 2.90.1 Quantitative Imaging software. Summary data are presented as mean ± SD. *P<0.05; **P<0.01.
Figure 2.
Fpn1Tek/Tek mice have liver iron loading but decreased Bmp6 and Hamp1 expression.
(A) Liver and spleen non-heme iron concentrations. (B) Serum iron concentration (SI, µg/dL) and percent Tf saturation (TS%). (C–E) Liver mRNA levels of Hamp1 (C), Bmp6 (D) and Epo (E). (F) Liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels were measured in 13–15-day-old male Fpn1flox/flox and Fpn1Tek/Tek mice (n = 6–7 per group), (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. *P<0.05; **P<0.01.
Figure 3.
Bmp6 and Hamp1 expression decreases in young Fpn1Alb/Alb;LysM/LysM mice despite liver iron loading.
(A) Liver and spleen non-heme iron concentrations. (B) SI and TS% levels. (C–E) Liver mRNA levels of Hamp1 (C), Bmp6 (D) and Epo (E). (F) Liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels were measured in 3-week-old male Fpn1flox/flox and Fpn1Alb/Alb;LysM/LysM mice (n = 5 per group). (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. *P<0.05; **P<0.01.
Figure 4.
Bmp6 and Hamp1 expression decreases in the livers of Fpn1Alb/Alb mice with high iron demand.
(A) Liver and spleen non-heme iron concentrations. (B) SI and TS%. (C–E) Liver mRNA levels of Hamp1 (C), Bmp6 (D), Epo (E). and (F) Liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels were measured in 3-week-old male Fpn1flox/flox and Fpn1Alb/Alb mice fed an iron-rich diet for one week, and then transferred to an iron-deficient diet for one month (n = 5 per group). (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. *P<0.05; **P<0.01.
Figure 5.
Changes in serum Tf-bound iron levels are consistent with p-smad1/5/8 and Hamp1 levels in Fpn1Alb/Alb;LysM/LysM mice given an iron-deficient diet.
(A) Time course of liver and spleen non-heme iron concentrations after switching to an iron-deficient diet. (B) Time course of SI and TS% levels. (C–E) Time course of liver mRNA levels of Hamp1 (C), Bmp6 (D), Epo (E). (F) Time course of liver p-Smad1/5/8, Smad1, p-Erk1/2, Erk1/2, p-Stat3, Stat3 and β-actin protein levels measured in 2-month-old male Fpn1Alb/Alb;LysM/LysM mice fed an iron-deficient diet for 0, 2, 4 or 8 days (n = 5 mice per time point). (G) Summary of the results in (F), quantitated using densitometry. Summary data are presented as mean ± SD. *P<0.05; **P<0.01.