Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice

Abstract

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Figure 1

Tf treatment of wild-type and hpx mice. Eight-week-old wild-type (wt) and hpx mice were injected intraperitoneally with 10 mg human Tf (TF; 0.3 mL total volume) every other day for 3 weeks. Reticulocyte counts (A), spleen masses (as percent of total body mass; B), hemoglobin levels (C), liver hepcidin levels relative to β-actin levels as measured by quantitative polymerase chain reaction (QPCR; D), total spleen iron (Fe) levels (E), and liver Fe concentrations (F) were determined from animals harvested at 0, 1, 2, and 3 weeks. Total spleen Fe levels were determined by multiplying spleen mass by spleen iron concentration. Each data point represents 3 mice. A ‘#’ above a data point indicates that the values for wild-type and hpx mice differ significantly for that time point (t test, P < .05); a ‘*’ above a data point indicates that the value at that time point differs significantly from the value at day 0 for that genotype or treatment group. Bars indicate 1 SD.

Figure 2

Doxorubicin and carboplatin treatment of wild-type mice. Eight-week-old C57BL6/J, 129/SvEvTac, and BALBc/J mice were treated with or without 0.25 mg doxorubicin or 2.5 mg carboplatin and then harvested 3 days later. Mice were analyzed for reticulocyte counts (A), serum iron (Fe) concentrations (B), Tf saturations (C), spleen masses (D), hemoglobin levels (E), and liver hepcidin levels relative to β-actin levels as measured by QPCR (F). Each value represents 5 mice. A ‘*’ above a bar indicates that the value differs significantly relative to untreated mice of the same strain (t test, P < .05).

Figure 3

Doxorubicin treatment of wild-type and hpx mice. Eight-week-old wild-type (wt) and hpx mice were injected intraperitoneally with 0.25 mg doxorubicin and harvested and analyzed at 0, 3, 6 and 8 days for reticulocyte counts (A), spleen masses (B), hemoglobin levels (C), serum iron (Fe) concentrations (D), total iron-binding capacities (E), and liver hepcidin levels relative to β-actin levels as measured by QPCR (F). Each point represents 4 to 6 mice. The symbols ‘#’ and ‘*’ indicate statistical significance as in Figure 1.

Figure 4

Further analysis of the effect of chemotherapeutic agents on hpx mice. (A-B) Splenic Gdf15 (A) and Twsg1 (B) levels were measured by QPCR and expressed as a ratio to β-actin levels in wild-type (wt) mice, untreated hpx mice (–), or hpx mice treated with 1.5 mg human Tf (TF) or 0.5 mL wild-type mouse serum (serum) every other day for 3 weeks or 0.25 mg doxorubicin (dox) for 6 days. Each bar represents 3 to 6 mice. Black and gray asterisks indicate statistical significance between the labeled value and wild-type or hpx mouse value, respectively. (C) Hepatic Bmp6 levels relative to β-actin levels were measured by QPCR in samples from doxorubicin-treated wild-type and hpx mice from Figure 3. The symbols ‘#’ and ‘*’ indicate statistical significance as in Figure 1. (D) Hepatic Bmp6 levels relative to β-actin levels were measured by QPCR in samples from wild-type mice treated with doxorubicin or carboplatin from Figure 2. A ‘*’ indicates statistical significance relative to untreated mice as in Figure 2.

Figure 5

Transfusion of hpx mice with wild-type RBCs. Hpx mice were transfused with 0.5 mL PBS or washed wild-type RBCs every other day for 1 week then harvested on day 7 and analyzed for hemoglobin levels (A), reticulocyte counts (B), spleen masses (C), splenic Gdf15 levels (D), and hepatic Bmp6 (E) and hepcidin (F) levels. Gene expression levels were measured by QPCR and expressed as a ratio to β-actin levels. A ‘*’ indicates statistical significance (t test, P < .05) relative to values for PBS-transfused hpx mice.

Figure 6

Doxorubicin and Tf treatment of hpx mice. Eight-week-old hpx mice were treated with or without 0.25 mg doxorubicin (+dox and -dox) on day 0; mice were then treated with or without 10 mg human Tf (TF) or 2.5 mg mouse Tf (Tf) on day 3 and 5. Mice were then harvested on day 6 and analyzed for reticulocyte counts (A), spleen masses (B), hemoglobin levels (C), serum iron (Fe) levels (D), Tf saturations (E), and hepcidin levels relative to β-actin levels by QPCR (F). Untreated wild-type (wt) mice, indicated in parentheses, were included for reference. Each bar represents 4 to 5 mice. Numbers indicate outcome of t test between bracketed values.

Figure 7

Model of Tf-dependent regulation of hepcidin expression. Tf-dependent regulation of hepcidin expression occurs via two axes in hpx mice. In the erythroid-regulator axis, Tf delivers iron to the erythron, leading to effective erythropoiesis; with effective erythropoiesis comes decreased activity of the erythroid-regulator and decreased inhibition of hepcidin expression. In the stores-regulator axis, Tf leads to stimulation of hepcidin expression in a process requiring hemochromatosis genes such as Bmp6, Hfe, Hjv and Tfr2. Treatment of hpx mice with exogenous Tf leads to effective erythropoiesis and decreased erythroid-regulator activity via the erythroid-regulator axis and stimulation of hepcidin expression via the stores-regulator axis. Treatment of hpx mice with wild-type RBCs leads to decreased erythroid-regulator activity without the need for endogenous erythropoiesis. Treatment of hpx mice with doxorubicin then Tf leads to stimulation of hepcidin expression via the stores-regulator axis.