Translational repression of HIF2α expression in mice with Chuvash polycythemia reverses polycythemia

Abstract

Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1-knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.

Keywords: Drug therapy; Hematology; Pharmacology; Translation.

Figure 1. Vhl^R200W mice developed erythrocytosis/polycythemia at early ages.

(A) Hematocrit levels as determined by capillary tube centrifugation, (B) hemoglobin levels, and (C) RBC counts of Vhl^R200W mice (red circles) were significantly higher than those of their WT littermates (black circles) as early as 7 weeks of age. (A–C) P < 0.0001. To determine the P values, statistical analyses were performed using a 2-tailed paired t test. The mean ± SD is indicated in each panel.

Figure 2. Dietary Tempol supplementation prevented plethora, splenomegaly, and erythrocytosis/polycythemia in Vhl^R200W mice.

(A) The snouts and paws (indicated by white arrows) of Vhl^R200W mice appeared more reddish in color than did those of their WT littermates. The redness disappeared when the mutant mice were fed a Tempol-supplemented diet for 3 months. (B) Splenomegaly of Vhl^R200W mice was rescued by dietary Tempol supplementation, and the color of the spleens became lighter, suggesting that Tempol supplementation ameliorated the extramedullary erythropoiesis. (C and D) Hematocrit levels, as determined by capillary tube centrifugation, and (E) hemoglobin and (F) RBC levels of 5- to 8-month-old WT and Vhl^R200W mice fed a control or Tempol-supplemented diet showed that with the control diet, these parameters were elevated in Vhl^R200W mice, and Tempol supplementation significantly reduced hematocrit, hemoglobin, and RBC levels in the mutant mice. (D–F) ***P < 0.001, by ordinary 1-way ANOVA (multiple comparisons).

Figure 3. Decreased erythropoiesis in Tempol-treated Vhl^R200W mice was the result of EPO downregulation caused by translational repression of HIF2α, which was a consequence of the increased HIF2α IRE–binding activity of Irp1.

(A) Serum EPO levels measured by ELISA were significantly increased in control diet–fed Vhl^R200W mice, and Tempol treatment reduced EPO levels in these mutant mice. (B) Immunoblot analyses of nuclear fractions from kidney lysates showed increased HIF2α protein levels in Vhl^R200W mice fed a control diet, and the HIF2α protein levels decreased when these mutant mice were fed a Tempol-supplemented diet. (C) Quantification of these HIF2α protein levels. (D) RNA mobility shift assays for ferritin IRE and HIF2α IRE binding of renal lysates in the absence or presence of β mercaptoethanol (ME) (which converts Irp1 from cytosolic aconitase to the IRE-binding form) showed that most of the IRE-binding activity was contributed by Irp1, and Tempol treatment significantly increased the IRE binding of Irp1 in Vhl^R200W mice. Quantifications of the Irp1-binding activities of (E) ferritin IRE and (F) HIF2α IRE. (G) Western blot analyses of kidney lysates revealed that Irp1 protein levels remained unchanged in Tempol diet–fed Vhl^R200W mice. Data in A, C, E, and F represent the mean ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001, by unpaired, 2-tailed t test.

Figure 4. Genetic ablation of Irp1 significantly increased polycythemia inVhl^R200W mice, which became refractory to Tempol, confirming that Tempol activates Irp1 to ameliorate the polycythemia in Vhl^R200W mice, and Tempol also partially protects WT mice from hypoxic polycythemia/erythrocytosis.

(A) Hematocrit (determined by capillary tube centrifugation), (B) hemoglobin, and (C) RBC levels of 5- to 8-month-old WT, Irp1^–/–, and Vhl^R200W Irp1^–/– mice fed a control or Tempol-supplemented diet revealed that Tempol did not decrease erythropoiesis when Irp1 was ablated in the mice. (D) Serum EPO levels of control diet–fed Vhl^R200W Irp1^–/– mice were 3.3-fold higher than were those of WT mice (data for WT mice are the same as those in Figure 3A). (E) Hematocrit, (F) hemoglobin, and (G) RBC levels of 10-month-old mice on a control diet or Tempol diet after placing them in normoxic or hypoxic (10% oxygen) conditions showed that blood parameters significantly increased in the hypoxic control diet–fed mice and that Tempol supplementation partially prevented erythrocytosis/polycythemia in these mice. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001, by 1-way ANOVA.

Figure 5. The reduced life expectancy of Vhl^R200W mice was alleviated by Tempol supplementation when Irp1 was present.

The survival curves of WT, Vhl^R200W, and Vhl^R200W Irp1^–/– mice on a control or Tempol-supplemented diet showed that Vhl^R200W mice on a control diet died at significantly higher survival rates than did WT mice on a control diet, whereas no significant difference was observed in the survival rates between Tempol-supplemented Vhl^R200W and WT mice (on either a control or Tempol-supplemented diet). Moreover, the beneficial effect of Tempol was lost when Irp1 was absent in these Vhl^R200W mice. n = 180 WT mice on a control diet; n = 34 WT mice on a Tempol diet; n = 76 Vhl^R200W mice on a control diet; n = 43 Vhl^R200W mice on a Tempol diet; n = 35 Vhl^R200W Irp1^–/– mice on a control diet; and n = 18 Vhl^R200W Irp1^–/– mice on a Tempol diet . Statistical analyses of mouse survival curves were performed with a log-rank Mantel-Cox test using GraphPad Prism software.

Figure 6. Model for the mechanism of ameliorative action of Tempol in Vhl^R200W mice.

The expression of HIF2α protein is regulated at multiple levels. The Vhl protein promotes the degradation of HIF2α under normoxic conditions. However, Vhl^R200W does not promote HIF2α degradation, which leads to high levels of HIF2α and augmented levels of translational targets, including EPO. HIF2α is also translationally regulated by the Irp IRE regulatory system. When Tempol increases the IRE-binding activity of Irp1, HIF2α expression diminishes, which leads to diminished erythropoietin expression and restoration of normal hematocrit levels.