von Hippel-Lindau mutation in mice recapitulates Chuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenic erythropoiesis

Abstract

The R200W mutation in the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) is unique in that it is not associated with tumor development, but rather with Chuvash polycythemia, a heritable disease characterized by elevated hematocrit and increased serum levels of erythropoietin and VEGF. Previous studies have implicated hypoxia-inducible factor-1alpha (HIF-1alpha) signaling in this disorder, although the effects of this mutation on pVHL function are not fully understood. In order to explore the mechanisms underlying the development of this polycythemia, we generated mice homozygous for the R200W mutation (Vhl(R/R)). Vhl(R/R) mice developed polycythemia highly similar to the human disease. The activity of HIF proteins, specifically the HIF-2alpha isoform, was upregulated in ES cells and tissues from Vhl(R/R) mice. Furthermore, we observed a striking phenotype in Vhl(R/R) spleens, with greater numbers of erythroid progenitors and megakaryocytes and increased erythroid differentiation of Vhl(R/R) splenic cells in vitro. These findings suggest that enhanced expression of key HIF-2alpha genes promotes splenic erythropoiesis, resulting in the development of polycythemia in Vhl(R/R) mice. This mouse model is a faithful recapitulation of this VHL-associated syndrome and represents a useful tool for studying polycythemias and investigating potential therapeutics.

Figure 1. Generation of Vhl^R/R ES cells.

(A) Schematic representation of human pVHL showing the location of the R200W mutation (asterisk). (B) Targeting construct used to introduce the R200W mutation in murine ES cells by homologous recombination. (C) Southern blot genotyping of ES cells using the probe indicated in B, showing heterozygous mutant Vhl^R/+ (R/+) and homozygous mutant Vhl^R/R (R/R) clones. (D) Northern blot analysis using the probe indicated in B, confirming that the Vhl transcript in Vhl^R/R cells was the same size as that in WT cells. (E) Western blot demonstrating that expression of pVHL in Vhl^R/R cells was decreased compared with WT cells. As quantitated below, mutant pVHL levels were 30%–40% those of WT. *ns, nonspecific band for loading control. (F) Expression of mutant pVHL was also significantly decreased in the kidney, liver, and spleen of Vhl^R/R mice compared with WT and Vhl^R/+ littermates.

Figure 2. HIF activity is increased in Vhl^R/R ES cells.

(A) Western blot analysis for HIF-1α and HIF-2α in WT or 2 Vhl^R/R clones (R/R¹ and R/R²) grown under either normoxia (N) or 1.5% hypoxia (H). HIF-1α protein was not detectably stabilized under normoxia; however, normoxic HIF-2α protein levels were greater in Vhl^R/R cells than in WT cells. (B) Stable clones of 786-O human renal carcinoma cells expressing murine WT Vhl or Vhl^R200W (R200W) were generated and screened by Western blot for equivalent expression of WT and R200W pVHL. These samples were run on the same gel but were noncontiguous, as indicated by black lines. Normoxic levels of HIF-2α were increased in R200W, but not WT, clones. (C) Secretion of VEGF by ES cells as measured by ELISA. Hypoxic induction of VEGF protein was maintained in Vhl^R/R cells, similar to control heterozygous (+/–) cells. However, normoxic VEGF protein levels were increased 2- to 3-fold in 3 independent Vhl^R/R clones compared with control heterozygous cells. **P < 0.0006. (D) WT or Vhl^R/R ES cells were grown in methylcellulose and allowed to differentiate into EBs for 9 days. The expression of Vegf and Pgk was analyzed by TaqMan real-time PCR. Vegf mRNA levels were increased 1.5- to 2-fold in Vhl^R/R EBs over WT, while Pgk was upregulated 1.3-fold. *P < 0.03.

Figure 3. Vhl^R/R mice develop polycythemia.

(A) The snout and paws of Vhl^R/R mice appeared redder in color than WT littermates. (B) There was no difference in the hematocrit levels based on genotype at 5–6 weeks; however, the hematocrit of Vhl^R/R mice became significantly greater than WT and Vhl^R/+ mice with increasing age (9% higher at 14–16 weeks and 11.8% higher at 26–29 weeks). *P < 0.0005. (C and D) Total red blood cell number (C) and hemoglobin concentration (D) were 14% and 22% greater, respectively, in Vhl^R/R mice than in WT mice. *P < 0.004; **P < 0.0007. (E) Total white blood cell number was also increased by 36% in Vhl^R/R mice. ^#P < 0.059 (trending toward significance). (F) The number of platelets was not significantly increased in Vhl^R/R mice. Results in C–F represent pooled data from mice aged 9–15 months.

Figure 4. HIF target gene expression is increased in Vhl^R/R mice.

(A and B) Serum levels of VEGF and Epo were determined by ELISA; pooled results from mice of varying ages are shown. (A) VEGF levels were 1.3-fold greater in Vhl^R/R mice than in WT and Vhl^R/+ mice. *P < 0.002. (B) Epo levels were 1.8-fold greater in Vhl^R/R mice (P < 0.08). (C–F) TaqMan real-time PCR analysis of HIF gene expression in kidney (C and D), liver (E), and spleen (F). The expression of several genes, most notably HIF-2α–preferential targets such as Glut-1, PAI-1, and VEGF, was increased in tissues isolated from Vhl^R/R mice compared with those of WT mice. *P < 0.05; **P < 0.007; ^#P < 0.059 (trending toward significance). Renal Epo mRNA expression was increased 1.3-fold (P < 0.09) in Vhl^R/R mice with increased serum Epo levels (D).

Figure 5. Bone marrow is not the source of the increased erythroid progenitors in Vhl^R/R mice.

(A) Flow cytometry demonstrated a 2-fold increase in the percentage of CD71⁺Ter119⁺ erythroid precursor cells in the blood of Vhl^R/R mice. *P < 0.03. (B) Representative flow cytometry plots for WT and Vhl^R/R blood stained for CD71 and Ter119. (C) The percentage of CD71⁺Ter119⁺ cells was not significantly different in Vhl^R/R bone marrow. (D) Representative flow cytometry plots for WT and Vhl^R/R bone marrow stained for CD71 and Ter119. (E) Representative sections of WT and Vhl^R/R bone marrow stained with H&E revealed no dramatic differences in bone marrow architecture. Original magnification, ×40. Scale bars: 20 μm.

Figure 6. Both erythroid progenitors and megakaryocytes are increased in Vhl^R/R spleens.

(A) Vhl^R/R spleens appeared slightly darker in color than WT and Vhl^R/+ littermates. Scale bar: 1 cm. (B) Representative sections of WT and Vhl^R/R spleens stained with H&E revealed a disruption of the normal splenic architecture in Vhl^R/R mice. Erythroid cells are indicated by white arrows, and megakaryocytes by black arrows. Original magnification, ×5 (top); ×40 (bottom). Scale bars: 200 μm; 20 μm. (C) Quantification of the increase in megakaryocytes in Vhl^R/R mice. *P < 0.006. (D) Representative flow cytometry plots of WT and Vhl^R/R spleens stained for CD71 and Ter119. (E and F) The percentage of CD71⁺Ter119⁺ erythroid progenitors was increased 6-fold (12%) in Vhl^R/R spleens (E), corresponding to a 9% (1.2-fold) decrease in CD19⁺ cells (F). *P < 0.05. (G) The percentage of CD41⁺ cells was also greater in Vhl^R/R spleens (2.5-fold). *P < 0.05.

Figure 7. Characterization of stress-induced erythropoiesis in Vhl^R/R mice.

(A) Hematocrit of Vhl^R/R mice, similar to that of WT mice, increased in response to PHZ (injected on days 1 and 2). However, the erythropoietic response was not downregulated appropriately in Vhl^R/R mice, and hematocrits continued to rise beyond WT levels to reach Vhl^R/R pre-PHZ levels. The result of 1 representative experiment is shown; similar results were obtained in separate experiments. (B) Spleen/body weight ratios in untreated mice and mice one month after PHZ injection; pooled results from multiple experiments are shown. Although the spleens of untreated Vhl^R/R mice were slightly larger (0.38% versus 0.3%), this difference was enhanced following PHZ, resulting in a greater increase in the size of mutant spleens compared with WT spleens.

Figure 8. Erythropoiesis is enhanced in Vhl^R/R spleens in vitro.

(A–C) Erythroid differentiation of Vhl^R/R bone marrow cells was not significantly different from WT (A). Similarly, there was only a small (but not statistically significant) increase in the number of nonerythroid colonies formed by Vhl^R/R bone marrow, most notably CFU-G (B) and CFU-M (C). (D–F) In contrast, Vhl^R/R splenic cells had 3.5-fold higher potential to generate erythroid colonies (D), as well as increased potential to form nonerythroid colonies such as CFU-G and CFU-GEMM (E) and CFU-M (F). *P < 0.02; **P < 0.003; ^#P < 0.059 (trending toward significance). (G and H) Epo dose-response curves of WT and Vhl^R/R cells showing the number of BFU-E colonies formed. Both bone marrow (G) and splenic (H) cells were hypersensitive to Epo.