Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins

Abstract

Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins, which tag hypoxia-inducible factor (HIF) alpha subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-alpha stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1(-/-) or Phd3(-/-) mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2alpha, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1alpha accumulated in both the kidney and liver. Elevated HIF-1alpha levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2alpha/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway.

Figure 1

Erythemic appearances in Phd1/3 DKO and Phd2 CKO mice. (A-D) Erythemic external appearance (reddish paw and snout, A and B, respectively), and enlarged liver (C) and spleen (D) in Phd1/3 DKO mice. WT, wild-type; DKO, Phd1/3 double knockout mice. (E-H) Phd2 CKO mice and Phd2^f/f control mice. Abdomens (E) and paws (F) at approximately 6 weeks after tamoxifen treatment of both CKO and Phd2^f/f mice. Note the necrotic intestine and red paw in Phd2 CKO mice. Left, Phd2^f/f; right, Phd2 CKO. (C) Spleens. Top, Phd2^f/f; bottom, Phd2 CKO. (D) Livers. Left, Phd2^f/f, right, Phd2 CKO. Images were taken with a CoolSNAP charge-coupled device digital camera (Photometrics-Roger Scientific, Tucson, AZ) attached to a Leica MZFLIII stereomicroscope (Leica Microsystems, Heerbrugg, Switzerland). A single objective lens (Leica Plan 1.0, 10× magnification, Leica Microsystems) was used to obtain the images. Openlab (Improvision, Lexington, MA) was used for initial image acquisition, and Photoshop 7.0 (Adobe Systems, San Jose, CA) was used for subsequent processing.

Figure 2

Hematologic parameters of peripheral blood from Phd1^−/−, Phd3^−/−, and Phd1/3 DKO mice. (A-E) Phd1/3 DKO and wild-type control mice. RBC indicates red blood cells; Hb, hemoglobin; Hct, hemtocrit; and WBC, white blood cells. n = 6. (E) ELISA for serum Epo levels. n = 4. **P < .01; *P < .05. (F-J) Normal blood properties in Phd1^−/− or Phd3^−/− mice. (A-I) Hematologic parameters of peripheral blood indicated in figures from wild-type (WT), Phd1^−/−, or Phd3^−/− mice. P > .05 for all pairs of comparisons. n = 6. (J) ELISA for serum Epo levels in WT, Phd1^−/−, or Phd3^−/− mice. n = 6. Error bars represent SEM.

Figure 3

Flow cytometry analyses of hematopoietic progenitors in Phd1/3 DKO mice. (A) Erythroid (Ter119⁺); (B) myeloid (CD11b⁺); (C) HSC (Lin⁻ Sca-1⁺ CD117⁺). Bone marrow (BM), spleen, and liver samples were analyzed. Total cell numbers in each organ are shown; n = 6 to 7. P values are indicated above the bars. Error bars represent SEM.

Figure 4

Abnormal hematologic parameters of peripheral blood from Phd2 CKO mice. (A-D), Phd2 CKO and Phd2^f/f control mice were all treated with tamoxifen at 6 weeks of age and analyzed after another 6 weeks. Red blood cells, hemoglobin concentrations, hematocrit values, and white blood cells were all significantly increased. n = 6. **, P < 0.01. (E) Time course of serum Epo level increases as measured by ELISA. n = 3 at each time point. (F) Time course of increases in hemoglobin concentration. n = 3 at each time point. Error bars represent SEM.

Figure 5

Analysis for hematopoietic progenitors and hematopoietic stem cells in Phd2 CKO mice. (A-C) Flow cytometry analyses. (A) Erythroid (Ter119⁺); (B) myeloid (CD11b⁺); (C) HSC (Lin⁻ Sca-1⁺ CD117⁺). Bone marrow (BM), spleen, and liver samples were analyzed. Total cell numbers in each organ are shown. n = 6 to 7. P values are indicated above the bars. (D) Methylcellulose colony formation assay. Genotypes are indicated at the bottom. f/f and CKO refer to Phd2^f/f and Phd2 CKO mice, respectively. Number of colonies per 10⁵ cells are presented, n = 6; P values are indicated in the figure. Error bars represent SEM.

Figure 6

Normal hematologic parameters in mice heterozygous for Phd2 knockout. (A-E) Phd2 conditional knockout mice and controls. (A) Hematocrit values. (B) Hemoglobin concentrations. (C) Number of red blood cells. (D) White blood cells. (E) Platelets. Two weeks before tamoxifen administration, baseline measurements were performed (Tam, −). Measurements were taken again 2 weeks after tamoxifen administration (Tam, +). CreERT2 status is indicated as Cre − or +, representing absence or presence, respectively. Phd2^f/− mice carrying CreERT2 and fed with tamoxifen are also designated as CKO, for conditional knockout. These are similar to Phd2 CKO mice used in other experiments except that one of the allele here was − before tamoxifen treatment; it was inherited as a germline mutation. For (A) to (E), n = 3 to 7; *P < .05. (F) Hematologic analysis of germline heterozygotes at more than 20 weeks of age. n = 5 to 6. Error bars represent SEM.

Figure 7

Molecular analysis for Phd2 CKO and Phd1/3 DKO mice. (A) Autoradiography of [³⁵S]methionine-labeled PHD1, 2 and 3 produced by IVTT. Free, unincorporated [³⁵S]methionine. (B) Western blotting analysis for PHD1, -2, and -3 in various tissues from wild-type adult mice. Equal amounts of IVTT-produced PHD1, -2, and -3 proteins were loaded as controls. Note PHD1 had 2 bands because of alternative translational initiation. (C) Anti-PHD2 Western blots for Phd1/3 DKO tissues. (D) Anti-HIF-1α and HIF-2α Western blots for Phd2 CKO and Phd1/3 DKO mice. (E,F) Q-PCR analysis for Epo mRNA. f/f, Phd2^f/f; CKO, Phd2 CKO; WT, wild-type; DKO, Phd1/3 DKO. n = 6. (F) The scale in panel E was reduced. **P < .01; *P < .05. Error bars represent SEM.