Integrity of the prolyl hydroxylase domain protein 2:erythropoietin pathway in aging mice

Abstract

The central transcriptional response to hypoxia is mediated by the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. In this pathway, PHD prolyl hydroxylates and thereby negatively regulates the alpha-subunit of the transcription factor HIF (HIF-alpha). An important HIF target gene is that for erythropoietin (EPO), which controls red cell mass. Recent studies have identified PHD2 as the critical PHD isoform regulating the EPO gene. Other studies have shown that the inducibility of the HIF pathway diminishes as a function of age. Thus, an important question is whether the PHD2:EPO pathway is altered in the aging. Here, we employed a mouse line with a globally-inducible Phd2 conditional knockout allele to examine the integrity of the Phd2:Epo axis in young (six to eight months old) and aging (sixteen to twenty months old) mice. We find that acute global deletion of Phd2 results in a robust erythrocytosis in both young and aging mice, with both age groups showing marked extramedullary hematopoiesis in the spleen. Epo mRNA is dramatically upregulated in the kidney, but not in the liver, in both age groups. Conversely, other Hif targets, including Vegf, Pgk1, and Phd3 are upregulated in the liver but not in the kidney in both age groups. These findings have implications for targeting this pathway in the aging.

Fig. 1

Analysis of peripheral blood in young and aging mice. (A, E) hematocrit (Hct), (B, F) hemoglobin (Hb), (C, G) white blood cell count (WBC), and (D, H) Platelets were measured. For all panels, n = 6–8. For (A–D), genotypes are as follows. f/+ = Phd2 f/+; f/− = Phd2 f/−; Rosa26-CreER^T2. * indicates p < 0.05 and ** indicates p < 0.01 in comparing f/+ and f/− groups at a given age. For (E–H), genotypes are as follows. Controls (Con): Phd2 f/+; CKO: Phd2 f/−; Rosa26-CreER^T2. Control and Phd2 CKO mice were administered tamoxifen for five consecutive days. Four weeks after the initial tamoxifen dose, peripheral blood was collected. * indicates p < 0.05 and ** indicates p < 0.01 in comparing control and CKO groups at a given age.

Fig. 2

Phd2 exon 2 deletion efficiency in mice after tamoxifen induction. Both young and aging mice were treated with tamoxifen for five consecutive days. Four weeks after the initial tamoxifen dose, mice were sacrificed. RNA and DNA were then isolated from select mouse tissues. (A and B) Phd2 mRNA levels in liver and kidney were measured by RT-PCR in (A) young and (B) aging mice. n = 4. * indicates p < 0.05 in comparing control (Con, Phd2 f/+) and CKO (Phd2 f/−; Rosa26-CreER^T2) groups at a given age. (C) Phd2 exon 2 recombination efficiency was surveyed in Phd2 CKO mice in both the young and aging groups. A PCR reaction employing three primers was performed on genomic DNA obtained from the indicated tissues. The floxed Phd2 allele without recombination produces a 0.95 kb band, while the knockout Phd2 allele produces a 0.8 kb band. In each panel, the first lane shows results obtained with a control DNA from a Phd2 f/− mouse.

Fig. 3

Extramedullary hematopoiesis in Phd2 CKO mice. Both young and aging mice were treated with tamoxifen for five consecutive days. Four weeks after the initial tamoxifen dose, mice were sacrificed. Genotypes are as follows. Controls: Phd2 f/+; CKO: Phd2 f/−; Rosa26-CreER^T2. (A) Liver and (B) spleen weights were measured. For (A) and (B), n=6–8. ** indicates p < 0.01. (C–F) Photomicrographs of hematoxylin and eosin stained sections of spleen from young (C,D) and aging (E,F) mice. (Leica DM2500 microscope equipped with Leica FireCam digital capture software, magnification 100×). Bars in (C) and (D) indicate 100 µm. Megakaryocytes are present in the spleens of the Phd2 CKO mice (indicated by arrows). Examination of ten high power fields from two mice in each age group reveals a higher number of megakaryocytes in the spleens of young Phd2 CKO mice (6.3 ± 2.0) as compared to aging Phd2 CKO mice (3.5 ± 1.5; p < 0.01).

Fig. 4

Erythroid burst forming unit assays. (A) Young and aging mice were treated with tamoxifen for five consecutive days. Four weeks after the initial tamoxifen dose, cells were isolated from the spleen and BFU-E assays were performed in the presence of 3 U/ml EPO. Genotypes are as follows. Controls: Phd2 f/+; CKO: Phd2 f/−; Rosa26-CreER^T2. (B and C) Two month old mice were treated with tamoxifen for five consecutive days. Three weeks after the initial treatment, cells were harvested from the (B) spleen and (C) bone marrow and BFU-E assays were performed in the absence or presence of the indicated EPO concentrations.

Fig. 5

Aging mice were treated with tamoxifen for five consecutive days. Three weeks after the initial treatment, RNA was extracted from bone marrow. The mRNA levels of select genes were determined by Real Time PCR and normalized to that of β-actin. Abbreviations are as follows. Controls: Phd2 f/+ mice; CKO: Phd2 f/−; Rosa26-CreER^T2. For both groups, n = 3. ** indicates p < 0.01 in comparing control and CKO groups.

Fig. 6

Upregulation of Hif target genes in Phd2 CKO mice. Both young and aging mice were treated with tamoxifen for five consecutive days. Four weeks after the initial dose, the mice were sacrificed. (A) Plasma Epo levels were measured by ELISA. Epo levels in young and aging CKO mice were compared by unpaired Student’s t test and the difference was not found to be statistically significant (p > 0.05). (B to G) RNA was extracted from kidney and liver. The mRNA levels of select genes were determined by Real Time PCR and normalized to that of β-actin. Abbreviations are as follows. Controls: Phd2 f/+ mice; CKO: Phd2 f/−; Rosa26-CreER^T2; L: Liver; K: Kidney. For (A) n = 6–9; for (B to G) n = 4. * indicates p < 0.05 and ** indicates p < 0.01 in comparing control and CKO groups.