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 α-subunit of the transcription factor HIF (HIF-α). 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 month old) and aging (sixteen to twenty month 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.

The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α

“…11C and D). In light of prior work (16,29) and to address the possibility that Egln1 ZF/ZF mice have features of primary erythrocytosis, we examined the proliferation of bone marrow cells in response to increasing levels of EPO by BFU-E assay. We found, indeed, that larger numbers of BFU-E were observed from the bone marrow of Egln1 ZF/ZF than from Egln1 ϩ/ϩ mice at two different EPO concentrations (Fig.…”

“…Primer sequences for Epo, Egln3, Vegfa, and Pgk1 have also been described previously (29). For Cybrd1 (Dcytb), the following primers were used: SG-mDcytB 5=, GCA GCG GGC TCG AGT TTA, and SG-mDcytB 3=, TTC CAG GTC CAT GGC AGT CT. For Hamp1 (Hepc), the following primers were used: SG-mHEPC 5=, TGT CTC CTG CTT CTC CTC CT, and SG-mHEPC 3=, CTC TGT AGT CTC TCT CAT CTG TTG.…”

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism

“…Tamoxifen Administration-A solution of tamoxifen (MP Biomedicals) in corn oil at a final concentration of 10 mg/ml was prepared as described (32). At 6 -10 weeks of age, experimental and control mice received a 2-mg dose daily for 5 days by oral gavage.…”

“…Mice with a tamoxifen-inducible Cre recombinase (fused to the estrogen receptor) and floxed Phd2 alleles were obtained by crossing a mouse line with a floxed Phd2 exon 2 allele (17,19) with C57BL/6-Gt(ROSA)26Sor tm9(cre/Esr1)Arte mice (Taconic) and have been described (32). These mice, hereafter denoted Rosa26-CreER; Phd2 f/f , were maintained in a mixed background, as were mice that were derived from them by crossing with the Hif1a f/f or Hif2a f/f mice.…”

Mouse Knock-out of IOP1 Protein Reveals Its Essential Role in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis

“…Dissociation curve analysis revealed a single peak. The sequences of other real time PCR primers has been described (29). Relative quantification was performed employing the ⌬⌬Ct method and ␤-actin as the endogenous control.…”