An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the ␣ subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm Ϫ/Ϫ mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1␣ and Hif-2␣ protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm Ϫ/Ϫ mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2 gt/gt ) and Hif-p4h-3 Ϫ/Ϫ mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm Ϫ/Ϫ and wild-type mice, but caused higher increases in both values in the Hif-p4h-2 gt/gt mice and in hematocrit value in the Hif-p4h-3 Ϫ/Ϫ mice than in the wild-type. Hif-p4h-2 gt/gt /P4h-tm Ϫ/Ϫ double genemodified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2 gt/gt or P4h-tm Ϫ/Ϫ mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis. (Blood. 2012;120(16): 3336-3344)
IntroductionErythropoiesis is a tightly controlled process, its key regulator being erythropoietin (EPO). During embryonic development most of the EPO production occurs in the liver, whereas the major EPO source in adults is the kidney, although the liver maintains a capacity for its expression. 1,2 Hypoxia-inducible transcription factor (HIF) plays a pivotal role in the regulation of the transcription of the EPO gene and numerous other hypoxia-regulated genes, including many additional genes influencing erythropoiesis. [1][2][3][4] The HIF-␣ subunit isoforms HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of 2 critical prolines generates 4-hydroxyproline residues that target HIF-␣ for rapid degradation in normoxia. [5][6][7] In hypoxia, this hydroxylation is inhibited, so that HIF-␣ escapes degradation, translocates into the nucleus, and dimerizes with HIF-. 5-7 HIF-1␣ is expressed in all nucleated cells, whereas HIF-2␣ expression is restricted to specific cell types, including renal interstitial cells and hepatocytes. 1,2 Renal and hepatic EPO production in adults is primarily regulated by HIF-2␣, but HIF-1␣ also plays a role in several situations. 1,2 Erythropoiesis requires iron. Hepcidin, a 25-amino-acid peptide secreted predominantly from hepatocytes, is the central regulator of iron metabolism. It down-regulates ferroportin and thus inhibits the absorption of dietary iron and the release of iron form erythrocytes and macrophages. 8,9 Hepcidin expression is lowered by hypoxia and agents that stabilize HIF-2␣ leading to increased serum EPO concentration and erythropoiesis, its regulation also involving the hemojuvelin/...
