245 HIF-1, hypoxia inducible factor-1 as a therapeutic target

Abstract

This chapter focuses on oxygen-dependent regulation of erythropoiesis. Hypoxia inducible factor 1, subunit α (HIF-1α) is a central molecule in oxygen sensing and the response to hypoxia. HIF-1α is unstable in normoxic conditions: specific proline hydroxylases convert its proline residues 402 and 564 to hydroxyprolines, the hydroxylated HIF-1α is captured by the von Hippel Lindau protein (pVHL), ubiqutinated, and degraded in proteasomes. This chapter describes the methods for the quantitative detection of mRNA for hypoxia-regulated genes in the blood cells and for assessing the response of red blood cell progenitors to erythropoietin—a hormone playing an essential role in the adaptation to hypoxia. Real data of studies of the congenital defect of the germ-line mutation of VHL gene leading to the constitutive upregulation of hypoxia sensing, Chuvash polycythemia, are used as an example. Concepts related to the separation of plasma, platelets, mononuclear cells, and granulocytes from human peripheral blood, the preparation of EBV-transformed B lymphoblastoid cell lines (B-LCL), quantification of hypoxia-responsive gene expression by real-time RT-PCR is discussed along with the response of erythroid progenitors to erythropoietin in vitro.