Control of Erythropoiesis by the Oxygen Sensor PHD2

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PROJECT SUMMARY/ABSTRACT The central oxygen sensor that controls red cell mass is PHD2 (also known as EGLN1). In specialized interstitial cells of the renal cortex, PHD2 prolyl hydroxylates the transcription factor HIF-2a in an oxygen-dependent manner and targets it for degradation. Under hypoxic conditions, prolyl hydroxylation is arrested, leading to the stabilization of HIF-2a and the activation of the ERYTHROPOIETIN gene, leading to expansion of red cell mass. We are still at an early stage of understanding how PHD2 works. PHD2 has two domains, a catalytic domain and a zinc finger domain. The former catalyzes prolyl hydroxylation of HIF- 2a. The function of the latter has been elusive. A critical role for the latter is evidenced by patients with erythrocytosis who harbor loss of function mutations in the zinc finger of PHD2, and by mice with inactivating mutations of the zinc finger that display erythrocytosis. Previous in vitro studies have shown the zinc finger binds to a Pro-Xaa-Leu-Glu (PXLE) motif that is found in components of the HSP90 pathway, including p23, FKBP38, HSP90a, and HSP90a, as well as in the ribosomal chaperone NACA. The HIF pathway is present in all metazoans, and this PXLE motif shows strong conservation in these proteins across metazoan species. This leads to a model in which this motif recruits PHD2 to the translation and HSP90 pathways to facilitate hydroxylation of HIF-2a, which is a known client of the protein folding HSP90 pathway. In this manner, PHD2 can maintain tight control over HIF-2a levels. The aforementioned observations on erythrocytosis identify a critical role for the zinc finger. However, they do not identify which of these PXLE-containing proteins, if any, are essential for normal regulation of red cell mass by PHD2. To address this, we have recently generated mice with knockin missense mutations that ablate the Pro-Xaa-Leu-Glu motif in the p23, Fkbp38, Hsp90a, Hsp90b, and Naca genes to prevent the interaction of the respective proteins with PHD2. We will examine the individual knockin mice as well as combinations of knockins for hemoglobin concentration, hematocrit, red blood cell count, and serum Epo levels. We will determine the tissue source of Epo mRNA. We will also cross these mice with a Tibetan Phd2 knockin mouse that bears a double amino acid substitution in its zinc finger that selectively impairs interaction with p23 in order to independently assess the importance of these interactions. The proposed studies will identify a mechanism by which patients with PHD2 zinc finger mutations develop erythrocytosis. Importantly, these studies will also resolve the apparent paradox of how high altitude-adapted Tibetans can harbor mutations that impair PHD2 zinc finger function and at the same time avoid erythrocytosis. The studies will reveal unanticipated links between oxygen sensing and the translation and HSP90 pathways in the control of red cell mass.