Modulation of oxygen-regulated erythropoietin expression

Erythropoietin (Epo) is the key hormone regulating red blood cells homeostasis and is produced in the foetal liver and adult kidney. Epo plasma levels physiologically increase in response to hypoxia and anaemia, following the activation of the HIF (hypoxia-inducible factor) pathway. In pathological conditions, such as chronic kidney disease (CKD) or erythrocytosis, Epo levels markedly decrease or increase, respectively, leading to a variety of complications. Despite extensive efforts to isolate the peritubular interstitial renal cells responsible for oxygen-sensing and Epo production, no kidney-derived cellular model capable of expressing Epo in a hypoxia-inducible manner is available so far. Thus, only the well-established human hepatoma cell lines HepG2 and Hep3B and some neuronal cell lines (such as Kelly and SH-SY5Y) expressing oxygen-dependent Epo can be used to investigate Epo gene regulation in vitro. The lack of an appropriate renal cell model explains the poor understanding of the cis-acting element regulating kidney-specific Epo expression (kidney-inducible element or KIE), as defined by transgenic animal experiments. By comprehensive in silico analysis we identified a novel distal hypoxia response element (HRE) located upstream of the EPO gene, within the KIE, and named it Epo 5’-HRE, in contrast to the well-known 3’-HRE, located downstream of the gene and responsible for liver-specific Epo induction. Functional studies showed that the 5’-HRE is mainly responsive to HIF-2α isoform. We additionally implemented the innovative CRISPR/Cas technology to mutate the endogenous 5’- and 3’-HREs in Hep3B cells in order to evaluate the contributions of each element in regulating oxygen-dependent Epo expression. Furthermore, we screened a number of candidate factors, either known or novel regulators of the HIF pathway, for a potential effect on Epo expression using RNA interference-mediated or overexpression approaches in combination with newly developed Epo-reporter assays to study the 5’-HRE. In erythrocytosis, a disease characterized by inappropriately high Epo plasma levels, Epo synthesis appears dysregulated. Whereas in the majority of congenital secondary erythrocytosis patients the cause is unknown, a number of mutations in genes involved in the oxygen-sensing pathway, such as HIFs and prolyl-4-hydroxylase domain proteins (PHDs) have been identified. As part of a collaboration with the group of Dr. Betty Gardie (Nantes, France) we functionally characterized several novel mutations in the PHD2 gene causing familial erythrocytosis and could group these mutations into two main categories in terms of their effect on HIF regulation. Notably, some of the mutations did not lead to impaired PHD2 activity, at least in our in vitro assays. An additional layer of modulation of Epo production is represented by humoral factors, including peptides and hormones. We tested selected peptides in Hep3B for a potential role in Epo regulation and complemented this approach by performing a large-scale screening of a peptide library derived from human haemofiltrates using different HIF-reporter cell lines. Results from the first, complete screening are promising and showed the presence of both “inhibitory” and “activatory” fractions in terms of HIF functionality. In conclusion, this PhD work focused on the hypoxic regulation of the EPO gene on multiple levels and described for the first time a novel HRE, possibly representing the hypoxia-inducible element responsible for kidney-specific Epo induction. The newly identified Epo 5’-HRE represents the basis for subsequent studies to investigate the effect of a number of factors and peptides on oxygen-dependent Epo regulation