Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Abstract 344

Red blood cells (RBC) are produced at a rate of 2.3 × 10⁶ cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1^−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1^−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.