The Effects of Mitochondrial Ferritin Expression in Normal and Sideroblastic Erythropoiesis.
Authors: Porta Matteo G Della , Rosti Vittorio , Gallì Anna , Travaglino Erica , Santambrogio Paolo , Marseglia Carmela , Matti Valentina , Malcovati Luca , Bergamaschi Gaetano , Levi Sonia , Invernizzi Rosangela , Cazzola Mario 
Published: October 2019

Abstract
Abstract 736
In erythroid cells from patients with refractory anemia with ringed sideroblasts (RARS), the expression of mitochondrial ferritin (MtF) - encoded by the nuclear FTMT gene located on chromosome 5q21.3 - occurs at a very early stage of differentiation and is closely related to the sideroblastic phenotype [Blood. 2003 Mar 1;101(5):1996-2000]. On the other hand, in extra-hematopoietic tissues MtF expression is associated with high cellular metabolic activity and oxygen consumption, suggesting a possible role of this protein in protecting mitochondria from iron-dependent oxidative damage. It is known that reactive oxygen species (ROS) act as second messengers in the JAK/STAT pathway, which is involved in the regulation of erythropoiesis, and that ROS scavengers inhibit STAT5 phosphorylation. It was the aim of this study to investigate the influence of experimentally induced MtF over-expression on erythroid differentiation in normal hematopoietic progenitors, and in addition to define the relationship between MtF expression and erythroid maturation, apoptosis, and JAK-STAT pathway activation in RARS. A liquid culture model was adopted to expand erythroid progenitors from CD34+ cells in the presence of IL-3, IL-6, stem cell factor and Epo [Blood. 2005 Jul 1;106(1):247-53]. To study the effect of MtF induction in normal hematopoiesis, CD34+ cells from 5 healthy donors were transduced using lentivirus carrying cDNA of FTMT downstream to the ubiquitous PGK promoter (transduction efficiency equal to 30-40%), and then cultured for 21 days. To assess the effect of MtF expression in myelodysplastic syndromes (MDS), CD34+ cells from 24 patients with RARS, as well as cells from 20 patients with refractory anemia (RA) and from 8 healthy donors (as control groups) were cultured. Cytospins were performed for MtF, H-ferritin (HF) and L-ferritin (LF) immunocytochemical analysis, and samples of cultured cells were removed at various days of culture for biological studies. Lentivirus-mediated FTMT transduction of normal CD34+ progenitors did not inhibit cell growth nor prevent the differentiation of erythroid progenitors. By flow cytometry analysis, MtF-positive erythroid progenitors showed significantly reduced levels of HF and increased expression of transferrin receptor (CD71) compared with MtF-negative progenitors (P = .004 and P = .01, respectively). In this model, induction of MtF resulted in increased cellular apoptosis (median number of apoptotic cells by TUNEL assay at day 21 equal to 83% in MtF-positive cells vs 18% in MtF-negative cells, P < .001). A significantly lower proliferation rate and higher apoptotic index were observed in cultures from patients with RARS and RA with respect to healthy controls. FTMT mRNA was detected by RT-PCR in CD34+ progenitor cells from patients with RARS, while protein expression was observed only from day 4 of culture, with a significant increase in the percentage of MtF-positive cells during culture (median value from 5% at day 4 to 24% at day 21, P = .002). No MtF expression was observed in RA patients. In MtF-positive cells from RARS patients, an inverse relationship between MtF and HF cellular content, and a direct relationship between MtF and CD71 expression were observed. In erythroid progenitors from RARS patients, the apoptotic rate was higher in MtF-positive than in MtF-negative cells (median number of apoptotic cells equal to 17% vs 6%, P < .001). Finally, we analyzed by flow cytometry the relationship between MtF expression and STAT5 phosphorylation in cultured cells following Epo stimulation. Preliminary data from three RARS patients showed that p-STAT5 expression was lower in MtF-positive than in MtF-negative cells (P = .02). In conclusion, experimental overexpression of MtF in normal erythroid progenitors may reduce mitochondrial iron availability thus inducing apoptosis. In erythroid progenitors from RARS patients, the pathological expression of MtF is associated with increased apoptosis of immature red cells (ineffective erythropoiesis), which may be at least in part determined by reduced activation of the JAK-STAT pathway in response to Epo.

Disclosures:
No relevant conflicts of interest to declare.