ASPECTS OF CELLULAR IRON HOMEOSTASIS: NRAMP TRANSPORTER FUNCTION AND ERYPTOSIS

The present study focus attention on “Aspects of cellular iron homeostasis: NRAMP transporter function and eryptosis” combining aspects of both basic (first chapter: “Iron transporters NRAMP1 and NRAMP2 from Dictyostelium discoideum as a model of cellular iron homeostasis”) and applied physiology (second chapter: “Effects of xenobiotics on the suicidal death of erythrocytes”). Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species (ROS). SLC11 and SLC40 families are involved in iron transport and play an important role in the maintenance of iron homeostasis. The SLC11 family is comprised of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the phagolysosome of macrophages and in the tertiary granules of neutrophils and it contributes to the innate resistance against bacterial infection. SLC11A2 (also known as DMT1) is expressed in the proximal duodenum, immature erythroid cells, brain, placenta and kidney and is a key player in iron metabolism. Intestinal iron absorption is indeed mediated by SLC11A2 at the apical membrane of enterocytes and is followed by basolateral exit via SLC40A1. D. discoideum represents a model for the study of cellular iron homeostasis, showing subcellular localization of iron transporters resembling that of macrophages. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis; in particular, D. discoideum expresses the ortholog of SLC11A1 transporter in phagolysosomes and that of SLC11A2 in the contractile vacuole. To better understand the function of Dictyostelium NRAMP proteins, they were expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical techniques and by a novel assay based on metal-induced changes in calcein fluorescence. Radiochemical assays showed that NRAMP1 induced iron transport is proton-dependent and it is inhibited by Mn2+, Cd2+, Co2+, Ni2+, Cu2+ and to a lesser extent by Zn2+. In calcein-injected oocytes expressing NRAMP1 and analyzed using confocal microscopy, Fe2+, Mn2+ and but not Fe3+ or Cu2+ led to fluorescence quenching due to their transport and accumulation into the cytoplasm of the oocytes. Therefore Dictyostelium NRAMP1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of rat DMT1. NRAMP1 colocalizes with V-ATPase in the membrane of phagolysosomes. Thus, it exploits the proton gradient maintained by the V-ATPase to mediate the efflux of iron from the phagolysosomes to the cytosol after bacterial engulfment. Preliminary studies showed that D. discoideum NRAMP2 can transport ferrous iron at neutral pH and it appears independent from proton gradient but dependent on Na+, nevertheless its transport activity is strongly reduced compared with that observed for NRAMP1. The second topic of this PhD thesis is eryptosis, the suicidal death of erythrocytes. Mutations that reduce DMT1 activity in human are associated with a severe defect in erythroid iron utilization and are correlated with several diseases. DMT1 deficiency causes an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast, accelerated death of erythroid precursors and a decreased survival in the erythroid progenitors. Iron deficiency is associated with shortened life span of erythrocytes. The accelerated clearance of erythrocytes can be attributed to excessive hemolysis or induction of programmed cell death of erythrocytes, called eryptosis. Eryptosis is fostered by an increase in cytosolic calcium. Iron deficient erythrocytes when exposed to stress conditions has been demonstrated to activate Ca2+-permeable cation channel allowing Ca2+ entry. Ca2+ entry through this channels leads to activation of a scramblase with subsequent phosphatidylserine exposure and to activation of the Gardos channels leading to KCl loss and cell shrinkage. This study was conducted in order to investigate prospective antitumoral products which are employed against tumor growth in humans, in particular CA4P or Pazopanib, or compounds used in vitro, such as Nocodazole, Terfenadine, Piceatannol, Ceranib-2 and Sclareol, in order to unveil the effects on erythrocytes survival and to clarify the mechanisms and signalling involved in their action. Human erythrocytes drawn from healthy individual were incubated in vitro at a hematocrit of 0.4% in Ringer solution. Where indicated, RBCs were exposed for 48 hours to the drugs at the indicated concentrations. The main hallmarks of eryptosis were investigated by flow cytometry. Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCF-dependent fluorescence, GSH levels by CMF-dependent fluorescence and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. ATP levels following CA4P treatment were measured using luciferin-luciferase assay kit. For studying the effect of Nocodazole on tubulin in human erythrocytes, TubulinTracker™ Green reagent was used. A 48 hours exposure of human erythrocytes to CA4P, Pazopanib, Nocodazole, Terfenadine, Piceatannol, Ceranib-2 or Sclareol treatment increased the percentage of annexin-V-binding cells. Furthermore, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone and in the presence of casein kinase 1α inhibitor D4476. CA4P and Piceatannol significantly decreased forward scatter. CA4P, Nocodazole, Terfenadine, Ceranib-2 and Sclareol treatment significantly increased Fluo3-fluorescence. CA4P significantly decreased GSH abundance and ATP levels. In addition, Pazopanib, Terfenadine, Ceranib-2 and Sclareol further resulted in significant hemolysis. Pazopanib, Nocodazole and Ceranib-2 significantly increased DCF-fluorescence and ceramide abundance. Nocodazole treatment reduced total tubulin abundance. In conclusion, these xenobiotics trigger eryptosis through different mechanisms.