Page 82 - 2019_09-HaematologicaMondo-web

N. Maio et al.
IRE in the messenger RNA of transcripts involved in iron homeostasis.18 G1E-ER4 cells robustly activated the IRE- binding activities of Irp1 and Irp2 within 10 h after induc- tion of differentiation, with maximal activation at 24 h and 48 h (Figure 2A). By 72 h in differentiation medium, when cells had reached the orthochromatophilic ery- throid stage and fulfilled their extracellular iron demands, the IRE-binding activities of Irp1 and Irp2 diminished (Figure 2A). In contrast, in Abcb7-KD G1E-ER4 cells, Irp2 activation was elicited well before induction of differenti- ation and its levels remained undiminished throughout the time course (Figure 2A). Control cells differentiated for 72 h showed increased sequestration of radiolabeled iron into ferritin (55Fe-autoradiogram) (Figure 2B), as already reported.19 In control cells, iron was also success- fully incorporated into protoporphyrin IX (PPIX) by Fech, as radiolabeled hemoglobin (55Fe-Hb) increased signifi- cantly (Figure 2B). Compared to control (NT), cells lack- ing Abcb7 exhibited prolonged activation of the cytosolic iron starvation response with stabilization of Irp2, decreased cytosolic aconitase (Aco1) activity and increased IRE-binding activities of IRP (Figure 2A-C). Hyperactivation of IRP caused translational repression of ferritin (Figure 2B) and decreased radiolabeled-iron sequestration into the heteropolymeric storage form of the protein (Figure 2B). Under the transcriptional control of Gata-1,20 differentiating cells upregulated hemoglobin subunits (Hba-a2 and Hbb-b1) and Alas2 (Figure 2B,D, Online Supplementary Figure S3). Mfrn1 increased during differentiation to fulfill the exceptional iron demand for heme synthesis (Figure 2B and Online Supplementary Figure S3). Abcb7-depleted cells failed to increase levels of Alas2, due to translational repression by IRP at the 5’-IRE present in its transcript21,22 (Figure 2D), even though Alas2 transcript levels were over 50-fold higher than those in undifferentiated cells (Online Supplementary Figure S3). Importantly, levels of Bach1, an erythroid transcriptional repressor that is rapidly degraded under conditions of heme sufficiency,23 increased upon KD of Abcb7 (Figure 2D), and levels of Hbb-b1 and Hba-a2, which are Bach1 targets,24 were repressed (Figure 2B and Online Supplementary Figure S3). Notably, Tfrc and Mfrn1 levels were elevated in Abcb7-KD cells prior to differentiation (Figure 2B and Online Supplementary Figure S4A,B). Total cellular iron content in early erythroid progenitors deplet- ed of Abcb7 was about 7-fold higher than that in controls (Online Supplementary Figure S5A) and iron accumulated in mitochondria (Figure 2E), whereas the pool of available cytosolic labile iron was significantly reduced (Figure 2F), indicating a condition of cytosolic functional iron defi- ciency. Mitochondrial iron overload increased the pro- duction of reactive oxygen species by 30% (Mitosox+ cells) (Figure 2G,H), which damaged plasma membranes, as shown by the increased percentage of annexin V+ cells (Figure 2G,H and Online Supplementary Figure S5B), even though both mitochondrial and cytosolic superoxide dis- mutase enzymes (Sod2 and Sod1) were activated (Figure 2I), and were transcriptionally upregulated about 2- and 5-fold, respectively (Online Supplementary Figure S6A-C). Unexpectedly, most of the genes involved in the antioxi- dant response were downregulated in Abcb7-KD cells (Online Supplementary Figure S6A). Manganese accumulat- ed in mitochondria of Abcb7-KD cells (Online Supplementary Figure S5C), raising the possibility that metalation of Sod2 accounted for the increase in man-
ganese. KD of ABCB7 in HEK293T cells also caused mito- chondrial iron accumulation (Online Supplementary Figure S5D), and activation of SOD enzymes (Online Supplementary Figure S5E,F). As MFRN2 was significantly upregulated upon silencing of ABCB7 in the non-ery- throid HEK293T cells (Figure 1A), we knocked down its expression and found that cells depleted of MFRN2 main- tained intact mitochondrial function (Online Supplementary Figure S5D,G). KD of MFRN2 in ABCB7- depleted cells lowered mitochondrial iron accumulation (Online Supplementary Figure S5D), but levels and activities of Fe-S-dependent enzymes did not return to normal (Online Supplementary Figure S5H), suggesting that mito- chondrial iron overload was not the primary cause of the compromise of Fe-S proteins in the matrix.
Heme biosynthesis defect in cells lacking ABCB7
Loss of Abcb7 during erythroid differentiation led to an 80% reduction in the levels of 55Fe-Hb, to the IRP-mediat- ed translational repression of Alas2 and to Bach1-mediat- ed blockage of Hba-a2 and Hbb-b1 transcription, pointing to a potential defect in heme biosynthesis. Treatment with succinylacetone, a potent inhibitor of Alad, con- firmed that the defect of 55Fe-Hb synthesis caused by KD of Abcb7 during differentiation was comparable to the inhibition of the heme biosynthetic pathway (Figure 3A, 55Fe-Hb autoradiogram, lanes 5 and 6). Alas2, Fech and Alad protein levels decreased in cells lacking Abcb7 (Figure 3B and Online Supplementary Figure S7A), whereas levels of the heme biosynthetic enzyme Cpox,25 of Sucla2 and Suclg2, which provide succinyl-CoA to the first rate- limiting step of the pathway.26 and of the unfoldase Clpx, required for the pyridoxal-phosphate-dependent activa- tion of Alas2,27 did not change (Online Supplementary Figure S7A). Abcb7-KD cells exhibited impaired hemoglo- binization (Figure 3C). Heme content decreased by 75% (Figure 3D,E) and activities of the heme-containing enzymes cytochrome c oxidase (Figure 3F) and peroxiso- mal catalase (Figure 3G and Online Supplementary Figure S7C-E) were reduced by more than 80%. Levels of respi- ratory complexes III and IV, which require three and two heme centers, respectively, were significantly reduced in Abcb7-KD cells (Figure 3F). Heme-bound cytochromes c (Cyc) and c1 (Cyc1) decreased (Holo-cyt) (Figure 3F), without changes in the levels of total apo-cytochromes (Online Supplementary Figure S7B). The heme-regulated eIF2a kinase (HRI), which inhibits the general translation initiation factor eIF2a under conditions of heme deficien- cy,28 may mediate the translational repression of the glo- bin chains, which are also transcriptionally repressed by Bach1 (Online Supplementary Figure S3), in Abcb7-depleted erythroid progenitors to prevent accumulation of globins in excess of heme, and may also repress translation of the heme biosynthetic enzymes Alas2 and Alad.28 A defect in heme biosynthesis was also observed in HEK293T and HeLa cells upon KD of ABCB7 (Online Supplementary Figure S7F-K).
Irp2 activation in Abcb7-depleted cells sustained mitochondrial iron overload mediated by mitoferrin-1 upregulation
We investigated the correlation between loss of Abcb7 and activation of IRP, by analyzing the effect of double KD of Abcb7 and Irp2 in developing erythroid cells. KD of Irp2 significantly diminished the stability of Tfrc
1760
haematologica | 2019; 104(9)