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. 2020 Sep:36:101670.
doi: 10.1016/j.redox.2020.101670. Epub 2020 Aug 3.

Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis

Dominik C Fuhrmann  1 , Antonia Mondorf  2 , Josefine Beifuß  1 , Michaela Jung  1 , Bernhard Brüne  3
Affiliations

Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis

Dominik C Fuhrmann et al. Redox Biol. 2020 Sep.

Abstract

Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.

Keywords: FTMT; Ferritinophagy; Ferroptosis; Hypoxia; Iron; JNK; Macrophages; NCOA4; miR-6862-5p.

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Conflict of interest statement

All authors concur with this submission. Data have neither been submitted previously nor are under consideration for publication elsewhere. We declare no conflict of interest.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Regulation of iron and associated genes under hypoxia. A. Primary human macrophages were incubated under hypoxia (1% O2, 16 h). Cellular iron was determined by atomic absorption spectroscopy. Data were normalized to the normoxic control (n = 6–7). B. Primary human macrophages were incubated for 16 h under hypoxia (1% O2) and iron in the cell supernatants was analyzed by atomic absorption spectroscopy (n = 6–7). Data were normalized to the normoxic control. C-L. Human macrophages were incubated for indicated times under hypoxia (1% O2), RNA of divalent metal transporter 1 (DMT1), transferrin receptor (TfR), ceruloplasmin (CP), ferroportin (FPN), ferritin heavy chain (FTH), ferritin light chain (FTL), mitochondrial ferritin (FTMT), poly(rC)-binding protein 1 (PCBP1), ubiquitin ligase E3 ubiquitin-protein ligase HERC2 (HERC2), and nuclear receptor coactivator 4 (NCOA4) was analyzed by qPCR, and normalized to TATA box binding protein (TBP). Date were normalized to the normoxic control (n = 7–8). Data are mean values with SEM. Students t-test values p < 0.05 were considered as significant.
Fig. 2
Fig. 2
Protein expression of ferritinophagy related proteins. A-D. Primary human macrophages were incubated for indicated times under hypoxia (1% O2), total protein was stained, and Western analysis was performed for nuclear receptor coactivator 4 (NCOA4), ferritin heavy chain (FTH), ferritin light chain (FTL), and mitochondrial ferritin (FTMT). For quantification, the lane normalization factor (LNF) was calculated and the normoxic control was set to 1 (n = 3–4). E. Macrophages were transfected with a siRNA against NCOA4 (siNCOA4) or an untargeted control (NTC). Total protein was stained and Western analysis for FTMT, FTH, and FTL was performed. For quantification, the intensity was normalized to LNF and NTC was set to 1 (n = 4–9). Complete total protein stains are collectively shown in Fig. S1. All data are depicted with SEM. Students t-test values p < 0.05 were considered as significant.
Fig. 3
Fig. 3
NCOA4 mediates degradation of FTMT under hypoxia. A. Primary human macrophages were treated with SP600125 (10 μM), LY294002 (15 μM), SB203580 (10 μM), AKT VIII (1 μM), or Rapamycin (100 nM) 1 h prior to hypoxic incubation (1% O2, 24 h). The mRNA of nuclear receptor coactivator 4 (NCOA4) was analyzed by qPCR and normalized to TBP. The DMSO control was set to 1 (n = 6–8). B. Primary human macrophages were treated with DMSO, SP600125, or LY294002 1 h prior to hypoxic incubation (1% O2, 24 h). Total protein was stained and NCOA4 was analyzed by Western blotting. For quantification, the lane normalization factor (LNF) was calculated and the normoxic DMSO control was set to 1 (n = 3). C. Cells were incubated under hypoxia (1% O2, 8 h) and chromatin immunoprecipitation was performed for immune globulin G (IgG), histon H3 (H3), and polymerase II (Pol II). The precipitate was probed for NCOA4 and data were expressed as percent of input (n = 3). D. Cells were incubated with SP600125 for 24 h and micro RNA (miR) 6862-5p was analyzed by qPCR. Data were normalized to SNORD44 and DMSO control was set to 1 (n = 6). E. Macrophages were transfected with an antagomir for miR-6862-5p (25 nM) and NCOA4 mRNA was analyzed (n = 4). F-G. Primary human macrophages were treated with SP600125 1 h prior to hypoxic incubation (1% O2, 24 h). Total protein was stained and mitochondrial ferritin (FTMT) and ferritin heavy chain (FTH) were analyzed by Western blotting. For quantification, protein expression was normalized to LNF and the normoxic control was set to 1 (n = 6–8). For reasons of clarity the blot was cut at the dashed line. H. Cells were transfected with an siRNA against NCOA4 for 24 h and treated with SP600125 1 h prior to hypoxic incubations (1% O2, 24 h). Western analysis was performed for FTMT and protein expression was normalized to LNF. DMSO control was set to 1 (n = 4). Complete total protein stains are collected in Fig. S1. I. Schematic outline of results. All data are depicted with SEM. Students t-test or ANOVA values p < 0.05 were considered as significant.
Fig. 4
Fig. 4
Ferroptosis in human macrophages. A-B. Cells were treated with RSL-3 (10 μM) and Liproxstatin-1 (Lip, 1 μM) and incubated under hypoxia (1% O2, 24 h). Expression of mitochondrial ferritin (FTMT) and ferritin heavy chain (FTH) were analyzed by Western blotting and normalized to the lane normalization factor (LNF). The DMSO control was set to 1 (n = 7–8). C. Macrophages were treated as described for A-B and vitality was analyzed after 1 h incubation with Cell titer blue (n = 8). The DMSO control was set to 100%. D. Cells were treated as mentioned in A and malondialdehyde (MDA) was measured (n = 3). E. Human macrophages were transfected with a siRNA against FTMT and/or FTH or an untargeted control, treated with RSL-3, and incubated under normoxia/hypoxia (1% O2, 24 h). Vitality of the cells was analyzed by CellTiter-Blue staining. The normoxic DMSO control was set to 100% (n = 5–8). F. Cells were transfected with siRNAs against FTMT and FTH or a nontargeting control and treated as mentioned in A. Vitality of the cells was analyzed by CellTiter-Blue staining. The normoxic DMSO control was set to 100% (n = 4–5). Complete total protein stains are collected in Fig. S1. All data are depicted with SEM. Students t-test or ANOVA values p < 0.05 were considered as significant.
Fig. 5
Fig. 5
Proposed mechanism of NCOA4 regulation under hypoxia. In human macrophages nuclear receptor coactivator 4 (NCOA4) mRNA is reduced under hypoxia by decreased transcription and in parallel by micro RNA (miR)-6862-5p, which is regulated by c-Jun N-terminal kinase (JNK). As result, NCOA4 protein and consequently ferritinophagy are decreased. In turn, this increases FTMT abundance under hypoxia, which protects from ferroptosis.
Fig. 6
Fig. 6
Regulation of ferritinophagy in HT1080 cells. A. Primary human macrophages and HT1080 cells were incubated for 24 h under hypoxia: FTMT, FTH, and NCOA4 were analyzed by Western blotting. For quantification, intensities were normalized to lane normalization factor (LNF) (n = 3). B–C. HT1080 cells were transfected with a siRNA against NCOA4 and Western analysis was performed for FTMT and FTH. Intensities were normalized to LNF (n = 3). D-E. HT1080 cells were incubated for 24 h under hypoxia and treated with RSL-3 (1 μM) and Liproxstatin-1 (Lip, 1 μM) within the last 4 h of incubation. Western analysis was performed for FTH and FTMT (n = 3). F. HT1080 cells were treated as in D and vitality was assessed by CellTiter blue assay (n = 3). G. HT1080 cells were transfected with a siRNA against FTH and incubated for 24 h under hypoxia followed by CellTiter-Blue assays (n = 4–6). H. HT1080 cells were transfected with a siRNA against FTH and treated with Lip directly after transfection. Vitality was measured by the CellTiter-Blue assay (n = 3). I. Proposed mechanism of ferroptosis in human macrophages vs. HT1080 cells. For details see the text. Complete total protein stains are collected in Fig. S1. All data are depicted with SEM. Students t-test or ANOVA values p < 0.05 were considered as significant.
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