SpringerLink
Log in

Alleviated NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in lipopolysaccharide-induced inflammation under hypoxia

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Objective

Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of non-apoptotic cell death process. Previous studies have demonstrated that ferroptosis participates in the development of inflammatory arthritis. However, the role of ferroptosis in rheumatoid arthritis (RA) inflammatory hypoxic joints remains unclear. This study sought to explore the underlying mechanism of ferroptosis on lipopolysaccharide (LPS)-induced RA fibroblast-like synoviocytes (FLSs).

Methods

FLSs, isolated from patients with RA, were treated with LPS and ferroptosis inducer (erastin and RSL-3), and ferroptosis inhibitor (Fer-1 and DFO), respectively. The cell viability was measured by CCK-8. The cell death was detected by flow cytometer. The proteins level were tested by Western blot. The cytosolic ROS and lipid peroxidation were determined using DCFH-DA and C11-BODIPY581/591 fluorescence probes, respectively. The small interfering RNA (siRNA) was used to knock down related proteins. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, inflammatory cytokines (IL6 and IL8), and LDH were analyzed by commercial kits.

Results

Ferroptosis was activated by LPS in RA FLS with increased cellular damage, ROS and lipid peroxidation, intracellular Fe and IL8, which can be further amplified by ferroptosis inducer (erastin and RSL-3) and inhibited by ferroptosis inhibitor (Fer-1 and DFO). Mechanistically, LPS triggered ferroptosis via NCOA4-mediated ferritinophagy in RA FLSs, and knockdown of NCOA4 strikingly prevent the process of ferroptosis. Intriguingly, LPS-induced RA FLSs became insensitive to ferroptosis and NCOA4-mediated ferritinophagy under hypoxia compared with normoxia. Knockdown of HIF-1α reverted ferroptosis and ferritinophagy evoking by LPS-induced RA FLSs inflammation under hypoxia. In addition, low dose of auranofin (AUR) induced re-sensitization of ferroptosis and ferritinophagy through inhibiting the expression of HIF-1α under hypoxia.

Conclusions

NCOA4-mediated ferritinophagy was a key driver of ferroptosis in inflammatory RA FLSs. The suppression of NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in LPS-induced inflammation under hypoxia. Targeting HIF-1α/NCOA4 and ferroptosis could be an effective and valuable therapeutic strategy for synovium hyperplasia in the patients with RA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Brazil)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

4-HNE:

4-Hydroxynonenal

AUR:

Auranofin

CA9:

Carbonic anhydrase 9

DFO:

Deferoxamine mesylate

Fer-1:

Ferrostatin-1

FLSs:

Fibroblast-like synoviocytes

FPN:

Ferroportin

FTH:

Ferritin heavy chain

FTL:

Ferritin light chain

HIF-1:

Hypoxia-inducible factor-1

LPS:

Lipopolysaccharide

MDA:

Malondialdehyde

NCOA4:

Nuclear receptor coactivator 4

PDAC:

Pancreatic ductal adenocarcinomas

RA:

Rheumatoid arthritis

ROS:

Reactive oxygen species

siRNA:

Small interfering RNA

SLE:

Systemic lupus erythematosus

TfR:

Transferrin receptor

References

  1. Peters CL, Morris CJ, Mapp PI, Blake DR, Lewis CE, Winrow VR. The transcription factors hypoxia-inducible factor 1alpha and Ets-1 colocalize in the hypoxic synovium of inflamed joints in adjuvant-induced arthritis. Arthritis Rheum. 2004;50:291–6.

    Article  CAS  PubMed  Google Scholar 

  2. Konisti S, Kiriakidis S, Paleolog EM. Hypoxia–a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol. 2012;8(3):153–62.

    Article  CAS  PubMed  Google Scholar 

  3. Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol. 2021;18(5):280–96.

    Article  CAS  PubMed  Google Scholar 

  4. Wu X, Li Y, Zhang S, Zhou X. Ferroptosis as a novel therapeutic target for cardiovascular disease. Theranostics. 2021;11(7):3052–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang Y, Quan F, Cao Q, et al. Quercetin alleviates acute kidney injury by inhibiting ferroptosis. J Adv Res. 2020;28:231–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xu S, He Y, Lin L, Chen P, Chen M, Zhang S. The emerging role of ferroptosis in intestinal disease. Cell Death Dis. 2021;12(4):289.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ou M, Jiang Y, Ji Y, et al. Role and mechanism of ferroptosis in neurological diseases. Mol Metab. 2022;61: 101502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Li P, Jiang M, Li K, et al. Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity. Nat Immunol. 2021;22(9):1107–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Luoqian J, Yang W, Ding X, et al. Ferroptosis promotes T-cell activation-induced neurodegeneration in multiple sclerosis. Cell Mol Immunol. 2022;19(8):913–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhou R, Chen Y, Li S, et al. TRPM7 channel inhibition attenuates rheumatoid arthritis articular chondrocyte ferroptosis by suppression of the PKCα-NOX4 axis. Redox Biol. 2022;55: 102411.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. McGettrick AF, O’Neill LAJ. The role of HIF in immunity and inflammation. Cell Metab. 2020;32(4):524–36.

    Article  CAS  PubMed  Google Scholar 

  12. Dixon SJ, Stockwell BR. The role of iron and reactive oxygen species in cell death. Nat Chem Biol. 2014;10:9–17.

    Article  CAS  PubMed  Google Scholar 

  13. Cen Y, Wang P, Gao F, et al. Tetramethylpyrazine nitrone activates hypoxia-inducible factor and regulates iron homeostasis to improve renal anemia. Front Pharmacol. 2022;13:964234.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ni S, Yuan Y, Qian Z, et al. Hypoxia inhibits RANKL-induced ferritinophagy and protects osteoclasts from ferroptosis. Free Radic Biol Med. 2021;169:271–82.

    Article  CAS  PubMed  Google Scholar 

  15. Mancias JD, Wang X, Gygi SP, Harper JW, Kimmelman AC. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature. 2014;509(7498):105–9.

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  16. Ryu MS, Duck KA, Philpott CC. Ferritin iron regulators, PCBP1 and NCOA4, respond to cellular iron status in develo** red cells. Blood Cells Mol Dis. 2018;69:75–81.

    Article  CAS  PubMed  Google Scholar 

  17. Dowdle WE, Nyfeler B, Nagel J, et al. Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. Nat Cell Biol. 2014;16(11):1069–79.

    Article  CAS  PubMed  Google Scholar 

  18. Dong W, Tan Y, Qin Q, et al. Polybrominated diphenyl ethers quinone induces NCOA4-mediated ferritinophagy through selectively autophagic degradation of ferritin. Chem Res Toxicol. 2019;32(12):2509–16.

    Article  CAS  PubMed  Google Scholar 

  19. Li Q, Chen H, Huang X, Costa M. Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1alpha) and HIF-regulated genes. Toxicol Appl Pharmacol. 2006;213(3):245–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Suarez-Almazor ME, Spooner CH, Belseck E. Shea B (2000) Auranofin versus placebo in rheumatoid arthritis. Cochrane Database Syst Rev. 2000;2:CD002048.

    Google Scholar 

  21. Freire Boullosa L, Van Loenhout J, Flieswasser T, et al. Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer. Redox Biol. 2021;42:101949.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Van Loenhout J, Freire Boullosa L, Quatannens D, et al. Auranofin and cold atmospheric plasma synergize to trigger distinct cell death mechanisms and immunogenic responses in glioblastoma. Cells. 2021;10(11):2936.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Kato I, Kasukabe T, Kumakura S. Menin-MLL inhibitors induce ferroptosis and enhance the anti-proliferative activity of auranofin in several types of cancer cells. Int J Oncol. 2020;57(4):1057–71.

    CAS  PubMed  Google Scholar 

  24. You S, Koh JH, Leng L, Kim WU, Bucala R. The tumor-like phenotype of rheumatoid synovium: molecular profiling and prospects for precision medicine. Arthritis Rheumatol. 2018;70(5):637–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu Y, Wei W, Wang Y, et al. TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis. Inflamm Res. 2019;68(7):597–611.

    Article  CAS  PubMed  Google Scholar 

  26. Luo H, Zhang R. Icariin enhances cell survival in lipopolysaccharide-induced synoviocytes by suppressing ferroptosis via the Xc-/GPX4 axis. Exp Ther Med. 2021;21(1):72.

    Article  CAS  PubMed  Google Scholar 

  27. Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Van der Paal J, Neyts EC, Verlackt CCW, Bogaerts A. Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress. Chem Sci. 2016;7(1):489–98.

    Article  PubMed  Google Scholar 

  29. Gryzik M, Srivastava A, Longhi G, et al. Expression and characterization of the ferritin binding domain of nuclear receptor coactivator-4 (NCOA4. Biochim Biophys Acta Gen Subj. 2017;1861((11 Pt A)):2710–6.

    Article  CAS  PubMed  Google Scholar 

  30. Fuhrmann DC, Mondorf A, Beifuß J, Jung M, Brüne B. Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis. Redox Biol. 2020;36:101670.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lin Z, Song J, Gao Y, et al. Hypoxia-induced HIF-1α/lncRNA-PMAN inhibits ferroptosis by promoting the cytoplasmic translocation of ELAVL1 in peritoneal dissemination from gastric cancer. Redox Biol. 2022;52:102312.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mao H, Zhao Y, Li H, Lei L. Ferroptosis as an emerging target in inflammatory diseases. Prog Biophys Mol Biol. 2020;155:20–8.

    Article  CAS  PubMed  Google Scholar 

  33. Wu J, Feng Z, Chen L, et al. TNF antagonist sensitizes synovial fibroblasts to ferroptotic cell death in collagen-induced arthritis mouse models. Nat Commun. 2022;13(1):676.

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  34. Ling H, Li M, Yang C, et al. Glycine increased ferroptosis via SAM-mediated GPX4 promoter methylation in rheumatoid arthritis. Rheumatology (Oxford). 2022;61(11):4521–34.

    Article  CAS  PubMed  Google Scholar 

  35. Ganz T, Nemeth E. Iron homeostasis in host defence and inflammation. Nat Rev Immunol. 2015;15(8):500–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tefanova KI, Delcheva GT, Maneva AI, et al. Pathobiochemical mechanisms relating iron homeostasis to parameters of inflammatory activity and autoimmune disorders in rheumatoid arthritis. Folia Med (Plovdiv). 2016;58(4):257–63.

    Article  Google Scholar 

  37. **e Z, Hou H, Luo D, An R, Zhao Y, Qiu C. ROS-dependent lipid peroxidation and reliant antioxidant ferroptosis-suppressor-protein 1 in rheumatoid arthritis: a covert clue for potential therapy. Inflammation. 2021;44(1):35–47.

    Article  CAS  PubMed  Google Scholar 

  38. Mancias JD, Pontano Vaites L, Nissim S, et al. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. Elife. 2015;4:e10308.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Santana-Codina N, Del Rey MQ, Kapner KS, et al. NCOA4-mediated ferritinophagy is a pancreatic cancer dependency via maintenance of iron bioavailability for iron-sulfur cluster proteins. Cancer Discov. 2022;12(9):2180–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li N, Wang W, Zhou H, et al. Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. Free Radic Biol Med. 2020;160:303–18.

    Article  CAS  PubMed  Google Scholar 

  41. Feng X, Wang S, Sun Z, et al. Ferroptosis enhanced diabetic renal tubular Injury via HIF-1α/HO-1 pathway in db/db Mice. Front Endocrinol (Lausanne). 2021;12:626390.

    Article  PubMed  Google Scholar 

  42. Yuan S, Wei C, Liu G, et al. Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF-1α/SLC7A11 pathway. Cell Prolif. 2022;55(1):e13158.

    Article  CAS  PubMed  Google Scholar 

  43. Singhal R, Mitta SR, Das NK, et al. HIF-2α activation potentiates oxidative cell death in colorectal cancers by increasing cellular iron. J Clin Invest. 2021;131(12):e143691.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang Y, Zhang L, Zhou X. Activation of Nrf2 signaling protects hypoxia-induced HTR-8/SVneo cells against ferroptosis. J Obstet Gynaecol Res. 2021;47(11):3797–806.

    Article  CAS  PubMed  Google Scholar 

  45. Liu XJ, Lv YF, Cui WZ, et al. Icariin inhibits hypoxia/reoxygenation-induced ferroptosis of cardiomyocytes via regulation of the Nrf2/HO-1 signaling pathway. FEBS Open Bio. 2021;11(11):2966–76.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Li Z, Jiang L, Chew SH, Hirayama T, Sekido Y, Toyokuni S. Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia. Redox Biol. 2019;26:101297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Stephenson J, Nutma E, van der Valk P, Amor S. Inflammation in CNS neurodegenerative diseases. Immunology. 2018;154(2):204–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yumnamcha T, Devi TS, Singh LP. Auranofin mediates mitochondrial dysregulation and inflammatory cell death in human retinal pigment epithelial cells: implications of retinal neurodegenerative diseases. Front Neurosci. 2019;13:1065.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Yang L, Wang H, Yang X, et al. Auranofin mitigates systemic iron overload and induces ferroptosis via distinct mechanisms. Signal Transduct Target Ther. 2020;5(1):138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gnanapradeepan K, Basu S, Barnoud T, Budina-Kolomets A, Kung CP, Murphy ME. The p53 tumor suppressor in the control of metabolism and ferroptosis. Front Endocrinol (Lausanne). 2018;9:124.

    Article  PubMed  Google Scholar 

  51. Hassannia B, Vandenabeele P, Vanden BT. Targeting ferroptosis to iron out cancer. Cancer Cell. 2019;35(6):830–49.

    Article  CAS  PubMed  Google Scholar 

  52. Liu J, Zhang C, Wang J, Hu W, Feng Z. The regulation of ferroptosis by tumor suppressor p53 and its pathway. Int J Mol Sci. 2020;21(21):8387.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rios Perez MV, Roife D, Dai B, et al. Antineoplastic effects of auranofin in human pancreatic adenocarcinoma preclinical models. Surg Open Sci. 2019;1(2):56–63.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by Special Fund for Discipline Construction of Tian** Medical University during the 13th Five-year Plan Period (2017XK030605; to F.Z), Natural Science Foundation of Tian** (20JCYBJC00700; to F.Z), and Integrative Medicine Foundation of Tian** Administration of Traditional Chinese Medicine (2021030; to H.D).

Funding

Integrative Medicine Foundation of Tian** Administration of Traditional Chinese Medicine, 2021030, Natural Science Foundation of Tian** Municipality, 20JCYBJC00700.

Author information

Author notes

  1. Yang Wang, Hongmei Ding and Yuqun Zheng contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Immunology, School of Medical Laboratory, Tian** Medical University, Tian**, China

    Yang Wang, Yuqun Zheng, **nyue Wei, **aoting Yang, Huan Wei, Yanshuang Tian, Xuguo Sun, Derun Tian & Fang Zheng

  2. Department of Health Statistics, College of Public Health, Tian** Medical University, Tian**, China

    Jun Ma

  3. Department of Rheumatology, General Hospital, Tian** Medical University, Tian**, China

    Wei Wei

  4. Department of Clinical Laboratory, Tian** Hospital, Tian** University, Tian**, China

    Yang Wang & Hongmei Ding

  5. Department of Clinical Laboratory, Tian** First Central Hospital, School of Medicine, Nankai University, Tian**, China

    Yang Wang & **nyue Wei

Authors
  1. Yang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongmei Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuqun Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. **nyue Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. **aoting Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huan Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yanshuang Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xuguo Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jun Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Derun Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Fang Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y W and H M D contributed to conceptualization, methodology, investigation, and writing––review and editing. Y Q Z was responsible for investigation, methodology, and software. X Y W and X T Y were involved in conceptualization, methodology, and writing––review and editing. H W performed data curation and writing––review and editing. Y S T was responsible for data curation, methodology, and writing––review and editing. W W was involved in methodology and supervision. J M performed supervision and project administration. F Z and D R T performed funding acquisition, supervision, and project administration.

Corresponding authors

Correspondence to Wei Wei, Jun Ma, Derun Tian or Fang Zheng.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Responsible Editor: John Di Battista.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Y., Ding, H., Zheng, Y. et al. Alleviated NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in lipopolysaccharide-induced inflammation under hypoxia. Inflamm. Res. 73, 363–379 (2024). https://doi.org/10.1007/s00011-023-01842-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-023-01842-9

Keywords

Search

Navigation