Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts

Our plain-English summary of Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts, by Q. Dang, C. Huang, Y. Liang, et al (Respiratory Research, 2026).

The study

• Authors: Q. Dang, C. Huang, Y. Liang, A. Surendran, D. Muthiah, K. Vaddadi, S. Hewawasam, T.W. Mills, L. Liu
• Journal: Respiratory Research
• Published: 2026;27(1):91
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Iron is an essential nutrient for almost all organisms. However, excess iron generates reactive oxygen species and causes tissue injuries. Iron is implicated in idiopathic pulmonary fibrosis (IPF). In this study, we examined iron accumulation in fibrotic lung fibroblasts and the underlying mechanisms. We hypothesize that the downregulation of Solute Carrier Family 40 Member 1 (SLC40A1) results in iron accumulation in lung fibroblasts of IPF patients. Using a Prussian Blue iron staining, we found that iron accumulated in the fibrotic region of the lungs from IPF patients and bleomycin- and asbestos-induced lung fibrosis mice. Iron was partially co-localized with the fibroblast marker vimentin in IPF lungs. By using publicly available single cell RNA sequencing datasets, we identified SLC40A1, the only known gene involved in iron export, as a downregulated gene in alveolar fibroblasts of IPF. The downregulation of SLC40A1 was confirmed in the lung fibroblasts isolated from IPF patients and bleomycin-treated mice. The treatment of human lung fibroblasts with transforming growth factor-β1 (TGF-β1), a major cytokine elevated in IPF, reduced SLC40A1 mRNA and protein expression. TGF-β1 downregulated SLC40A1 expression via SMAD3 as determined by chromatin immunoprecipitation and luciferase promoter reporter assays. Knockdown of SLC40A1 using lentiviral shRNAs or TGF-β1 treatment induced iron accumulation in human lung fibroblasts as determined by live cell ferrous dye staining using a SiRhoNox-1 probe. Knockdown of SLC40A1 enhanced the iron-induced lung fibroblast activation. In summary, we conclude that the downregulation of SLC40A1 caused by TGF-β1 induces iron accumulation in lung fibroblasts, resulting in fibroblast activation.

© 2025. The Author(s).

In plain English

This laboratory study examined why iron accumulates in lung fibroblasts in idiopathic pulmonary fibrosis (IPF). The authors stained lung sections from IPF patients and from mice given bleomycin (1 U/kg, 14 days) or crocidolite asbestos (200 µg per mouse, 21 days, with TiO2 as the control particle) using DAB-enhanced Prussian blue. They reported that iron was significantly increased in fibrotic regions compared with non-fibrotic regions in the IPF lungs and in both mouse models; in IPF lung sections, iron partially overlapped with the fibroblast marker vimentin. Using publicly available single cell RNA-sequencing datasets, the iron export gene SLC40A1 was identified as downregulated in IPF fibroblasts, and this was confirmed by real-time PCR in fibroblasts from IPF patients and bleomycin-treated mice. TGF-β1 (5 ng/ml, 48 h) reduced SLC40A1 mRNA to 0.15 plus or minus 0.09 of control, acting through SMAD3. SLC40A1 knockdown or TGF-β1 treatment raised cellular ferrous iron in human lung fibroblasts given added iron. Knockdown lowered baseline alpha smooth muscle actin, an activation marker. However, the rise in this marker with added iron, measured against each group's own no-iron level, was significantly greater in knockdown cells than in control cells.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining human and mouse lung tissue staining, mouse fibrosis models, analysis of published single cell RNA-sequencing datasets, and human lung fibroblast cell-culture experiments
• Who was studied: IPF patient lung tissue sections (Lung Tissue Research Consortium); human pulmonary fibroblasts from 5 healthy subjects and 4 IPF patients; lung fibroblasts from 9 PBS-treated and 9 bleomycin-treated mice; 8-week-old C57BL/6 and C57BL/6J mice; commercial human pulmonary fibroblast and HEK 293 T cell lines
• Exposure: In mice, intratracheal crocidolite asbestos 200 µg per mouse (TiO2 200 µg as control particle) or bleomycin 1 U/kg. In cells, TGF-β1 and other cytokines, ferric ammonium citrate, ferrous ammonium sulfate, deferoxamine, and lentiviral shRNA knockdown of SLC40A1, SLC39A8, SMAD2 or SMAD3
• Main results:
  • Iron staining was significantly increased in fibrotic regions compared with non-fibrotic regions in IPF lungs and in the bleomycin and asbestos mouse models
  • In human IPF lung sections, iron co-staining with the fibroblast marker vimentin showed partial overlap
  • SLC40A1 mRNA was significantly lower in fibroblasts from IPF patients and from bleomycin-treated mice than in control fibroblasts
  • In human pulmonary fibroblasts, TGF-β1 reduced SLC40A1 mRNA to 0.15 plus or minus 0.09 of the control; TNF-α reduced it to a lesser extent, and other cytokines had no significant effect
  • Knockdown of SMAD3, but not SMAD2, partially reversed the TGF-β1-induced downregulation of SLC40A1, and ChIP showed that TGF-β1 induced SMAD3 binding to the SLC40A1 promoter
  • In human pulmonary fibroblasts, iron supplementation (FAC) increased SLC40A1 mRNA, while the chelator deferoxamine reduced SLC40A1 mRNA
  • Lentiviral shRNA knockdown of SLC40A1 lowered SLC40A1 protein and increased ferrous iron levels induced by FAC, FAC plus ascorbic acid or FAS in human pulmonary fibroblasts
  • TGF-β1 treatment increased cellular ferrous iron levels induced by all three iron compounds in human pulmonary fibroblasts
  • SLC40A1 knockdown decreased alpha smooth muscle actin expression without FAC
  • Iron-induced alpha smooth muscle actin mRNA was significantly higher in SLC40A1 knockdown fibroblasts than in shCON cells when each was normalized to its own 0 FAC level
  • FAC at 250 µM for 48 h had no significant effect on the senescence markers P53, P16 and P21 in human pulmonary fibroblasts
• The authors' conclusion: The authors concluded that TGF-β1-SMAD3 signalling downregulates SLC40A1 in fibrotic lung fibroblasts, which induces iron accumulation and fibroblast activation
• Limitations the authors state:
  • The authors stated that iron accumulation may trigger ferroptotic signalling in fibroblasts, and that future work with ferroptosis markers is needed to distinguish fibroblast activation from ferroptotic stress
  • The authors stated that the specific role of SLC40A1 in myofibroblast activation remains to be elucidated

Topics

Medical terms in this study, explained in our Lexicon: Fibroblast, Reactive Oxygen Species (ROS), Idiopathic, Pulmonary Fibrosis, Fibrosis, Cytokine.

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