Introduction
Idiopathic pulmonary fibrosis (IPF ) is a deadly disease with 3–5 years average survival after diagnosis [ 1 ]. It usually starts from the edge of the lungs and progresses to the whole lungs. Once fibrosis occurs, gas exchange of the patients is impaired. Along with dry cough, patients have difficulties to breathe, which affects the quality of life of the patients [ 2 ]. It is now believed that repeated alveolar epithelial cell injuries contribute to the progression of IPF. The injuries can be caused by many factors such as environmental exposures, genetic mutations, epigenetic changes, ageing and senescence [ 3 ]. Pirfenidone and nintedanib were approved for treating IPF in 2014 by the Food and Drug Administration. These two drugs slow the decrease in forced vital capacity and the progression of IPF but cannot cure the disease [ 4 , 5 ]. There is still no therapy to reverse pulmonary fibrosis. The molecular mechanisms leading to IPF are still unknown and need further studies to identify new therapeutic targets.
Several reports have shown that the bleomycin-induced lung fibrosis in mice is reduced by iron depletion using deferoxamine administered intranasally or clioquinol administered orally [ 6 – 8 ]. Airway fibrosis are also observed in the lungs of two iron-overload mouse models of Transferrin receptor 2 Y245X mutant ( Tfr2 mut/mut ) and hemochromatosis protein gene ( Hfe )–deficient mice ( Hfe −/− ) [ 6 ]. These studies suggest the role of iron in IPF.
Iron is a trace element that is indispensable to nearly all living organisms because it is required for the function of many proteins involved in a variety of biological processes [ 9 ]. Excess iron can result in tissue damage because of its role in the formation of reactive oxygen species [ 10 ]. Iron homeostasis is highly regulated via iron metabolism genes, which are controlled by iron regulatory proteins (IRPs) via the binding of iron responsive elements (IREs) in their 5'- or 3'-untransltional regions (UTRs) [ 11 – 13 ]. Iron is imported into cells by the transferrin receptor (TFRC) for transferrin-bound iron and by divalent metal transporter 1, ZIP 8 and 14 for non-transferrin bound iron, and iron is exported by SLC40A1 . TFRC functions as a sensor of circulating transferrin-bound iron, while hemochromatosis protein (HFE) interacts with transferrin receptors to modulate cellular iron uptake and also influences systemic hepcidin signaling. Cellular iron exists in a labile reactive iron pool and ferritin-bound nonreactive storage pool [ 11 – 13 ].
Solute Carrier Family 40 Member 1 (SLC40A1, also named ferroportin) is a transmembrane protein with 12 predicted transmembrane domains [ 14 ]. It is the only known gene involved in iron export. SLC40A1 contains an iron response element (IRE) on its 5’-UTR. When the intracellular iron level is high, the binding of iron with IRPs causes the release of IRP from IRE and thus an increase in SLC40A1 mRNA translation, resulting a reduction in the intercellular iron level [ 15 ]. Hepcidin is the major cytokine that regulates SLC40A1 level. It binds SLC40A1 to trigger its internalization and degradation [ 16 ]. The mutation of SLC40A1 causes impaired iron export and iron overload in cells [ 17 ]. Iron overload was observed in alveolar macrophages, lung epithelium cells and vascular smooth muscle cells in homozygous Slc40a1 C326S/C326S mice that harbor a point mutation of Slc40a1 [ 18 ].
Iron levels are elevated in the lungs of animals exposed to agents that cause lung fibrosis, including bleomycin, silica, and asbestos [ 19 , 20 ]. Iron accumulates in the alveolar epithelial lining fluid and alveolar macrophages in patients with IPF [ 21 , 22 ]. An increased proportion of airway macrophages lacking transferrin receptor was found in patients with IPF and was correlated with reduced survival [ 23 ], suggesting that other iron transporters may be responsible for iron accumulation. However, the mechanisms by which iron accumulates in fibrotic lungs are still unclear. Furthermore, iron accumulation in other lung cell types has not been carefully examined.
The objective of this study is to identify iron metabolism gene(s) that is responsible for iron accumulation in fibrotic lung fibroblasts. We found that iron accumulated and SLC40A1 was downregulated in human and mouse fibrotic lung fibroblasts. Transforming growth factor-β1 (TGF-β1), a major cytokine elevated in IPF, reduced SLC40A1 mRNA expression through the SMAD3 pathway. The knockdown of SLC40A1 increased the cellular ferrous iron level and the iron-induced fibroblast activation. We concluded that a reduction in SLC40A1 results in the iron accumulation in fibrotic lung fibroblasts and thus fibroblast activation.
Materials and methods
Cell culture
Human embryonic kidney 293 T (HEK 293 T) cells were purchased from American Type Culture Collection (Catalog no. CRL-1573; ATCC, Manassas, VA, USA). The gender, age, passage and source of healthy and IPF human pulmonary fibroblasts (HPF) that were used for iron metabolism gene validation (Fig. 2 ) were listed in Supplementary Table 1. For all other figures, experiments were conducted using a single HPF line (Catalog no, CC-2512; Lonza, Basel, Switzerland) and repeated 3–4 times across different passages. All cells were grown and maintained with the following media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S): HPF, Roswell Park Memorial Institute (RPMI)−1640 medium and HEK297T, Dulbecco's Modified Eagle (DMEM) medium.
Human lung tissues
IPF patient lung tissue sections were obtained from the Lung Tissue Research Consortium (LTRC).
Bleomycin and asbestos mouse models
The bleomycin and asbestos mouse models were performed as previously described [ 24 ]. Eight-week-old C57BL/6 mice were randomly assigned to two treatment groups: phosphate-buffered saline (PBS) or bleomycin. On Day 1, mice received an intratracheal instillation of either 60 µL bleomycin (1 U/kg body weight; Catalog no. B8416-15UN, Sigma, St. Louis, MO, USA) or PBS.
For the asbestos model, eight-week-old C57BL/6J mice were administered intratracheal instillations of either crocidolite asbestos (200 µg per mouse; kindly provided by Dr. Andy Ghio, U.S. Environmental Protection Agency) or the control particle, TiO₂ (200 µg per mouse; Sigma-Aldrich, St. Louis, MO, Cat. No. 1667585).
Lung tissues were collected on Day 14 for the bleomycin-treated mice and on Day 21 for the asbestos-treated mice for subsequent analyses.
Isolation of mouse lung fibroblasts
Mouse lung fibroblasts were isolated according to the previous report [ 25 , 26 ].The lungs were trimmed to remove major airways, rinsed with calcium- and magnesium-free Hanks' balanced salt solution (HBSS) and minced into 1 to 2 mm pieces. The lung tissues were then incubated in 0.25% trypsin in a water bath maintained at 37 °C for 30 min. At the end of the digestion, the dissociated cells were collected by centrifugation (250g at 4 °C for 10 min) and plated in a 100 mm cell culture dish. The cells were cultured in DMEM containing 10% FBS and 1% P/S for 5 days and used at passages 1 for RNA isolation.
Single cell RNA-sequencing (scRNA-seq) datasets
Publicly available single cell RNA-sequencing (scRNA-seq) datasets [ 27 ] ( http://ipfcellatlas.com/ ) were used to select the iron metabolism candidate genes that are changed in the lungs of IPF patients.
Real-time PCR
Total RNAs were extracted from cells using TRI Reagent® (Catalog no, TR 118; Molecular Research Center, Cincinnati, OH, USA). DNase-treated total RNAs (500 ng) were reverse-transcribed into single-strand complementary DNA using the M-MLV reverse transcriptase kit (Catalog no. 28025013; Invitrogen, Grand Island, NY, USA) with random primers and oligo dT. mRNA expression was determined using SYBR Green I–based real-time PCR. The real-time PCR thermal conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 60 sec. The primers used are listed in Supplementary Table E2. Data were analyzed using 2 -ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ).
Construction of plasmids
shRNA vectors: All the shRNAs were designed by the BLOCK-iT™ RNAi Designer software from Invitrogen. The shRNA sequences are listed in Supplementary Table E3. SLC40A1 , SLC39A8 , SMAD2 and SMAD3 shRNAs were inserted into the lentiviral pSIH-H1 vector (Catalog no. SI500A-1; System Biosciences, Mountain View, CA, USA) at BamHI and EcoRI, which utilizes the H1 promoter to drive shRNA expression. A control vector containing scrambled shRNA was purchased from System Biosciences.
SLC40A1 promoter reporter vector: The 5′-flanking region of SLC40A1 (−1883 to −11) was amplified by PCR using specific primers (Supplementary Table E3) from human genomic DNA. The fragments were inserted into the luciferase reporter vector pGL3-Basic (Catalog no. E1751; Promega, Madison, WI, USA) at XhoI and HindIII.
All vector sequences were confirmed by sequencing (DNA Protein Facility, Oklahoma State University, OK, USA).
Lentivirus production
Lentiviruses were produced in HEK293T cells [ 20 ]. The cells were transfected with 6 µg of packaging plasmid psPAX2 (Catalog no. 12260; Addgene, Watertown, MA, USA) and 3 µg of the envelope plasmid pMD2.G (Catalog no. 12259; Addgene) using 63 μl of 1 µg/µl polyethyleneimine (PEI). After 48 h of transfection, media containing viruses were collected. For titer determination, HEK 293 T cells were seeded at a density of 6 x 10 5 cells per well in a 12-well plate and were infected with serial dilutions of lentivirus particles. After 48 h of infection, virus titers were determined by counting green fluorescent protein (GFP) positive cells. For knockdown experiments, the viruses from 3 shRNAs were mixed equally and used to infect cells.
Iron staining
Iron staining was performed using a 3,3’-diaminobenzidine (DAB) enhanced Prussian blue stain as previously described [ 28 ]. Human IPF lung sections were obtained from the Lung Tissue Research Consortium. Bleomycin- (1 U/kg body weight for 14 days) and asbestos- or TiO 2 - (200 µg/mouse for 21 days) treated lung sections were from previous studies [ 20 , 29 ]. Human and mouse lung sections were dewaxed and rehydrated. The sections were then incubated in 1% H 2 O 2 for 15 min to remove endogenous peroxidase. After being washed for 15 min in running tap water, the sections were immersed in 2% HCl and 2% potassium ferrocyanide 1:1 at room temperature using the Prussian Blue Stain kit (ab150674, Abcam) for 30 min. The sections were washed three times, 10 min each, in distilled water. Sections were immersed in 0.05% DAB in PBS, pH 7.4, for 10 min, followed by immersion in 0.033% H 2 O 2 in 0.05% DAB in PBS, pH 7.4, for 10 min. When dual straining is needed, the slides were rinsed in water 2 times and PBS one time, 10 min each and blocked with 10% goat serum and 1% BSA in PBS for 1 h at room temperature. After removing the block solution, each slide was incubated in 200 µl anti-vimentin antibodies (1:100, ab92547, Abcam) in 1% BSA in PBS for 1 h at room temperature. After being washed three times with PBS for 5 min each, the slide was incubated with 200 µl of goat anti-Rabbit IgG cross-adsorbed secondary antibodies, Alexa Fluor 488 (1:100. A-1108, Thermo Fisher Scientific) for 45 min at room temperature. The slide was counterstained with hematoxylin Solution (Mayer's, Modified, ab220365, Abcam) for 2 min and mounted with proLong gold antifade mountant ( P36935 , ThermoFisher). The images were taken by a fluorescent microscope using 20x or 40x objective.
Western blot
Total proteins were harvested from cells using RIPA lysis buffer (Catalog no. PI89900, ThermoFisher Scientific, Waltham, MA, USA) supplemented with Protease and Phosphatase Inhibitor Cocktail (100X) (78440, Thermo Fisher Scientific). The protein samples were then subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Catalog no. 1620115; Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat dry milk dissolved in Tris-buffered saline with 0.1% Tween® 20 Detergent buffer (TTBS) for 1 h and then probed with antibodies of interest in 4℃ overnight. After being washing with TBST buffer for three times, 5 min each, the blots were probed with secondary antibodies and visualized by Super Signal West Pico Luminol Enhancer solution and Super Signal West Pico Stable Peroxidase solution (ThermoFisher Scientific). Primary antibodies and dilutions used for western blotting are: GAPDH (GTX100118, 1:5000, GeneTex), SLC40A1 (NBP1-21502, 1:1000, Novus Biologicals, Centennial, CO), and Smad2/3 (#8685, 1:1000, Cell Signaling). Secondary antibodies and dilutions used for western blotting are Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (#111-035-003, 1:5000, Jackson Immuno Research Laboratories, West Grove, PA, USA), and Peroxidase AffiniPure Goat Anti-mouse IgG (H+L) (#111-035-003, 1:5000, Jackson Immuno Research Laboratories).
Cytokine screen
HPFs (1x10 5 /well) were seeded into 12-well plates and treated with different cytokines and other compounds for 48 h: TNF-α (10 ng/ml, #300-01A, PeproTech, Cranbury, NJ, USA), lipopolysaccharides (LPS) (1,000 ng/ml, #00–4976-93, Invitrogen), TGF-β1 (5 ng/ml, #240-B, R&D Systems, Minneapolis, MN, USA), IL-4 (20 ng/ml, #200-04, PeproTech), IFN-γ (20 ng/ml, #300-02, PeproTech), IL-21 (25 ng/ml, #200-21, PeproTech), IL-13 (25 ng/ml, #200-13, PeproTech), IFN-β (1 ng/mL, #300-02BC, PeproTech), IL-25 (25 ng/ml, #1258-IL, R&D Systems), IL-33 (30 ng/ml, #200-33, PeproTech), WNT-1 (50 ng/ml, #120-17, PeproTech), IL-1β (10 ng/ml, #200-01B, PeproTech), IL-5 (20 ng/ml, #200-05, PeproTech), WNT-3a (50 ng/mL, #5036-WN, R&D Systems), WNT-5a (50 g/mL, #645-WN, R&D Systems), IL-6 (50 ng/ml, #200-06, Gibco), thrombin (10 nM, #RP-43100, Invitrogen), factor Xa (100 nM, #RP-43114, Invitrogen), ferric ammonium citrate (FAC) (200 µM, # RES20400 -A7, Sigma), deferoxamine (DFO) (10 µM #D9533, Sigma). For thrombin and factor Xa, HPFs serum-starved for 4 h or 24 h, respectively, before the treatment. SLC40A1 mRNA levels were determined by real-time PCR.
Promoter reporter assay
HPFs (5,000 per well) in 96-well plates were co-transfected with the pGL3-Basic vector containing the SLC40A1 promoter fragment and firefly luciferase gene (180 ng) along with pRL-TK plasmid containing Renilla luciferase gene (20 ng) using Lipofectamine™ 3000 (Invitrogen). After a 24-h culture, cells were treated with 100 μM FAC or 5 ng/ml TGF-β1 for 48 h. Firefly and Renilla luciferase activities in cell lysates were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). The relative firefly luciferase activities were normalized to the respective Renilla luciferase activities in each sample. The results are presented as the fold change of the ratio of firefly to Renilla luciferase activities.
Cellular ferrous iron measurement
Cellular ferrous iron levels were measured using the BioTracker Far-red Labile Fe 2+ Dye (Catalog no. SCT037; Sigma, St. Louis, MO, USA) live cell ferrous staining [ 30 ]. HPFs (0.5 x 10 5 /well) were seeded into 24-well plates and treated with shSLC40A1, shSLC39A8, or its control vector shCON lentivirus at an MOI of 50 for 72 h or TGF-β1 for 48 h. The cells were cultured in iron-free and serum-free RPMI-1640 media for 1 h and treated with 100 µM FAC, 100 µM ferrous ammonium sulfate (FAS) or 100 µM FAC plus 5 mM ascorbic acid for 3 h. The cells were washed with HBSS twice and incubated for 1 h with fresh 5 µM BioTracker Far-red Labile Fe 2+ Dye in HBSS made from 1 mM stock in DMSO, followed by incubation with 1 μg/ml DAPI for 20 min in the dark. After staining, the cell images were taken using Nikon AX R microscope under a Cy5 filter for red fluorescence. Integrated fluorescence intensity was analyzed by ImageJ software.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP Assay Kit (Catalog no. 17–295; Sigma, St. Louis, MO, USA) according to the instruction manual of the manufacturer. In brief, 1x10 6 HPFs in 100 mm cell culture dish were treated with TGF-β1 (5 ng/ml) for 48 h and cross-linked with 1% formaldehyde at room temperature for 10 min. The reaction was stopped by washing the cells with PBS twice and the cells were harvested using cell scrapers. The cells were then centrifuged, lysed with 300 µl SDS lysis buffer on ice and sonicated with 20% power and 3 s on and 10 s off mode for 18 times to achieve an average size between 200–1000 bp. The cell lysates were pre-cleaned with sonicated salmon sperm DNA and protein G agarose slurry (50%) for 2 h at 4°C. The pre-cleaned DNA suspensions were incubated with 3 μg of anti-Smad3 antibodies (Catalog no, ab208182; Abcam, Waltham, MA, USA) or 3 µg normal rabbit IgG as the negative control at 4 °C overnight. 100 µl salmon sperm DNA/protein G agarose slurry was added into the DNA suspensions and incubated for 1 h at room temperature. The agarose pellets were collected and washed. Smad3-containing chromatins were eluted with 250 μl elution buffer (1% SDS in 0.1 M NaHCO 3 ) and the crosslinks were reversed at 65 °C for 7 h. The chromatins were then digested with proteinase K (Catalog no. EO0492, ThermoFisher Scientific, Waltham, MA, USA) at 45 °C for 2 h. Chromatin DNAs were purified using QIAquick Spin Columns (Catalog no, 28115; Qiagene, Hilden, Germany) and eluted in 50 μl water. DNAs (10 μl/well) were used for real-time PCR using primers targeting the SMAD3 binding region on SLC40A1 (Supplementary Table E3).
Bioinformatics analysis
The SLC40A1 chromosome localization and promoter sequence were analyzed using the Genome browser ( http://genome.ucsc.edu ). Prediction of the SMAD3 binding site on SLC40A1 promoter was performed using version 8.3 of TRANSFAC software ( http://alggen.lsi.upc.es/ ).
Statistical analysis
The data presented in the figures represent the means ± standard error (SE). Statistical analyses were performed using Student’s t-test for two-group comparisons, and one-way analysis of variance (ANOVA), followed by Fisher's LSD test for multiple comparisons. A p-value <0.05 was considered significant.
Results
Iron accumulates in lung fibroblasts of IPF patients and bleomycin- and asbestos-induced lung fibrosis mouse models
To determine if iron accumulates in the lungs of IPF, we performed iron staining in the lung sections of human IPF patients or bleomycin- and asbestos-induced lung fibrosis mouse models. Compared to the non-fibrotic region, iron was significantly increased in the fibrotic regions of the lungs of IPF patients and two mouse lung fibrosis models (Fig. 1 a). By co-staining the human fibrotic region with iron and vimentin, a fibroblast marker, a partial overlap of iron and vimentin was observed (Fig. 1 b). The results indicate that iron accumulates in fibroblasts from fibrotic lungs.
SLC40A1 is identified as a downregulated gene in the fibroblasts isolated from the lungs of IPF and bleomycin-treated mice
To identify iron metabolic genes that are altered in IPF lung fibroblasts, we used publicly available single cell RNA-sequencing (scRNA-seq) datasets to select the candidate genes in myofibroblasts ( http://ipfcellatlas.com/ ). The iron metabolic genes can be classed as follows: iron uptake gene: transferrin receptor (TFRC), divalent metal transporter (SLC11A2), solute carrier family 39 member 8 (SLC39A8), solute carrier family 39 member 14 (SLC39A14), solute carrier family 11 member 1 (SLC11A1), hemoglobin scavenger receptor (CD163), solute carrier family 48 member 1 (SLC48A1), LDL receptor related protein 1 (LRP1), FLVCR heme transporter 2 (FLVCR2), scavenger receptor class A member 5 (SCARA5), lipocalin 2 (LCN2), transferrin (TF), actotransferrin (LTF). Iron export: SLC40A1 (ferroportin). Iron Storage: ferritin light chain (FTL), ferritin heavy chain 1 (FTH1). Among these genes, two iron uptake genes, SLC39A8 and SLC39A14 are highly upregulated and the iron export gene SLC40A1 is downregulated in IPF lung fibroblasts (Supplementary Figure 1a). Based on the expression level of these three genes across the human tissue ( https://gtexportal.org/ ), these three genes are relatively highly expressed in lung tissue (Supplementary Figure 1d). In alveolar epithelial type II cells and alveolar macrophages, none of the iron uptake genes were upregulated in IPF, except for CD163, which showed a slight increase in IPF alveolar macrophages. There were no differences in the expression levels of iron export or storage genes between control and IPF alveolar epithelial cells or alveolar macrophages (Supplementary Figure 1b, c).
To validate the identified candidate target genes, we performed real-time PCR analysis on the lung fibroblasts isolated from IPF patients and bleomycin-treated mice. We found that the iron export gene, SLC40A1 mRNA was significantly downregulated in the human and mouse fibrotic fibroblasts compared to the control group (Fig. 2 ). However, there were no differences in SLC39A8 and SLC39A14 mRNA expression between normal and fibrotic fibroblasts in both humans and mice except the SLC39A8 mRNA was increased in mouse fibrotic fibroblasts. The results suggest that the iron export gene, rather than iron uptake genes may be responsible for the iron accumulation in fibrotic fibroblasts.
TGF-β1 downregulates SLC40A1 via SMAD3
To identify the pathways for downregulating the SLC40A1 gene, we selected several cytokines to test their effects on SLC40A1 expression in human lung fibroblasts. The first group we selected is the cytokines related to cell proliferation, migration, and proliferation. TGF-β1 is the key factor elevated in IPF and involved in the fibroblast activation during the IPF progression [ 31 ]. WNT-1, WNT-3a and WNT-5a are the growth factors that play a key role in lung development and are highly expressed in IPF lung epithelium cells [ 32 ]. The second group we selected is the cytokines related to inflammation and immune response. IFN-γ, TNF-α, LPS and IFN-β have been shown to regulate the fibrocyte differentiation [ 33 ]. The IL-1β, IL-4, IL-5, IL-13, IL-21, IL-25 and IL-23 are all belongs to the interleukin family that are directly or indirectly involved in fibroblast activation and fibrosis development [ 34 , 35 ]. We found that TGF-β1 reduced SLC40A1 mRNA expression to 0.15 ± 0.09 of the control (Fig. 3 a). TNF-α also significantly decreased SLC40A1 mRNA expression although to less extent. Other cytokines had no significant effects on SLC40A1 mRNA levels.
Additionally, we examined the effects of other mediators known to induce fibroblast activation including thrombin, factor Xa and IL-6 [ 36 – 38 ]. However, none of these factors affected SLC40A1 expression (Fig. 3 b).
We then determined whether TGF-β1 affects the SLC40A1 promoter activity using a promoter luciferase reporter assay. HPFs were transfected with a SLC40A1 promoter luciferase reporter plasmid and treated with 5 ng/ml TGF-β1 for 48 h. The luciferase assay showed that TGF-β1 decreased the SLC40A1 promoter activity (Fig. 3 c).
The extracellular TGF-β1 binds the TGF-β receptor 1/2 (TGFR1/2) on the target cell membrane and induces the SMAD2/3 phosphorylation to regulate the expression of genes involved in fibroblast activation and collagen deposition [ 39 ]. Thus, we determined whether the TGF-β1-mediated SLC40A1 downregulation is through the SMAD pathway. We knocked down SMAD2 or SMAD3 in HPFs using a lentiviral shRNA and examined SLC40A1 mRNA expression upon TGF-β1 stimulation. shRNA SMAD2 reduced SMAD2 protein levels but had no effects on SMAD3 protein levels (Fig. 3 d). Similarly, SMAD3 only reduced SMAD3 protein levels, but not SMAD2, indicating a good knockdown efficiency and specificity of our shRNAs. Next, we treated the SMAD2- or SMAD3-knocked down HPFs with 5 ng/ml TGF-β1 for 48 h. The knockdown of SMAD3 but not SMAD2 partially reversed the TGF-β1-induced SLC40A1 downregulation (Fig. 3 e). Consistently, treatment with SIS3, a SMAD3 inhibitor also reversed TGF-β1-induced reduction in SLC40A1 expression (Fig. 3 f).
Using TRANSFAC software (version 8.3), we identified a SMAD3 binding site (−208, −199) on the SLC40A1 promoter. To confirm that SMAD3 can directly bind the SLC40A1 promoter, we performed ChIP assay. HPFs treated with 5 ng/ml TGF-β1 for 48 h were cross-linked with 1% formaldehyde and sonicated. The sonicated chromatin was immunoprecipitated with anti-SMAD3 or normal IgG. Real-time PCR analysis using the primers for the SMAD3 binding site on the SLC40A1 promoter revealed that TGF-β1 induced the binding of SMAD3 to the SLC40A1 promoter (Fig. 3 g). These results suggest that TGF-β1 downregulates SLC40A1 expression in HPFs through the TGF-β1-SMAD3 pathway.
Iron regulates SLC40A1 transcriptionally
SLC40A1 contains IREs in its 5’-UTR and thus is regulated by iron via protein translation [ 40 ]. Whether SLC40A1 is regulated at the transcriptional level is unknown. We supplement HPFs with iron using ferric ammonium citrate (FAC) or deplete iron with the iron chelator, deferoxamine (DFO) and examined the SLC40A1 mRNA levels in these cells. FAC treatment markedly increased SLC40A1 mRNA expression. In contrast, DFO treatment significantly reduced SLC40A1 mRNA levels (Fig. 4 a). FAC also increased the SLC40A1 promoter activities in HPFs as determined by a promoter luciferase reporter (Fig. 4 b). These results indicate that iron can directly regulate SLC40A1 gene expression at the transcriptional level.
Previous studies have shown that SLC40A1 has 6 different mRNA variants: I, IIA, IIB, IIIA. IIIB, and IIIC [ 41 , 42 ]. The main difference among the variants is at its 5’-UTR (Fig. 4 c). Only variant I (VI) contains IRE in its 5’-UTR. For the studies in Figs. 2 and 3 above, we used common primers located in coding region and thus can detect all the isoforms. To determine which isoforms are expressed in HPFs and affected by iron treatment, we designed specific primers targeting the unique region of each isoform to distinguish these isoforms (Fig. 4 c). Real-Time PCR analysis revealed that variant I is the major isoform in HPFs and induced by iron treatment (Fig. 4 d). Variant IIA was detectable but was much lower compared to Variant I. Variant IIA was also increased by iron treatment.
We further determined the combined effects of TGFβ1 and FAC on SLC40A1 expression. As expected, As TGF-β1 reduced and FAC increased SLC40A1 level. However, the addition of FAC did not overcome the TGF-β1’s downregulation effects (Fig. 4 e).
Knockdown of SLC40A1 increases cellular ferrous iron levels
To assess whether SLC40A1 downregulation can result in iron accumulation in lung fibroblasts, we performed live cell ferrous iron dye staining using a SiRhoNox-1 probe, which turns into red fluorescence when it binds ferrous iron in live cells [ 30 ]. FAC is an iron compound in ferric form. Extracellular ferric iron is covered into ferrous iron by duodenal cytochrome b (DcytB) and then enters cells via DMT1 [ 43 ]. Ferric iron can also be converted into ferrous iron by ascorbic acid (ASC) in vitro [ 44 ]. We supplemented HPFs with iron by the addition of FAC (ferric iron) or FAC and ascorbic acid (ASC) (ferrous iron converted from ferric iron) and measured cellular ferrous iron levels. After FAC treatment, ferrous iron in HPFs gradually increased and reached a peak at 180 min. Pretreatment of FAC with ASC significantly increased cellular ferrous iron levels at all the time points (Supplementary Figure 2). Based on these results, we chose 180 min incubation time for further experiments.
We then compared the cellular ferrous iron levels in HPFs supplemented with FAC/ASC, FAC and ferrous ammonium sulfate (FAS, ferrous iron). As shown in Supplemental Figure 3, The treatment of HPFs with FAS/ASC yield a highest level of ferrous iron while these with FAC and FAS are comparable.
We next knocked down SLC40A1 and examined its effects on cellular ferrous iron levels. The treatment of HPFs with lentiviral shRNAs resulted in a reduction in the SLC40A1 protein levels (Fig. 5 a). The decrease in SLC40A1 protein levels increased FAC, FAC/ASC or FAS-induced ferrous iron levels in HPFs (Fig. 5 b).
Because TGF-β1 decreased SLC40A1 protein levels HPFs (Fig. 3 b), we examined whether TGF-β1 can increase the cellular ferrous iron levels. Indeed, the treatment of HPFs with TGF-β1 increased the cellular ferrous iron levels induced by all the three iron compounds (Fig. 5 c).
Knockdown of SLC40A1 enhances iron-induced fibroblast activation
As the downregulation of SLC40A1 increases cellular ferrous iron levels, we determined the effects of SLC40A1 knockdown on fibroblast activation. We knocked down SLC40A1 in HPFs using shRNA lentivirus, treated cells with different doses of FAC and determined the mRNA levels of alpha smooth muscle actin (α-SMA), a fibroblast activation marker. Knockdown of SLC40A1 decreased the α-SMA expression in the absence of FAC (Fig. 6 a). Iron-induced α-SMA expression was significantly higher in the SLC40A1 knockdown cells compared with the shCON control cells when they were normalized to respective 0 FAC (Fig. 6 b). These results suggest that SLC40A1 knockdown can increase the sensitivity of cells in response to iron for fibroblast activation.
It has been reported that iron accumulation results in cell senescence [ 45 ]. We therefore determined the effect of FAC on cell senescence marker expression. As shown in Fig. 6 c, the treatment of HPFs with 250 µM FAC for 48 h had no significant effects on the cell senescence markers, P53 , P16 and P21 , suggesting the effect of SLC40A1 on lung fibroblast activation is not likely via cell senescence.
Discussion
Iron homeostasis is important for maintaining cellular activities. Increasing evidence suggests that iron contributes to IPF progression [ 6 – 8 ]. However, how iron accumulates in the lungs is unclear. In this study, we found that iron was significantly increased in lung fibroblasts of IPF and bleomycin- and asbestos-induced lung fibrosis mouse models. TGF-β1 downregulated SLC40A1 through the SMAD3 pathway. On the other hand, iron itself upregulated SLC40A1 transcriptionally to form a feedback loop. The downregulation of SLC40A1 increased the cellular ferrous iron levels, which resulted in fibroblast activation (Fig. 7 ).
In our studies, we found that iron accumulated in the fibrotic foci of the lungs from IPF patient and bleomycin- or asbestos-treated mice models. By co-staining iron with the fibroblast marker, vimentin, we confirmed that iron accumulated in lung fibroblasts of IPF patients. Earlier studies have shown that iron accumulates in alveolar macrophages in patients with IPF [ 21 , 22 ]. One recent study indicates that the labile iron pool is increased in the lung fibroblasts of bleomycin-treated mice [ 7 ]. Another study also demonstrates that ferrous iron accumulates in α-SMA-positive fibroblasts of bleomycin-treated mice for 7 days and 14 days [ 8 ]. Both observations are consistent with our current studies. An additional study shows that iron is increased in alveolar macrophages and epithelial type II cells of bleomycin-treated mice model [ 46 ]. It seems that iron can accumulate in multiple cell types in fibrotic lungs.
SLC40A1 is found in the apical membrane of human airway epithelial cells and expressed in human pulmonary artery smooth muscle cells [ 47 ]. In the current studies, we found that SLC40A1 was downregulated in alveolar fibroblasts from scRNA-seq data of IPF patients, which was further validated in the lung fibroblasts isolated from IPF patients and bleomycin-treated mice by real-time PCR. We also show that knockdown of SLC40A1 increased cellular ferrous iron in human lung fibroblasts, suggesting that downregulation of SLC40A1 may be responsible for the iron accumulation in fibrotic lung fibroblasts. A previous study has shown that CRISPR-mediated SLC40A1 knock out increases liable ferrous iron pool, the active form of iron in multiple myeloma cells [ 48 ].
SLC40A1 is a transmembrane protein located on cell surface and acts as the only known iron exporter to export non-heme iron [ 49 ]. The SLC40A1 expression is known to be regulated by the IRP/IRE system at the post-transcriptional level [ 15 ], and through hepcidin-triggered SLC40A1 internalization and degradation at the post-translational level [ 16 ]. In this study, we reported two new regulatory mechanisms of SLC40A1 at the transcriptional level. One is the downregulation by TGF-β1 and another is the upregulation by iron. TGF-β1 down-regulates SLC40A1 expression by inhibiting its transcription through the SMAD3 binding of its promoter. This is supported by a decrease in mRNA level, an increase in the promoter activity and an enhancement in the SMAD3 binding to the SLC40A1 promoter upon the TGF-β1 treatment. TGF-β1 has been shown to upregulates the iron uptake gene TFRC through TAZ-TEAD pathway in lung fibroblast [ 8 ].
Iron has been reported to increase the SLC40A1 protein levels by releasing IRPs from its 5’-UTR and thus relieving its inhibition on translation. Our current studies revealed that iron also increased the transcription of SLC40A1 because iron supplementation increased mRNA levels and promoter activities of SLC40A1. The mechanism by which iron induces the transcription SLC40A1 regulation needs further studies.
Iron plays a crucial role in many fundamental cellular tasks such as DNA replication and repair, mitochondrial function involving OXPHOS and enzymatic reactions that rely on iron as a cofactor. For example, iron overload reduces muscle cells insulin sensitivity and triggers insulin resistance, further decreasing glucose uptake by increasing ROS or decreasing autophagy [ 50 ]. Iron is also involved in the process of biosynthesis. Dioxygenase prolyl-4-hydroxylase is an important enzyme for collagen synthesis contain irons [ 51 ]. Our current studies showed that knockdown of SLC40A1 increased the sensitivity of iron-induced fibroblast activation. This is likely due to the increased ferrous iron levels.
There are two main limitations in the current studies. First, iron accumulation may trigger ferroptotic signaling in fibroblasts. Future research incorporating ferroptosis markers such as lipid peroxidation assays, GPX4 expression, and system Xc⁻ activity will be essential to distinguish between fibroblast activation and potential ferroptotic stress under conditions of SLC40A1 downregulation. Second, although current studies focus on the regulation of SLC40A1, the specific role of SLC40A1 in myofibroblast activation remains to be elucidated.
In conclusion, we identified SLC40A1 as a downregulated gene via the TGF-β1-SMAD3 signaling in fibrotic lung fibroblasts, which induces iron accumulation and fibroblast activation.
Supplementary Information
Supplementary Material 1.
Supplementary Material 2.
Supplementary Material 3.
Authors’ contributions
Quanjin Dang performed experiments, analyzed data and wrote the manuscript. Chaoqun Huang, Yurong Liang, Akshaya Surendran, Dharanya Muthiah, Kishore Vaddadi, and Sankha Hewawasam performed experiments and analyzed data. Tingting W Mills provided human lung fibroblasts and critically reviewed the manuscript. Lin Liu designed the concept of the paper, provided resources, analyzed data, and wrote the manuscript.
Funding
This work was supported by the National Institutes of Health grants, R01HL157450 and P30GM149368, and the Lundberg-Kienlen Endowment fund (to LL) and National Institutes of Health grant, R56AG076144 (to TM).
Data availability
All data generated or analyzed during this study are included in this article and its supplementary material files. Further enquiries can be directed to the corresponding author.
Declarations
Ethics approval and consent to participate
All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Oklahoma State University (Approval number: VM-21-81).
De-identified human lung samples from IPF patients were obtained through the lung transplantation programs at Memorial Hermann Hospital and The Houston Methodist Hospital. Collection of these tissues was approved by the respective institutional review boards under protocol numbers HSC-MS-15-1049 and HSC-MS-08-0354.
Consent for publication
All authors have read the final version of the manuscript and agree to the publication.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
Supplementary Materials
Supplementary Material 1.
Supplementary Material 2.
Supplementary Material 3.
Data Availability Statement
All data generated or analyzed during this study are included in this article and its supplementary material files. Further enquiries can be directed to the corresponding author.