Abstract
A hallmark of pulmonary fibrosis is the aberrant activation of lung fibroblasts into pathological fibroblasts that produce excessive extracellular matrix1,2,3. Thus, the identification of key regulators that promote the generation of pathological fibroblasts can inform the development of effective countermeasures against disease progression. Here we use two mouse models of pulmonary fibrosis to show that LEPR+ fibroblasts that arise during alveologenesis include SCUBE2+ alveolar fibroblasts as a major constituent. These alveolar fibroblasts in turn contribute substantially to CTHRC1+POSTN+ pathological fibroblasts. Genetic ablation of POSTN+ pathological fibroblasts attenuates fibrosis. Comprehensive analyses of scRNA-seq and scATAC-seq data reveal that RUNX2 is a key regulator of the expression of fibrotic genes. Consistently, conditional deletion of Runx2 with LeprcreERT2 or Scube2creERT2 reduces the generation of pathological fibroblasts, extracellular matrix deposition and pulmonary fibrosis. Therefore, LEPR+ cells that include SCUBE2+ alveolar fibroblasts are a key source of pathological fibroblasts, and targeting Runx2 provides a potential treatment option for pulmonary fibrosis.
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Data availability
The scRNA-seq and scATAC-seq data generated in this study have been deposited into the GEO (accession numbers GSE229523, GSE276546 and GSE278419). The publicly available scRNA-seq data for mouse lung development (accession numbers GSE160876 and GSE165063), normal adult mouse lungs (accession numbers GSE132771, GSE201698 and GSE211713), bleomycin-challenged mouse lungs (accession numbers GSE131800, GSE132771, GSE183545 and GSE201698), silica-exposed mouse lungs (accession GSE184854), CCl4-treated mouse liver (accession number GSE171904), human patients with IPF (accession number GSE136831) and bulk RNA-seq data for human patients with IPF (accession numbers GSE124685 and GSE134692) were used for analyses. Source data are provided with this paper.
Code availability
The codes used in this study for scRNA-seq and scATAC-seq analyses are available from the corresponding authors upon request. No custom code was generated.
Change history
17 June 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41586-025-09254-x
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Acknowledgements
We thank the colleagues in the Que Laboratory for their critical input to the study. Runx2flox/flox mouse strain (acc.no. CDB0832K) was kindly obtained from RIKEN BioResource Research Center (www2.clst.riken.jp/arg/mutant%20mice%20list.html). This work is partly supported by NIH grants R01HL152293, R01HL159675 and Department of Defense grant W81XWH2110196 (to J.Q.), NIH grants R01HL172990 and P01HL108793 (to D.J.), American Heart Association award 24CDA1268568 and the Pulmonary Fibrosis Foundation Scholars Program 1272558 (to X.L.). Flow cytometry was performed at the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core at Columbia University Medical Center, supported in part by the Office of the Director, National Institutes of Health under the awards S10RR027050 and S10OD020056. The CCHD microscopy core is supported by S10 OD032447 from the NIH. This research was also funded in part through the NIH/NCI Cancer Center Support Grant P30CA013696 and used the Genetically Modified Mouse Models/GMMMSR Core and the Genomics and High Throughput Screening Shared Resource.
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Contributions
Y.F. and J.Q. designed experiments, analysed data and wrote the manuscript. Y.F., C.-S.L. and J.Q. generated the LeprcreERT2 and Higd1bcreERT2 knock-in mouse lines. Y.F. performed immunostaining, imaging, flow cytometry and mouse genetics. S.S.W.C. performed immunostaining, imaging and mouse genetics. Y.F. and C.X. performed scRNA-seq analyses. L.X., R.L. and Y.K. performed scATAC-seq analyses. K.Y., Y.H., X.L., D.J., T. Takarada, T. Tsukui and D.S. provided materials. A.S. and H.H. oversaw and performed tissue collection. X.S. and J.Q. supervised this work. All authors reviewed and approved the final manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Lepr+ mesenchymal cells in the developing and adult mouse lung.
a-b, UMAP plot showing the different cell populations (a) in the lungs collected at E18.5, P0, P3, P7 and P14 (b). Re-analysis of the datasets GSE160876 and GSE165063. c, UMAP plot showing expression of Lepr in the developing lungs. d, UMAP plot showing the integration of lung mesenchymal cells at E18.5, P0, P3, P7 and P14. e, Dot plot showing the representative markers for each mesenchymal cell population. f, tdTomato (tdT) expression in the lungs of Lepr-Cre;R26tdT mice at different postnatal stages. P: Postnatal. g, tdT and α-SMA expression in the lungs of Lepr-Cre;R26tdT mice at different postnatal stages. The arrows indicate rare tdT+ smooth muscle cells. aw: airway, bv: blood vessel. h, Schematic depicting the generation of Lepr-CreERT2 mouse strain. i-j, Schematic diagram for tamoxifen injection and the representative immunostaining image of tdT in the lungs of Lepr-CreERT2;R26tdT mice at P14 (i) and P20 (j). k, Schematic depicting the use of tamoxifen-containing chow to feed Lepr-CreERT2;R26tdT mice and representative immunostaining images of surfactant protein C (SPC) and tdT in lung tissues. l, UMAP plot showing different mesenchymal populations from the adult control lungs that integrate the datasets GSE132771, GSE201698 and GSE211713. m, Dot plot showing the representative markers for each mesenchymal population. n-o, UMAP plot showing expression level of Lepr (n) and Scube2 (o). p, Blended feature plots showing co-expression of Lepr and Scube2. q, Representative images of tdT immunostaining and Scube2 RNA in situ hybridization. r-s, Quantification analysis showing the percentage of Scube2+ tdT+ cells in total tdT+ cells (r, n = 5) and in Scube2+ alveolar fibroblasts (s, n = 5). Data are representative of at least three independent experiments. Data are mean ± SEM. Scale bars: 100 µm (20 µm in magnified views).
Extended Data Fig. 2 Lepr-Cre labeled cells contribute to pathological fibroblasts.
a, Representative images of migrated tdT+EGFP+ and tdT-EGFP+ lung mesenchymal cells triggered by PDGF-BB. b, Quantification of the migrated cells (DAPI+) in a (tdT+EGFP+: n = 3, tdT-EGFP+: n = 3). c, Relative expression of matrix genes in tdT+EGFP+ and tdT-EGFP+ lung mesenchyme (RT-qPCR) (tdT+EGFP+: n = 3, tdT-EGFP+: n = 3). d, Expression of Acta2, Col1a1 and Ccn2 in tdT+EGFP+ and tdT-EGFP+ lung mesenchymal cells treated with/without TGF-β1 (RT-qPCR) (n = 3 for each group). e, tdT and EGFP expression in the lungs of Lepr-Cre;R26tdT;Col1a1-EGFP mice challenged with saline or bleomycin. f, Quantification of tdT+ cells (Saline: n = 6, Bleomycin: n = 6). g-h, Quantification of the proportion of α-SMA+tdT+ cells among α-SMA+ cells (g) and among tdT+ cells (h) (Saline: n = 6, Bleomycin: n = 6). i, Gating strategy to identify α-SMA+ tdT+ cells in live mesenchymal cells. j, FACS analysis of tdT+ and α-SMA+ cells isolated from the lung of Lepr-Cre;R26tdT mice treated with saline or bleomycin. k, Flow cytometric quantification for the percentage of α-SMA+ tdT+ cells in the lungs of Lepr-Cre;R26tdT mice treated with saline or bleomycin (Saline: n = 3, Bleomycin: n = 4). l, Experimental schematic and representative H&E images. m, α-SMA+ tdT+ cells in the lungs of Lepr-Cre;R26tdT mice exposed to saline or silica. n, Quantification of α-SMA+ tdT+ cells (Saline: n = 6, Silica: n = 6). o, Experimental schematic and representative H&E images. p, α-SMA and tdT expression in lung tissues. q, Quantification of α-SMA+ tdT+ cells in the lungs of Lepr-CreERT2;R26tdT mice following silica exposure (Saline: n = 6, Silica: n = 6). Data are representative of at least three independent experiments. Data are mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test with Welch’s correction (b, c, f, g, h, k, n, q) and two-way analysis of variance with multiple comparisons test with Sidak’s correction (d). Scale bars: l, o: 200 µm, a, e, m, p: 100 µm (e, m, p: 20 µm in magnified views).
Extended Data Fig. 3 scRNA-seq reveals the contribution of Lepr-Cre labeled alveolar fibroblasts to pathological fibroblasts.
a, Schematic diagram for scRNA-seq experimental design. b, UMAP plot showing the transcript levels of tdTomato. c, UMAP plot showing different cell populations. d, Red dots showing the cells from saline-treated lungs and blue dots showing the cells from bleomycin-treated lungs. e, Dot plot showing the representative markers for each population. f, UMAP plot showing four different fibroblast populations. g, Red and blue dots showing the cells from saline- and bleomycin-treated lungs, respectively. h, Dot plot showing the representative markers for each fibroblast population. i, Frequency of each fibroblast population in saline and bleomycin-treated lungs. j, UMAP plot showing the score of alveolar fibroblast signature. k-l, UMAP plot showing the expression level of the alveolar fibroblast markers Npnt (k) and Scube2 (l). m, UMAP plot showing the adventitial fibroblast signature score. n, UMAP plot showing the expression level of the adventitial fibroblast marker Pi16. o, Representative immunostaining image of Pi16 and tdT in the adventitial cuffs. aw: airway. p, UMAP plot showing the pathological fibroblast signature score. q, UMAP plot showing the expression level of the pathological fibroblast marker Cthrc1. r, Representative immunostaining image of Postn and tdT in the lungs of Lepr-Cre;R26tdT mice treated with bleomycin. s, UMAP plot showing pseudotime analysis with Monocle3. t-u, Trajectory analysis with Monocle2 showing cells ordered by pseudotime (t) and cell type (u). v, UMAP plot showing the expression of Lepr in the mesenchymal cells of the lungs isolated from Lepr-Cre;R26tdT mice challenged with saline or bleomycin. Data are representative of at least three independent experiments. Scale bars: o and r: 100 µm (o: 20 µm in magnified views).
Extended Data Fig. 4 scRNA-seq analysis reveals the heterogeneous lung fibroblasts labeled by Lepr-CreERT2.
a, Gating strategy to sort tdT+ lung mesenchymal cells (CD45-EpCAM-CD31-) from the lungs of Lepr-CreERT2;R26tdT mice treated with bleomycin or saline for scRNA-seq. b, tdTomato transcript levels are shown by UMAP plot. c, UMAP plot showing multiple Lepr-CreERT2 labeled cell populations. d, Red and blue dots showing the cells from saline- and bleomycin-treated lungs, respectively. e, Dot plot showing the representative markers for each population. f, Violin plot showing the significantly increased fibrosis-associated genes upon bleomycin challenge. g, 3D UMAP plot showing four different Lepr-CreERT2 labeled fibroblast populations. h-i, UMAP plot showing the expression level of the alveolar fibroblast markers Npnt (h) and Inmt (i). j, UMAP plot showing the adventitial fibroblast signature score. k-l, UMAP plot showing the expression level of the adventitial fibroblast markers Dcn (k) and Pi16 (l). m, Schematic depicting for the use of tamoxifen (Tmx)-containing chow to feed Lepr-CreERT2;R26tdT mice and representative immunostaining image of Pi16 and tdT in the adventitial cuffs. aw: airway. bv: blood vessel. n, Schematic depicting for the use of Tmx-containing chow and bleomycin challenge of Lepr-CreERT2;R26tdT mice, and representative images of Postn+ tdT+ pathological fibroblasts in lung tissues. Data are representative of at least three independent experiments. Statistical analysis was performed using two-sided Wilcoxon Rank Sum test (f). Scale bars: m and n: 100 µm (20 µm in magnified views).
Extended Data Fig. 5 Lepr-CreERT2 labeled alveolar fibroblasts generate pathological fibroblasts.
a, The cell distribution of Lepr-CreERT2 labeled fibroblast clusters along with the color-coded pseudotime (upper panel). Heatmap showing dynamic gene expression in fibroblast clusters (lower panel). Representative genes (left) and enriched pathways (right) in alveolar fibroblasts and pathological fibroblasts are shown, respectively. b, The expression levels of the alveolar fibroblast makers Scube2, Npnt, Inmt and the pathological fibroblast markers Cthrc1, Postn along pseudotime analysis. c-d, The top ten GO terms (c) and KEGG pathways (d) associated with genes enriched in pathological fibroblasts. e, UMAP plot showing the enrichment of Tgfβ1 transcript in pathological fibroblasts. f, UMAP plot showing the expression of Lepr transcript in lung mesenchymal cells of Lepr-CreERT2;R26tdT mice challenged with saline or bleomycin.
Extended Data Fig. 6 A novel Higd1b-CreERT2 mouse strain specifically labels pericytes which rarely contribute to pathological fibroblasts during lung fibrosis.
a, UMAP plot showing different mesenchymal populations from the adult control lungs that integrate the datasets GSE132771, GSE201698 and GSE211713. b-c, UMAP plot showing the expression levels of Higd1b (b) and Cox4i2 (c) in pericytes. Of note, 88% pericytes expressed Higd1b. d, Schematic depicting the generation of Higd1b-CreERT2 mouse strain. e, Schematic diagram for Tmx injection of adult Higd1b-CreERT2;R26-tdT mice and representative images showing immunostaining of pericyte marker NG2 and tdT. f, Schematic diagram for Tmx injection and bleomycin challenge of adult Higd1b-CreERT2;R26-tdT mice and representative images showing immunostaining of α-SMA, ERG and tdT. g, Representative images of two examples showing immunostaining of Cthrc1 and tdT in the lungs of Higd1b-CreERT2;R26-tdT mice challenged with bleomycin. h, Quantification analysis showing the rare contribution of pericytes to Cthrc1+ pathological fibroblasts in the lungs of Higd1b-CreERT2;R26-tdT mice (n = 6). Data are representative of at least three independent experiments. Data are mean ± SEM. i-j, Representative images showing immunostaining of tdT, Collagen I (i) and Ki67 (j) in the lungs of Higd1b-CreERT2;R26-tdT mice challenged with bleomycin. Scale bars: 100 µm (20 µm in magnified views).
Extended Data Fig. 7 Postn+ pathological fibroblasts are identified in silica-challenged lungs.
a, UMAP plot showing four different lung fibroblast populations in silica-treated lungs. The results were generated by re-analysis of the dataset GSE184854. b, Dot plot showing the representative markers for each fibroblast population. c-d, Postn expression shown by UMAP plot (c) and Violin Plot (d). e, Schematic diagram for Tmx injection and silica exposure of Postn-CreER;R26tdT mice and representative images showing immunostaining of α-SMA and tdT in lung tissues. f, Quantification analysis showing increased tdT+ cells upon silica exposure (Saline: n = 6, Silica: n = 6). g, Representative images showing immunostaining of α-SMA, ERG and Endomucin in lung tissues. Data are representative of at least three independent experiments. Data are mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test with Welch’s correction. Scale bars: 100 µm (20 µm in magnified views).
Extended Data Fig. 8 The TF-binding motif enrichment identified by scATAC-seq.
a, UMAP plot showing the expression of Runx2 transcripts in lung mesenchymal cells from Lepr-Cre;R26tdT mice challenged with saline or bleomycin. b, Violin plot showing the increased expression of Runx2 in the lungs of Lepr-CreERT2;R26tdT mice challenged with bleomycin. c, Violin plot showing the enrichment of Runx2 in pathological fibroblasts and proliferating fibroblasts of bleomycin-treated Lepr-CreERT2;R26tdT mouse lungs. d, Schematic depicting the experimental design for scATAC-seq. e, UMAP plot of scATAC-seq showing various lung mesenchymal populations. f, Blue and red dots showing the cells from saline- and bleomycin-treated lungs, respectively. g, Heatmap showing the representative markers for each lung mesenchymal population. h-i, UMAP plot showing various lung mesenchymal populations (h) from bleomycin-treated (blue dots) and control (red dots) lungs (i) that integrate the published scRNA-seq datasets GSE131800, GSE132771, GSE183545 and GSE201698. j, Dot plot showing the representative markers for each fibroblast population. k-l, UMAP plot (k) and Violin Plot (l) showing the enrichment of Runx2 transcripts in pathological fibroblasts. m, RT-qPCR analysis of Runx2 transcripts in FACs-sorted tdT+ cells isolated from the lungs of Lepr-Cre;R26tdT mice treated with bleomycin or saline (Saline: n = 3, Bleomycin: n = 3). n-o, UMAP plot showing the quiescent and active hepatic stellate cells (n) of control (red dots) and CCl4-treated (blue dots) mice (o). The dataset GSE171904 was used for re-analysis. HSC: Hepatic stellate cell. p-q. UMAP plot showing the expression of Cthrc1 (p) and Runx2 (q) in hepatic stellate cells. Data are representative of at least three independent experiments. Data are mean ± SEM. Statistical analysis was performed using two-sided Wilcoxon Rank Sum test (b) and unpaired two-tailed t-test with Welch’s correction (m).
Extended Data Fig. 9 Conditional deletion of Runx2 attenuates bleomycin-induced lung fibrosis.
a,b, FACS analysis to quantify bone marrow monocytes among CD45+ cells (Lepr-Cre;Runx2+/+: n = 3, Lepr-Cre;Runx2f/f: n = 3). c,d, FACS analysis to quantify bone marrow neutrophils among CD45+ cells (Lepr-Cre;Runx2+/+: n = 3, Lepr-Cre;Runx2f/f: n = 3). e, Representative H&E staining images of untreated lungs from Lepr-Cre;Runx2+/+ and Lepr-Cre;Runx2f/f mice. f, Experimental schematic and representative H&E staining images of the lungs from Lepr-Cre;Runx2+/+ and Lepr-Cre;Runx2f/f mice after bleomycin challenge. g, Quantification of fibrotic areas in the lungs of Lepr-Cre;Runx2+/+ (n = 9) and Lepr-Cre;Runx2f/f mice (n = 9). h, Hydroxyproline content in the lungs of Lepr-Cre;Runx2+/+ (n = 7) and Lepr-Cre;Runx2f/f mice (n = 7) treated with bleomycin. i, α-SMA and SPC expression in the lungs of Lepr-Cre;Runx2+/+ and Lepr-Cre;Runx2f/f mice after bleomycin challenge. j, Quantification of α-SMA+ cells in the lungs of Lepr-Cre;Runx2+/+ (n = 6) and Lepr-Cre;Runx2f/f mice (n = 6). k, Experimental schematic and representative Picro-Sirius Red staining images. l, α-SMA and tdT expression in the lungs of Lepr-CreERT2;Runx2+/+;R26tdT and Lepr-CreERT2;Runx2f/f;R26tdT mice. m, Quantification of α-SMA+ cells in the lungs of Lepr-CreERT2;Runx2+/+;R26tdT (n = 6) and Lepr-CreERT2;Runx2f/f;R26tdT mice (n = 6). n, Experimental schematic and representative Picro-Sirius Red staining images. o, α-SMA and tdT expression in the lungs of Scube2-CreERT2;Runx2+/+;R26tdT and Scube2-CreERT2;Runx2f/f;R26tdT mice. p, Quantification of α-SMA+ cells in the lungs of Scube2-CreERT2;Runx2+/+;R26tdT (n = 6) and Scube2-CreERT2;Runx2f/f;R26tdT mice (n = 6). Data are representative of at least three independent experiments. Data are mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test with Welch’s correction. Scale bars: e, f, k and n:1 mm (e and f: 200 µm in magnified views, k and n: 100 µm in magnified views), i, l and o: 100 µm (20 µm in magnified views).
Extended Data Fig. 10 Increased expression of RUNX2 in IPF lungs.
a, Separated UMAP plots showing fibroblasts from normal human lungs and IPF lungs. These results were generated through the re-analysis of the dataset GSE136831. b, Dot plot of scRNA-seq showing the representative markers for each fibroblast population. c-f, UMAP plot showing the expression levels of LEPR (c), SCUBE2 (d), COL1A1 (e) and ACTA2 (f). g, Violin plot of scRNA-seq showing the expression levels of ECM associated genes. h, Venn diagram showing the common and differential genes expressed in pathological fibroblasts of human IPF and bleomycin-treated mouse lungs. i, Increased transcript levels of RUNX2 in IPF lungs revealed by re-analyzing the bulk RNA seq dataset GSE134692 (Normal: n = 26, IPF: n = 46). Data are mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test with Welch’s correction.
Supplementary information
Supplementary Fig. 1
Gating strategy used to identify CD45+Ly6G–CD11b+Ly6C+ monocytes and CD45+Ly6G–CD11b+Ly6C+ neutrophils in mouse bone marrow.
Supplementary Table 1
Top 100 differentially expressed genes in Leprcre-labelled lung fibroblast populations.
Supplementary Table 2
Top 100 differentially expressed genes in LeprcreERT2-labelled lung fibroblast populations.
Supplementary Table 3
Marker genes of pathological fibroblast in human IPF and mouse bleomycin-treated lungs.
Supplementary Table 4
Primers used for RT–qPCR.
Supplementary Table 5
Oligonucleotide sequences of siRNA.
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Fang, Y., Chung, S.S.W., Xu, L. et al. RUNX2 promotes fibrosis via an alveolar-to-pathological fibroblast transition. Nature 640, 221–230 (2025). https://doi.org/10.1038/s41586-024-08542-2
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DOI: https://doi.org/10.1038/s41586-024-08542-2
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