Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun 1;128(6):2389-2405.
doi: 10.1172/JCI87631. Epub 2018 May 7.

FOXM1 is a critical driver of lung fibroblast activation and fibrogenesis

Loka R Penke  1 , Jennifer M Speth  1 , Vijaya L Dommeti  2 , Eric S White  1 , Ingrid L Bergin  3 , Marc Peters-Golden  1
Affiliations

FOXM1 is a critical driver of lung fibroblast activation and fibrogenesis

Loka R Penke et al. J Clin Invest. .

Abstract

While the transcription factor forkhead box M1 (FOXM1) is well known as a proto-oncogene, its potential role in lung fibroblast activation has never been explored. Here, we show that FOXM1 is more highly expressed in fibrotic than in normal lung fibroblasts in humans and mice. FOXM1 was required not only for cell proliferation in response to mitogens, but also for myofibroblast differentiation and apoptosis resistance elicited by TGF-β. The lipid mediator PGE2, acting via cAMP signaling, was identified as an endogenous negative regulator of FOXM1. Finally, genetic deletion of FOXM1 in fibroblasts or administration of the FOXM1 inhibitor Siomycin A in a therapeutic protocol attenuated bleomycin-induced pulmonary fibrosis. Our results identify FOXM1 as a driver of lung fibroblast activation and underscore the therapeutic potential of targeting FOXM1 for pulmonary fibrosis.

Keywords: Drug therapy; Fibrosis; Pulmonology; Signal transduction.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Fibrotic fibroblasts exhibit increased FOXM1 expression.
(A and B) Basal FOXM1 expression in fibroblasts grown from lungs of patients with IPF and control nonfibrotic lungs by qPCR analysis (A) and Western blot analysis (B). (C and D) Basal FOXM1 expression in fibroblasts grown from lungs of bleomycin- and saline-treated mice by qPCR analysis (C) and Western blot analysis (D). Each numeral in A and B denotes a single patient–derived cell line. In C, each symbol represents an individual murine-derived line of fibroblasts. Bars represent mean ± SEM. In B and D, each lane represents an individual patient- or murine-derived line of fibroblasts. *P < 0.05.
Figure 2
Figure 2. FOXM1 is critical for fibroblast proliferation.
(A) Time-dependent induction of FOXM1 mRNA (analyzed by qPCR) in CCL210 cells by FGF2 stimulation in the presence and absence of actinomycin D. (B) Representative Western blot (from 1 of 3 independent experiments) of FOXM1 expression in CCL210 cells after 24 hours of treatment with and without FGF2. (C) qPCR analysis of FOXM1 mRNA expression in IPF and nonfibrotic fibroblasts treated for 48 hours with and without FGF2; cells from a given patient-derived line are denoted by a distinct numeral, and mean ± SEM relative values are depicted below the graphs. *P < 0.05; **P < 0.01 vs. no FGF2 control, 2-tailed paired t test. (D) Basal proliferation of fibroblasts from lungs of patients with IPF and control nonfibrotic lungs assayed by the CyQUANT NF Cell Proliferation Assay at 72 hours after culture. For C and D, values are expressed relative to those of normal fibroblasts. (E) Expression of cell cycle–regulated genes CCND1, CCNB1, PLK1, and BIRC5 determined by qPCR in CCL210 cells transfected with FOXM1 overexpression plasmid or control plasmid. (F and G) Effect of 24-hour pretreatment with FOXM1 or control (Cont) siRNA on FGF2-induced expression of FOXM1 (F) and cell cycle–regulated genes (G) as determined by qPCR. (H and I) Effect of treatment with 2.5 μM Sio A on FGF2-induced expression of FOXM1 (H) and cell cycle–regulated genes (I). (J) Effect of Sio A on basal and FGF2-induced cell proliferation, as determined by the CyQUANT NF Cell Proliferation Assay at 72 hours in culture. (K) Effect of Sio A and the known proteasome inhibitor MG132 on proteasome activity in lung fibroblasts, as determined by the 20S proteasome activity assay. For D and J, control value represents fluorescence value of cells initially seeded. *P < 0.05; **P < 0.01, 2-way ANOVA.
Figure 3
Figure 3. FGF2 upregulates FOXM1 expression via a PI3K/PDK1/AKT signaling pathway.
(A) Representative Western blot (from 1 of 3 independent experiments) showing p-ERK1/2 and p-AKT in CCL210 cells stimulated with FGF2 for 15 minutes and 30 minutes. (B) qPCR analysis of FGF2-induced FOXM1 expression in cells pretreated with 10 μM PI3K inhibitor (LY294002), 1 μM PDK1 inhibitor (GSK 2334470), 5 μM AKT inhibitor (triciribine), or 5 μM ERK1/2 inhibitor (U0126). (C) qPCR analysis of PIK3C isoform expression (relative to that of PIK3CA) in fibroblasts at baseline. (D) qPCR analysis of FGF2-induced FOXM1 expression in cells pretreated with and without 10 μM PI3Kα inhibitor. (E) mRNA expression of FOXM1 and cell cycle–regulated genes CCND1, CCNB1, PLK1, and BIRC5 in cells transfected with a Myr-AKT plasmid or control plasmid. *P < 0.05; **P < 0.01, 2-way ANOVA. (F) Schematic illustrating the signaling pathway by which FGF2 induces FOXM1 expression in fibroblasts.
Figure 4
Figure 4. PGE2 acts as an endogenous brake for FOXM1 expression in lung fibroblasts.
(A) Effect of pretreatment for 30 minutes with 0.1 mM aspirin or 0.5 μM PGE2 on FGF2-induced proliferation in CCL210 cells, as measured using the CyQUANT NF Cell Proliferation Assay at 72 hours. The control value represents the fluorescence value of cells initially seeded. (B and C) qPCR analysis of FGF2-induced expression of cell cycle–regulated genes (B) and FOXM1 (C) in cells pretreated for 30 minutes with and without 0.5 μM PGE2. (D) Representative Western blot showing basal and FGF2-induced FOXM1 expression in cells pretreated with and without 0.5 μM PGE2. (E) qPCR analysis of FGF2-induced FOXM1 expression in cells pretreated for 30 minutes with and without 10 μM forskolin or 10 μM butaprost. (F and G) Effect of pretreatment for 1 hour with 0.1 mM aspirin on FGF2-induced expression of FOXM1 and CCNB1 evaluated by qPCR (F) and by Western blot (G). Western blots in D and G are representative of 3 independent experiments. *P < 0.05; **P < 0.01, 2-way ANOVA.
Figure 5
Figure 5. PGE2 prevents FOXM1 binding to promoters of target genes.
(A and B) CCL210 cells transfected with Myr-AKT plasmid for 24 hours and then treated with and without 0.5 μM PGE2 for an additional 24 hours (A) or 48 hours (B) and analyzed by qPCR for FOXM1 expression (A) and expression of cell cycle–regulated genes CCND1, CCNB1, PLK1, and BIRC5 (B). (C) qPCR analysis of expression of cell cycle–regulated genes in cells transfected with FOXM1 overexpression plasmid for 24 hours and then treated with and without 0.5 μM PGE2 for an additional 48 hours. (D) Effects of 30-minute pretreatment with and without 0.5 M PGE2 on FGF2-induced FOXM1 binding to the CCNB1 (left), PLK1 (middle), and BIRC5 (right) promoters, determined by ChIP-qPCR analysis. (E) Western blot analysis of FOXO3A and p-FOXO3A in cells treated for 30 minutes with and without FGF2 and 0.5 μM PGE2. The Western blot is representative of 3 independent experiments. (F) Effect of prior transfection for 24 hours with FOXO3A or control siRNA on the ability of 0.5 μM PGE2 to inhibit CCNB1 mRNA levels in cells expressing Myr-AKT. *P < 0.05; **P < 0.01, 2-way ANOVA. (G) Schematic illustrating the multiple mechanisms by which PGE2 inhibits FOXM1 signaling in fibroblasts.
Figure 6
Figure 6. FOXM1 inhibition prevents and reverses TGF-β–induced myofibroblast differentiation and sensitizes myofibroblasts to FasL-induced apoptosis.
(A–C) Effects of a 1-hour pretreatment of CCL210 cells with 2.5 μM Sio A (prevention protocol) on TGF-β–induced expression of α-SMA mRNA (A) and protein (B, Western blot; C, immunofluorescence microscopy) as well as collagen I protein (B). (D) Basal levels of ACTA2 mRNA in lung fibroblasts isolated from IPF patients or nonfibrotic controls (n = 5). (E) Effect of transfection with FOXM1 siRNA or scrambled (control) siRNA (for 16 hours) (left) or 30-minute pretreatment with 2.5 μM Sio A (right) on ACTA2 mRNA levels in IPF or control lung fibroblasts (n = 5) stimulated with and without TGF-β for 24 hours. (F) Immunofluorescence microscopic analysis of α-SMA expression in TGF-β–generated myofibroblasts treated with 2.5 μM Sio A for 24 hours (reversal protocol). (G) Effect of transfection with FOXM1 siRNA or scrambled (control) siRNA (for 16 hours) on FasL-induced apoptosis and expression of apoptosis-associated genes FAS, CASP3, and BIRC5 by Western blot analysis of TGF-β–generated myofibroblasts. (H and I) Effects of a 1-hour pretreatment with 2.5 μM Sio A on FasL-induced apoptosis in TGF-β–generated myofibroblasts, as determined by cleaved PARP expression assessed by Western blot (H) and by the frequency of annexin V staining assessed by flow cytometric analysis (I). (J) Effect of Sio A treatment for 24 hours on BIRC5 expression in nonfibrotic control and IPF fibroblasts. *P < 0.05, 2-way ANOVA. Images in C and D are representative immunofluorescence images showing α-SMA (green) and DAPI (blue). Original magnification, ×200. Western blots in B, G, and H are representative of 3 independent experiments. *P < 0.05, 2-way ANOVA.
Figure 7
Figure 7. Fibroblast-specific deletion of FOXM1 in a prevention protocol protects mice from bleomycin-induced fibrosis.
(A) Schematic illustrating the timelines for in vivo administration of tamoxifen and bleomycin and determination of experimental end points in the bleomycin model of pulmonary fibrosis. (B) Digital images of Masson’s trichrome staining for collagen deposition (blue) at day 21. Original magnification, ×200. Scale bars: 500 μm. (C–H) Effect of conditional fibroblast-specific FOXM1 deletion in mice treated with and without bleomycin, as reflected by changes in the Ashcroft histology score (C), lung hydroxyproline content (D), and the mRNA expression of fibrotic markers (Acta2, Col1a1, Ctgf, and Tgfb1) (E–H). In C–H, each symbol represents an individual mouse. Values in each group represent results from 2 pooled independent experiments with a total of 5 to 8 mice per group. *P < 0.05; **P < 0.01, 2-way ANOVA with Tukey’s multiple comparisons test.
Figure 8
Figure 8. Fibroblast-specific deletion of FOXM1 in a therapeutic protocol protects mice from bleomycin-induced fibrosis.
(A) Schematic illustrating the timelines for in vivo administration of bleomycin and delayed administration of tamoxifen and determination of experimental end points in the bleomycin model of pulmonary fibrosis. (B) Digital images of Masson’s trichrome staining for collagen deposition (blue) at day 21. Original magnification, ×200. Scale bars: 500 μm. (C–F) Effect of delayed fibroblast-specific FOXM1 deletion in mice treated with and without bleomycin, as reflected by changes in the Ashcroft histology score (C), lung hydroxyproline content (D), and the expression of fibrotic markers Acta2 (E) and Tgfb1 (F) mRNA. In C–F, each symbol represents an individual mouse. Values in each group represent results from 2 pooled independent experiments with a total of 5 to 8 mice per group. *P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test.
Figure 9
Figure 9. Sio A treatment in a therapeutic protocol ameliorates bleomycin-induced fibrosis in mice.
(A) Schematic illustrating the timelines for in vivo administration of bleomycin and Sio A, for determination of experimental end points, and for the pertinent phases of the pulmonary response in the bleomycin model of pulmonary fibrosis. (B) Digital images of Masson’s trichrome staining for collagen deposition (blue) at days 14 (upper panels) and 21 (lower panels) in mice treated with and without bleomycin and Sio A. Original magnification, ×200. Scale bars: 500 μm. (C–F) Effect of Sio A treatment in mice treated with and without bleomycin, as reflected by values determined at days 14 and 21 for the Ashcroft histology score (C), lung hydroxyproline content (D), mRNA expression of the myofibroblast marker Acta2 (E), and levels of active TGF-β (F). (G) 20S proteasome activity in lung homogenates harvested at day 14 from mice treated with and without bleomycin and with and without Sio A. In C–G, each symbol represents an individual mouse. Values in each group represent results from 2 pooled independent experiments with a total of 5 to 8 mice per group. *P < 0.05; **P < 0.01, 2-way ANOVA with Tukey’s multiple comparisons test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med. 2012;18(7):1028–1040. doi: 10.1038/nm.2807. - DOI - PMC - PubMed
    1. Raghu G, Selman M. Nintedanib and pirfenidone. New antifibrotic treatments indicated for idiopathic pulmonary fibrosis offer hopes and raises questions. Am J Respir Crit Care Med. 2015;191(3):252–254. doi: 10.1164/rccm.201411-2044ED. - DOI - PubMed
    1. Thannickal VJ, et al. Matrix biology of idiopathic pulmonary fibrosis: a workshop report of the national heart, lung, and blood institute. Am J Pathol. 2014;184(6):1643–1651. doi: 10.1016/j.ajpath.2014.02.003. - DOI - PMC - PubMed
    1. Xue L, Chiang L, He B, Zhao YY, Winoto A. FoxM1, a forkhead transcription factor is a master cell cycle regulator for mouse mature T cells but not double positive thymocytes. PLoS ONE. 2010;5(2):e9229. doi: 10.1371/journal.pone.0009229. - DOI - PMC - PubMed
    1. Zhang H, et al. The FoxM1 transcription factor is required to maintain pancreatic beta-cell mass. Mol Endocrinol. 2006;20(8):1853–1866. doi: 10.1210/me.2006-0056. - DOI - PubMed

Publication types

MeSH terms