SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

TL;DR

This study shows that SIRT3 helps prevent lung fibrosis by controlling FoxM1 activity in lung cells, offering new treatment possibilities.

Contribution

The study identifies a novel SIRT3-FoxM1 regulatory axis in pulmonary fibrosis and suggests therapeutic strategies via SIRT3 activation.

Findings

SIRT3 deacetylates FoxM1, reducing its stability and preventing fibroblast activation.

Nicotinamide riboside protects against fibrosis by activating SIRT3.

FoxM1 nuclear translocation promotes resistance to apoptosis in fibroblasts.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a life-threatening interstitial lung disease characterized by the abnormal activation of pulmonary fibroblasts. In our study, we demonstrated that FoxM1 is highly expressed in activated pulmonary fibroblasts, and its nuclear translocation plays a crucial role in conferring resistance to FasL-induced apoptosis in pulmonary fibroblasts. Disruption of FoxM1 function was shown to restore the ability to resolve fibrosis in mice treated with bleomycin. Mechanistic investigations revealed that a decrease in SIRT3 expression leads to increased acetylation of FoxM1, which is essential for the activation of pulmonary fibroblasts in vitro. Further, downregulation of SIRT3 expression enhances the stability of FoxM1, thereby accelerating bleomycin-induced pulmonary fibrosis through the activation of pulmonary fibroblasts. Importantly, treatment with nicotinamide riboside was found to suppress the activation of pulmonary fibroblasts and protect mice from bleomycin-induced pulmonary fibrosis by activating SIRT3. In summary, our findings highlight a critical role of the SIRT3/FoxM1 axis in regulating the activation of pulmonary fibroblasts. These insights suggest potential therapeutic strategies against pulmonary fibrosis, focusing on modulating this pathway for effective treatment. This work opens new avenues for the development of targeted therapies aimed at mitigating the progression of IPF.

In the progression of pulmonary fibrosis, decreased SIRT3 expression leads to elevated FoxM1 acetylation, which promotes its nuclear translocation and subsequently mediates the activation of pulmonary fibroblasts. Conversely, activation of SIRT3 by nicotinamide riboside (NR) or inhibition of FoxM1 nuclear translocation by RCM-1 effectively suppresses the activation of pulmonary fibroblasts.Image 1

•Enhanced FoxM1 expression is responsible for the anti-apoptosis of activated pulmonary fibroblasts.•Sirt3-dependent deacetylation of FoxM1 regulates the stability of FoxM1.•SIRT3 deficiency promotes pulmonary fibroblasts activation and accelerates bleomycin-induced pulmonary fibrosis.•Nicotinamide riboside protects mice from bleomycin-induced pulmonary fibrosis via activation of SIRT3.

Enhanced FoxM1 expression is responsible for the anti-apoptosis of activated pulmonary fibroblasts.

Sirt3-dependent deacetylation of FoxM1 regulates the stability of FoxM1.

SIRT3 deficiency promotes pulmonary fibroblasts activation and accelerates bleomycin-induced pulmonary fibrosis.

Nicotinamide riboside protects mice from bleomycin-induced pulmonary fibrosis via activation of SIRT3.

Figures

Full text

Introduction

1

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by a diverse group of lung disorders, which culminate in the progressive and irreversible destruction of lung architecture and disruption of gas exchange [1]. Given the limited therapeutic options available, significant efforts have been directed toward elucidating the mechanisms underlying the pathogenesis of pulmonary fibrosis [2]. It is hypothesized that the development of this condition involves chronic injury to alveolar epithelial cells, triggering an aberrant reparative response mediated by the secretion of various growth factors [[3], [4], [5]]. This process leads to fibroblast expansion and excessive extracellular matrix (ECM) production, resulting in the architectural remodeling of lung tissues, which adversely affects pulmonary function [6]. The failure of fibroblasts to return to a quiescent state drives excessive extracellular matrix (ECM) production and remodeling [[7], [8], [9]], ultimately exacerbating fibrotic disease and accelerating its progression toward end-organ failure and mortality [10]. Thus, the illustration of pulmonary fibrosis pathophysiology and the identification of novel targets are urgently required to design more effective treatments for pulmonary fibrosis.

Recently, growing evidences identified a strong correlation between Forkhead Box M1 (FoxM1) and the activation of pulmonary fibroblasts in the context of pulmonary fibrogenesis [11]. FoxM1 is commonly overexpressed in a wide range of human malignancies and is critically involved in multiple biological processes, including mammalian development, DNA damage repair, monocyte/macrophage recruitment, and tumorigenesis [[12], [13], [14], [15]]. It has been reported that FoxM1 interacts with multiple signaling pathways, directly or indirectly activating the transcription of target genes involved in various aspects of cell fate during both physiological and pathological processes [16]. Elevated FoxM1 expression can induce epithelial-mesenchymal transition and fibroblast proliferation, contributing to the progression of pulmonary fibrosis [17]. In our study, we observed both elevated levels and enhanced nuclear translocation of FoxM1 in activated pulmonary fibroblasts. However, the molecular mechanisms underlying the elevation of FoxM1 require further investigation.

Sirtuin 3 (SIRT3) is a mitochondrial member of the sirtuin family of NAD-dependent deacetylases, known for its diverse subcellular localization and broad activity on target proteins [18]. SIRT3 levels is decreased in the lung tissues of IPF patients [19]. The deficiency of SIRT3 has been implicated in the acceleration of aging processes, the development of cancer, and the onset of age-related neurodegenerative disorders [20], potentially exacerbating pulmonary epithelial senescence by increasing the acetylation of p53 [21]. Conversely, the administration of SIRT3 agonists has been shown to effectively delay the progression of pulmonary fibrosis [22]. Studies have demonstrated that FoxM1 acetylation plays a pivotal role in regulating its transactivation function and promoting tumor progression. Acetylated FoxM1 enhances transcriptional activity through mechanisms involving increased DNA binding affinity and improved protein stability [23]. However, whether decreased SIRT3 expression is responsible for enhanced FoxM1 expression in activated remains unknown.

In our study, we investigated whether aberration of FoxM1 is a critical molecular mechanism for the activation of pulmonary fibroblasts and the subsequent progression of pulmonary fibrosis. Our work revealed that FoxM1 was aberrantly upregulated in activated pulmonary fibroblasts, which mediated the resistance of fibroblasts to FasL-induced apoptosis. In addition, we demonstrated that SIRT3-mediated deacetylation of FoxM1 could regulate the stability of FoxM1, and suppress the nuclear translocation of FoxM1 in pulmonary fibroblasts. Restoration of SIRT3 via nicotinamide could effectively protect mice from bleomycin-induced pulmonary fibrosis. Therefore, our findings identify a novel and potent mediator of fibrotic processes, highlighting the therapeutic potential of targeting the SIRT3/FoxM1 axis as a promising strategy for the treatment of pulmonary fibrosis.

Materials and methods

2

Animal study

2.1

C57BL/6J male mice aged 8-weeks and SIRT3^flox/flox^ mice were purchased from the Model Animal Research Center of Nanjing University. Mice were housed under controlled environmental conditions, including a temperature of 25 ± 2 °C and a 12-h light/dark cycle, with ad libitum access to food and water. All experimental protocols involving animals were conducted in compliance with ethical standards and were approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Drum Tower Hospital, Nanjing University Medical School. For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group. The control group received an equivalent volume of sterile saline following the same administration protocol.

To explore the role of FoxM1 in pulmonary fibrosis, mice were treated with RCM-1 (MedChem Express, San Diego, CA, USA), which is an inhibitor of FoxM1 [24]. RCM-1 (1 mg/kg) or vehicle was injected intraperitoneally every other day from Day 7 to Day 13. To investigate the role of SIRT3 in pulmonary fibrosis, SIRT3^flox/flox^ mice were intratracheally injected with 50 μl AAV-Cre (1 × 10^13^ v.g./ml) 14 days before BLM treatment. To further investigate the effect of NR on pulmonary fibrosis, mice were oral gavaged with NR at a dose of 400 mg/kg/d for 2 weeks. Mice were sacrificed 14 days after BLM injection, and lung tissues were collected for further analysis.

Primary pulmonary fibroblast isolation

2.2

Primary pulmonary fibroblasts were isolated using a previously established protocol [25]. Briefly, mice were euthanized via cervical dislocation, and lung tissues were dissected and minced into 1 mm^3^ fragments. The tissue fragments were enzymatically digested at 37 °C for 30 min under constant agitation using a solution containing 2.4 U/mL dispase (Sigma, St. Louis, USA), 0.2% collagenase I (Sigma), and 0.001% DNase (Sigma). The digested mixture was washed three times with 10 ml of warm DMEM/F12 medium supplemented with 15% fetal bovine serum (FBS, WISENT, Nanjing, China). The resulting pellet was resuspended in 10 ml of warm DMEM/F12 medium with 10% FBS and transferred to a 10 cm tissue culture dish. Cells were maintained in a humidified incubator at 37 °C with 5% CO_2_. After 14 days of culture, the cells were harvested and replated at a density of 5 × 10^5^ cells per plate in EMEM medium containing 10% FBS.

Cell viability assay

2.3

Pulmonary fibroblasts isolated from lung tissues of normal mice or bleomycin (BLM)-treated mice were seeded in 96-well plates at a density of 5 × 10^4^ cells/ml and cultured in serum-free medium with or without FasL (100 ng/mL) for 24 h. After treatment, 10 μl of CCK-8 solution (Beyotime Institute of Biotechnology, Shanghai, China) was added directly to each well, followed by incubation at 37 °C for 2–4 h. Absorbance was measured at 450 nm using a multi-detection microplate reader (BioTek Instruments, USA).

Caspase 3 activity, ROS and TUNEL assay

2.4

For Caspase 3 activity assay, pulmonary fibroblasts subjected to specified treatments, including TGF-β1 (10 ng/ml), LV-FoxM1 or si-FoxM1 transfection, were seeded in 6-well culture plate, along with or without FasL treatment for 24 h. Cells were lysed in ice-cold lysis buffer, and lysates were centrifuged at 12,000×g for 10 min at 4 °C. Supernatants were collected and incubated with the caspase-3-specific substrate Ac-DEVD-pNA (2 mM, Beyotime Institute of Biotechnology, Shanghai, China) in 96-well plates at 37 °C for 2 h. Caspase-3 activity was quantified by measuring absorbance at 405 nm using a multi-detection microplate reader (BioTek Instruments, USA).

Cellular ROS levels were assessed using the Highly Sensitive DCFH-DA ROS Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, pulmonary fibroblasts subjected to the indicated treatments were incubated with DCFH-DA dye (1 μmol/L) at 37 °C for 30 min. Following incubation, cells were washed twice with PBS and immediately imaged using a laser scanning confocal microscope (Olympus, Japan).

For the TUNEL assay, pulmonary fibroblasts were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in sodium citrate buffer for 2 min on ice. After washing with PBS, cells were incubated with the TUNEL reaction mixture (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 60 min in a humidified chamber protected from light. Nuclei were counterstained with DAPI for visualization.

EdU assay

2.5

Pulmonary fibroblasts with indicated treatment were harvested and seeded in six-well plates along with EdU (KeyGEN BIOTECH, Nanjing, China) at the concentration of 500 μM for 2 h. Then, cells were fixed with 4% paraformaldehyde and 0.5 mL click reaction solution was added to each well for EdU staining. The nucleus was stained with DAPI (Solarbio, Beijing, China). The images of cell proliferation were captured by confocal fluorescence microscopy (Olympus, Tokyo, Japan).

Co-immunoprecipitation (Co-IP)

2.6

Co-IP was carried out according to a previously established protocol [26]. The total cell lysate lysates were first subjected to immunoprecipitation with Acetylated-Lysine Antibody (CST, Boston, USA). Subsequently, the immunoprecipitated complexes were analyzed by western blotting with antibody against FoxM1 (Selleckchem, Houston, USA).

Western blot

2.7

Protein lysates were prepared by homogenizing samples in RIPA buffer containing a protease inhibitor cocktail on ice. Nuclear extracts were prepared by using a cytoplasmic and nuclear protein isolation kit (Beyotime, Shanghai, China). Western blotting was conducted following established protocols [27]. Briefly, proteins were separated by electrophoresis and subsequently transferred onto PVDF membranes (Millipore, Boston, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, followed by washing and incubation with the appropriate primary antibodies at 4 °C overnight. The primary antibodies used were: FoxM1, α-SMA, SIRT3 and Lamin B1 (Selleckchem, Houston, USA), Collagen I (Proteintech, Wuhan, China) and GAPDH (ABclonal, Wuhan, China). The second day, membranes were further incubated with horseradish peroxidase-conjugated IgG to mark the signals from the primary antibody. The blots were visualized with an ECL plus western blotting detection system (New Cell & Molecular Biotech, Suzhou, China), and the protein quantities were analyzed by ImageJ software. The relative protein levels were adjusted with the housekeeping protein GAPDH. The detail information of antibodies was provided in Supplement Table S2.

Quantitative RT–PCR

2.8

Total RNA was isolated from mouse lung tissues or cultured cells using TRIzol Reagent (Vazyme, Nanjing, China). Reverse transcription was performed using a HiScript 1st Strand cDNA Synthesis Kit (Vazyme) according to the manufacturer's instructions. Comparative quantitative PCR (q-PCR) was performed by using the NovoStart® Universal Fast SYBR qPCR SuperMix (Novoprotein, Shanghai, China). Primers were listed in Table S1. The Ct values were analyzed using the ΔΔCt method, and relative changes in mRNA levels were obtained by normalization to Gapdh relative to the control.

Histopathology and hydroxyproline assay

2.9

The lung tissues derived from experimental pulmonary fibrosis models were fixed in 4% paraformaldehyde solution overnight and embedded in paraffin before sectioning into 5 μm-thick slices. The lung sections were stained with hematoxylin-eosin (H&E) for structure observation or used for detection of collagen deposition by Masson's trichrome staining, which were visualized under an Olympus BX53F microscope. Lung fibrosis was assessed by analyzing six distinct tissue slices from each mouse using the Ashcroft scoring system [28], with the average score used to grade the severity of fibrosis. In addition, the hydroxyproline (HYP) content of mouse lung tissues were measured to quantify lung collagen contents and determined colourimetrically with a hydroxyproline assay kit (KGT030-2, KeyGen Biotech, Nanjing, China) following the manufacturer's instructions.

Immunofluorescence staining

2.10

For immunofluorescence analysis, lung tissue sections or cultured cells were permeabilized using 0.3% Triton X-100 (Sigma) for 5 min and subsequently blocked with 5% bovine serum albumin (BSA, Sigma) for 1 h at room temperature. Primary antibodies were applied to the samples and incubated overnight at 4 °C. After washing, the samples were incubated with fluorophore-conjugated secondary antibodies (Invitrogen, Carlsbad, USA) for 1 h at 37 °C. Nuclei were counterstained with DAPI (Sigma), and images were acquired using a confocal fluorescence microscope (Olympus, Tokyo, Japan). For the quantification of the numbers of FoxM1 and α-SMA double-positive cells, samples were captured at 6 random vision fields and the mean numbers of FoxM1 positive pulmonary fibroblasts were calculated by using imageJ software.

Statistical analysis

2.11

The data are presented as the mean values ± standard error of the mean (SEM) by using Prism 8 (GraphPad Software, La Jolla, CA). Statistical analyses were performed using Student's t-test for comparisons between two groups and one-way ANOVA followed by post-hoc tests for multiple group comparisons via SPASS (SPASS, Chicago, IL). P values were considered statistically significant at ∗P < 0.05.

Results

3

• 1.FoxM1 is highly expressed in fibroblasts of fibrotic lung tissues.

Recently, growing evidences have demonstrated that FoxM1 played an essential role in the progression of pulmonary fibrosis [17]. In our work, we observed significantly elevated protein levels of FoxM1 in lung tissues from IPF patients and mice treated with BLM (Fig. 1A–D). According to the RNA-seq results from dataset GSE24206, we found that FoxM1 expression was positively correlated with the expression of COL1A1, a hallmark of tissue fibrosis (Fig. 1E). Additionally, increased FoxM1 levels in pulmonary fibrosis mouse models were tightly associated with aggravated fibrosis, as indicated by Ashcroft scores (Fig. 1F). The public single-cell RNA sequencing dataset for IPF lung tissues (GSE122960) demonstrated that FOXM1 expression was significantly upregulated in fibroblasts derived from IPF lung tissues compared to those from donor lung tissues (Fig. S1). In fibrotic lungs, fibroblasts, which produce large quantities of extracellular matrix proteins such as collagens, are primarily implicated in tissue fibrosis. The results of immunofluorescence showed enhanced co-localization of α-SMA and FoxM1 in the fibrotic lung tissues (Fig. 1G, H, S2, S3). In vitro, the expression of FoxM1 was also profoundly increased in pulmonary fibroblasts isolated from the lung tissues of BLM-treated mice (Fig. 1I). Furthermore, we demonstrated that FoxM1 was significantly upregulated in TGF-β1-induced activated pulmonary fibroblasts (Fig. 1J). Overall, these data indicate that FoxM1 may be a relevant contributor to fibroblast activation and pulmonary fibrogenesis.

• 2.Enhanced FoxM1 expression is responsible for the resistance of fibroblasts to FasL-mediated apoptosis Fig. 1FoxM1 is highly expressed in fibroblasts of fibrotic lung tissues. (A, B) qPCR (n = 9) and Western blot (n = 6) analysis of the expression of FoxM1 in normal and IPF lung tissues. ∗P < 0.05. (C) qPCR analysis of the mRNA levels of FoxM1 in the lung tissues from BLM-treated mice. n = 3, ∗P < 0.05. (D) Western blot analysis of the protein levels of FoxM1, CTHRC1, α-SMA, and Collagen I in the lung tissues from bleomycin (BLM)-treated mice. n = 3, ∗P < 0.05. (E) The Pearson's correlation analysis of COL1A1 expression with FoxM1 expression based on the RNA-seq results of GSE24206 from GEO database. (F) The Pearson's correlation analysis of Ashcroft score with FoxM1 expression in the lung tissues from BLM-treated mice. (G) Representative images of co-immunostaining for α-SMA and FoxM1 in IPF lung tissues. White arrows indicate double-positive cells. (H) Representative images of co-immunostaining for α-SMA and FoxM1 in the lung tissues from BLM-treated mice. White arrows indicate double-positive cells. (I) Western blot analysis of FoxM1 expression in pulmonary fibroblasts isolated from mice subjected to BLM treatment. n = 3, ∗P < 0.05. (J) Western blot analysis of FoxM1, CTHRC1, α-SMA, and Collagen I expression in pulmonary fibroblasts treated with TGF-β1. n = 3, ∗P < 0.05. All data were presented as the means ± SEM. Paired t-test (A, B, I) and one-way ANOVA with Tukey's post-hoc test (C, D, J) were used for statistical analysis.Fig. 1

It is reported that the evasion of apoptosis by fibroblasts is a characteristic feature of fibrotic diseases [29]. In our study, fibroblasts were isolated and treated with FasL, a potent activator of the extrinsic apoptotic pathway [30]. Our results showed that FasL could extensively increase the ROS levels and induce apoptosis via enhancing the caspase 3 activity in normal pulmonary fibroblast, but not in fibroblasts isolated from lung tissues of BLM-treated mice (Fig. 2A and B, S4A). In addition, we demonstrated that TGF-β1 treatment extensively impaired FasL-induced elevation of ROS and apoptosis in pulmonary fibroblasts, which indicated that fibrotic pulmonary fibroblasts possess antioxidant and anti-apoptotic properties (Fig. 2C, D, S4B). To further investigate whether FoxM1 played an essential role in modulating the apoptotic response of fibroblasts to FasL, we conducted a gain-of-function and loss-of-function analysis of FoxM1 in pulmonary fibroblasts (Fig. 2E and F). We demonstrated that overexpression of FoxM1 could suppress FasL-induced elevation of ROS and apoptosis (Fig. 2G, H, S4C), whereas inhibition of FoxM1 could effectively impair the antioxidant and anti-apoptotic properties of fibrotic pulmonary fibroblasts (Fig. 2I, J, S4D). These results suggest that modulating FoxM1 prevents fibrotic pulmonary fibroblasts from evading apoptosis by compromising their antioxidant defenses during the progression of pulmonary fibrosis.

• 3.Impairing nuclear translocation of FoxM1 suppresses fibroblast activation and protects mice from bleomycin-induced pulmonary fibrosis Fig. 2Enhanced FoxM1 expression is responsible for the resistance of fibroblasts to FasL-mediated apoptosis.(A) Caspase 3 activity analysis in pulmonary fibroblasts isolated from bleomycin (BLM)-treated mice following FasL treatment. (B) Cell viability assessment using the CCK-8 assay in pulmonary fibroblasts isolated from BLM-treated mice after FasL treatment. (C) Caspase 3 activity analysis in TGF-β1-treated pulmonary fibroblasts, along with or without FasL treatment. (D) Cell viability assessment using the CCK-8 assay in TGF-β1-treated pulmonary fibroblasts, along with or without FasL treatment.(E) Western blot analysis of FoxM1 expression in pulmonary fibroblasts transfected with or without LV-FoxM1. (F) Western blot analysis of FoxM1 expression in pulmonary fibroblasts transfected with or without FoxM1 siRNA (si-FoxM1). (G, H) Caspase 3 activity and cell viability assessment in pulmonary fibroblasts transfected with or without LV-FoxM1, following with or without FasL treatment. (I, J) Caspase 3 activity and cell viability assessment in pulmonary fibroblasts transfected with or without si-FoxM1, following with or without FasL treatment. All data (n = 3, ∗P < 0.05) were presented as the means ± SEM. Paired t-test was used for statistical analysis.Fig. 2

Taking into account that FoxM1 was a typical proliferation-associated transcription factor, we measured the nuclear expression of FoxM1, and observed an enhanced nuclear FoxM1 levels in fibrotic pulmonary fibroblasts (Fig. 3A, S5A). To further confirm whether impairing the nuclear translocation of FoxM1 could suppress the activation of fibroblasts and attenuate pulmonary fibrogenesis, RCM-1, a nontoxic small molecule that could prevent the nuclear localization and increase the proteasome degradation of FoxM1, was used in our study. RCM-1 effectively decreased the nuclear levels of FoxM1 in pulmonary fibroblasts treated with TGF-β1 (Fig. 3B–S5B). RCM-1 treatment significantly suppressed TGF-β1-induced activation of pulmonary fibroblasts, as demonstrated by the marked reduction in the expression levels of CTHRC1, α-SMA and collagen I (Fig. 3B). Notably, FoxM1 inhibition led to a decline in the proliferation ability of pulmonary fibroblasts, which was exacerbated in the presence of TGF-β1 (Fig. 3C, D, S6). In vivo, we further demonstrated that RCM-1 evidently decreased the extent of lung lesions, attenuated collagen deposition and decreased hydroxyproline content (Fig. 3E–G, S7), which resulted in elevated survival of BLM-treated mice (Fig. 3H). Immunolabeling analysis revealed that RCM-1 treatment significantly reduced the expression of fibrotic markers, including CTHRC1, α-SMA and collagen I (Fig. 3I), along with decreased FoxM1 levels and impaired nuclear localization of FoxM1 (Fig. 3I–S8). These results demonstrated that blocking the nuclear translocation of FoxM1 with RCM-1 could suppress pulmonary fibroblast activation and protect mice from BLM-induced pulmonary fibrosis.

• 4.Acetylation of FoxM1 is required for the activation of pulmonary fibroblasts Fig. 3Impairing nuclear translocation of FoxM1 suppresses fibroblast activation and protects mice from bleomycin-induced pulmonary fibrosis. (A) Western blot analysis was performed to assess the nuclear expression levels of FoxM1 in pulmonary fibroblasts isolated from BLM-treated mice. n = 3, ∗P < 0.05. (B) Western blot analysis of nuclear FoxM1, CTHRC1, α-SMA, and Collagen I expression in TGF-β1-treated pulmonary fibroblasts accompany with or without RCM-1 treatment. n = 3, ∗P < 0.05. (C, D) EdU assay for the proliferation of TGF-β1-treated pulmonary fibroblasts accompany with or without RCM-1 treatment. n = 3, ∗P < 0.05. (E) Hematoxylin–eosin (H&E) and masson's trichrome staining for the lung tissues from BLM-treated mice injected with or without RCM-1. (F, G) The ashcroft score (n = 6, ∗P < 0.05.) and hydroxyproline contents (n = 6, ∗P < 0.05.) in the lung tissues of BLM-treated mice injected with or without RCM-1. (H) The survival of BLM-treated mice injected with or without RCM-1. n = 18. (I) Western blot analysis of FoxM1, CTHRC1, α-SMA, and Collagen I expression in the lung tissues from BLM-treated mice injected with or without RCM-1. n = 3, ∗P < 0.05. All data were presented as the means ± SEM. Paired t-test (A) and one-way ANOVA with Tukey's post-hoc test (B, D, F–I) were used for statistical analysis.Fig. 3

To further uncover the underlying mechanism of elevated FoxM1 expression in pulmonary fibrogenesis, we measured the nuclear expression of FoxM1 in fibroblasts treated with ribosome inhibitor CHX (10 μM) and proteasomal inhibitor MG132 (10 μM). Our results showed that inhibiting the degradation of FoxM1 could sustain the nuclear expression of FoxM1 (Fig. 4A and B). In addition, we observed that the half-life of FoxM1 was greatly prolonged in TGF-β1-treated fibroblasts (Fig. 4C), which indicated that post-transcriptional modification may enhance the stability of FoxM1 in activated fibroblasts. Previous reports indicate that acetylation could prevent protein ubiquitylation and thereby inhibit proteasome-dependent degradation [31]. As shown in Fig. 4D and E, the acetylation of FoxM1 was notably enhanced in fibroblasts isolated from fibrotic lung tissues and in fibroblasts treated with TGF-β1. Additionally, the NAM treatment, an inhibitor of the SIRT family deacetylases, significantly induced the acetylation and nuclear translocation of FoxM1, accompanied by elevated expression of CTHRC1, α-SMA and Collagen I (Fig. 4F, G, S9). These results indicated that FoxM1 acetylation plays a critical role in promoting the activation of pulmonary fibroblasts in vitro.

• 5.Sirt3-dependent deacetylation of FoxM1 regulates the stability of FoxM1 Fig. 4Acetylation of FoxM1 is required for the activation of pulmonary fibroblasts. (A, B) Western blot analysis of FoxM1 expression in the cytoplasm and nucleus of CHX-treated pulmonary fibroblasts along with or without MG132 treatment at indicated time. n = 3, ∗P < 0.05. (C) Western blot analysis of FoxM1 expression in CHX-treated pulmonary fibroblasts along with or without TGF-β1 treatment. n = 3, ∗P < 0.05. (D) Western blot analysis of the acetylation levels of FoxM1 in pulmonary fibroblasts isolated from bleomycin (BLM)-treated mice. n = 3, ∗P < 0.05. (E) Western blot analysis of the acetylation levels of FoxM1 in pulmonary fibroblasts treated with or without TGF-β1. n = 3, ∗P < 0.05. (F, G) Western blot analysis of FoxM1 acetylation, CTHRC1, α-SMA, and Collagen I expression in pulmonary fibroblasts treated with TSA (50 nM), or NAM (1 mM) for 24 h, or not. n = 3, ∗P < 0.05. All data were presented as the means ± SEM. Paired t-test (D, E) and one-way ANOVA with Tukey's post-hoc test (B, C, G) were used for statistical analysis.Fig. 4

Given the elevated acetylation of FoxM1 in nicotinamide-treated pulmonary fibroblasts, we examined the relationship between SIRTs expression and COL1A1 expression using data from GSE2052, which indicated that the expression of SIRT3 may be negatively correlated with COL1A1 expression (Fig. 5A). We demonstrated that the protein levels of SIRT3 was significantly attenuated in fibrotic lung tissues (Fig. 5B). Overexpression of SIRT3 effectively decreased the acetylation levels of FOXM1 in vitro (Fig. S10). Importantly, the acetylation of FoxM1 was markedly increased in SIRT3^flox/flox^ mice following intratracheal injection with AAV-Cre (Fig. 5C). Impairing the expression of SIRT3 dramatically increased the acetylation of FoxM1 and prolonged the half-life of FoxM1 in vitro (Fig. 5D, E, S11). Furthermore, our results demonstrated that SIRT3 inhibition significantly upregulated the expression of α-SMA and Collagen I, concomitant with enhanced proliferation of pulmonary fibroblasts (Fig. 5F, G, S12). Oppositely, overexpression of SIRT3 effectively suppressed TGF-β1-mediated acetylation of FoxM1 and extensively decreased the expression of CTHRC1, α-SMA and Collagen I (Fig. 5H), which indicated that upregulation of SIRT3 could inhibit TGF-β1-induced fibroblast activation via promoting the deacetylation of FoxM1.

• 6.Sirt3 knockdown accelerates BLM-induced pulmonary fibrosis via activation pulmonary fibroblasts in vivo. Fig. 5Sirt3-dependent deacetylation of FoxM1 regulates the stability of FoxM1. (A) The Pearson's correlation analysis of COL1A1 expression with SIRTs expression based on the RNA-seq results of GSE2052 from GEO database. (B) Western blot analysis of SIRT3 expression in the lung tissues from bleomycin (BLM)-treated mice. n = 3, ∗P < 0.05. (C) Western blot analysis of the acetylation levels of FoxM1 in SIRT3^flox/flox^ mice intratracheally injected with AAV-Cre. n = 3, ∗P < 0.05. (D) Western blot analysis was performed to assess the acetylation status of FoxM1 in pulmonary fibroblasts following transfection with Sirt3 siRNA (si-Sirt3). n = 3, ∗P < 0.05. (E) Western blot analysis of FoxM1 expression in CHX-treated pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗P < 0.05. (F) Western blot analysis of CTHRC1, α-SMA and Collagen I expression in pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗P < 0.05. (G) EdU assay for the proliferation of pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗P < 0.05. (H) Western blot analysis of FoxM1 acetylation, CTHRC1, α-SMA, and Collagen I expression in TGF-β1-treated pulmonary fibroblasts transfected with or without LV-Sirt3. n = 3, ∗P < 0.05. All data were presented as the means ± SEM. Paired t-test (B-D, F, G) and one-way ANOVA with Tukey's post-hoc test (E, H) were used for statistical analysis.Fig. 5

Next, we continued to evaluate the effect of SIRT3 knockdown on the progression of pulmonary fibrosis. SIRT3^flox/flox^ mice were intratracheally injected with AAV-Cre (Fig. S13A). Our results showed that the expression of SIRT3 was dramatically decreased (Fig. 6D), accompany with increased alveolar septal thickening and collagen deposition (Fig. 6A). In addition, our data revealed that depletion of SIRT3 aggravated the BLM-induced interstitial fibrosis based on HE staining and Masson's trichrome staining (Fig. 6A, B, S13B), along with elevated fibrotic markers, including CTHRC1, α-SMA, Collagen I and hydroxyproline expression (Fig. 6C and D). Considering that the AAV-Cre-mediated SIRT3 knockdown was not specific to pulmonary fibroblasts, we further isolated the fibroblasts from the lung tissues of SIRT3^flox/flox^ mice injected with AAV-Cre. We demonstrated that AAV-Cre significantly decreased SIRT3 expression in the pulmonary fibroblasts, which further resulted in elevated CTHRC1, α-SMA and Collagen I expression, and enhanced proliferation ability (Fig. 6E–G, S14). Taken together, these results indicated that the loss of SIRT3 could promote the activation of pulmonary fibroblasts, and exacerbated BLM-induced pulmonary fibrosis.

• 7.Nicotinamide riboside protects mice from bleomycin-induced pulmonary fibrosis via activation of SIRT3 Fig. 6Sirt3 knockdown accelerates BLM-induced pulmonary fibrosis via activation pulmonary fibroblasts in vivo. (A) Hematoxylin–eosin (H&E) and masson's trichrome staining for the lung tissues from SIRT3^flox/flox^ mice or BLM-treated SIRT3^flox/flox^ mice that intratracheally injected with or without AAV-Cre. (B, C) The ashcroft score (n = 6, ∗P < 0.05.) and the hydroxyproline contents (n = 6, ∗P < 0.05.) in the lung tissues of mice treated as in Fig. 6A. (D) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in the lung tissues from mice treated as in Fig. 6A n = 3, ∗P < 0.05. (E) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in pulmonary fibroblasts isolated from mice treated as in Fig. 6A n = 3, ∗P < 0.05. (F, G) EdU assay for the proliferation of pulmonary fibroblasts isolated from mice treated as in Fig. 6A. All data were presented as the means ± SEM. One-way ANOVA with Tukey's post-hoc test was used for statistical analysis.Fig. 6

To further confirm whether restoring SIRT3 could alleviate the development of pulmonary fibrosis, nicotinamide riboside (NR, 0.5 mM), identified as a NAD^+^ precursor, was used to activate SIRT3 in our study. In vitro, NR treatment could increase the expression of SIRT3 in the pulmonary fibroblasts treated with TGF-β1, which dramatically suppressed the FoxM1, CTHRC1, α-SMA and Collagen I expression (Fig. 7A). Notably, we demonstrated that TGF-β1-mediated proliferation of pulmonary fibroblasts was effectively impaired with the treatment of NR (Fig. 7B), which indicated that NR could suppress the activation of pulmonary fibroblast via activating SIRT3. In vivo, NR evidently increased the survival of mice suffering from BLM-induced pulmonary fibrosis (Fig. 7C–S15A), and decreased the extent of lung lesions and attenuated collagen deposition (Fig. 7D, E, S15B). Immunolabeling also showed that NR profoundly increased SIRT3 expression, along with decreased FoxM1 levels and attenuated expression of fibrotic markers CTHRC1, α-SMA and collagen I and hydroxyproline expression (Fig. 7F–S16). In addition, the fibroblasts isolated form lung tissues of NR-treated mice also exhibited elevated levels of SIRT3, decreased expression of FoxM1, α-SMA and collagen I (Fig. 7G), and impaired proliferation ability (Fig. S17). Overall, our results demonstrated that activation of SIRT3 by NR could protect mice from BLM-induced pulmonary fibrosis via inhibition of pulmonary fibroblast activation.Fig. 7Nicotinamide riboside protects mice from bleomycin-induced pulmonary fibrosis via activation of SIRT3. (A) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in TGF-β1-treated pulmonary fibroblasts accompany with or without NR treatment. n = 3, ∗P < 0.05. (B) Cell viability assessment using the CCK-8 assay in pulmonary fibroblasts treated as in Fig. 7A n = 6, ∗P < 0.05. (C) The survival of BLM-treated mice oral gavaged with or without NR. n = 18. (D) Hematoxylin–eosin (H&E) and masson's trichrome staining for the lung tissues from mice treated as in Fig. 7C. (E) The ashcroft score of mice treated as in Fig. 7C n = 6, ∗P < 0.05. (F) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in the lung tissues from mice treated as in Fig. 7C n = 3, ∗P < 0.05. (G) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in pulmonary fibroblasts isolated from the lung tissues of mice treated as in Fig. 7C n = 3, ∗P < 0.05. All data were presented as the means ± SEM. One-way ANOVA with Tukey's post-hoc test was used for statistical analysis.Fig. 7

Discussion

4

IPF is a progressive and fatal disease characterized by a poor prognosis and high mortality rate. To date, lung transplantation is thought to be the only effective treatment [1]. Current therapeutic strategies for IPF have shown limited efficacy in significantly improving patient survival, underscoring the urgent need for novel clinical interventions. IPF is pathologically characterized by the persistent activation of fibroblasts, which drive ECM deposition and profound tissue remodeling. [32]. During fibrotic lung remodeling, fibroblasts derived from multiple cellular sources contribute to the formation of fibrotic lesions, including the proliferation of resident lung fibroblasts, the transformation of epithelial cells into fibroblasts, and the mobilization of bone marrow-derived circulating fibrocyte precursors [1,33,34]. Extensive research efforts have been dedicated to elucidating the molecular mechanisms underlying fibroblast survival, proliferation, and sustained activation in fibrotic processes.

Recent studies have provided evidence that epithelial cells undergo differentiation into FoxM1-positive fibroblasts during the progression of pulmonary fibrosis [35]. Fully activated fibroblasts exhibit a highly contractile phenotype and play a critical role in remodeling the surrounding ECM [36]. Therefore, the potential role of FoxM1 in fibroblast activation and its contribution to the progression of pulmonary fibrosis remain poorly understood. In this study, we aimed to elucidate the functional involvement of FoxM1 in pulmonary fibrosis and to evaluate the therapeutic implications of targeting FoxM1 in modulating fibrotic progression. FoxM1 belongs to the forkhead box transcription factor family, which plays an important role in regulating embryonic development, carcinogenesis and organ regeneration by promoting cell cycle progression [37]. It was reported that expression of FoxM1 protein is low in quiescent cells, and aberrantly overexpressed FoxM1 has been implicated in several lung diseases [[38], [39], [40], [41], [42]]. Conditional knockout of the FoxM1 gene or pharmacological inhibition of FoxM1 protein activity in bronchiolar progenitor cells conferred protection against allergic responses in mice, leading to a significant attenuation of mucus hyperplasia and pulmonary inflammation [43]. FoxM1 inhibition could sensitize IPF fibroblasts to radiation-induced cell death [44], which could also ameliorate fibrosis by decreasing extracellular matrix and epithelial-mesenchymal transition [45]. In our study, we also confirmed that FoxM1 inhibition effectively abrogates the antioxidant capacity and resistance to apoptosis of activated pulmonary fibroblasts. However, it was also reported that loss of FoxM1 in macrophage could promote pulmonary fibrosis [46]. Here, we detected an extensive increase and nuclear translocation of FoxM1 in fibrotic lung tissues. Disruption of FoxM1 nuclear translocation elicited antifibrotic effects in BLM-induced pulmonary fibrosis, thereby establishing a rationale for the development of novel therapeutic strategies targeting FoxM1 inhibition.

Previous studies have demonstrated that FoxM1 expression initiates during the late G1 phase, peaks at the early S phase, and persists throughout the G2 phase and mitosis, with its activity being primarily regulated through phosphorylation-dependent mechanisms [[47], [48], [49]]. The anaphase‐promoting complex/cyclosome E3 ubiquitin ligase could induce the degradation of FoxM1 in late mitosis and early G1 phase [50]. In our study, we observed enhanced stability of FoxM1 in activated pulmonary fibroblasts, which indicated the post-translational modification may regulate the degradation of FoxM1 in the progression of pulmonary fibrosis. It is well-established that FoxM1 function is tightly regulated through multiple post-translational modifications [51,52]. FoxM1 acetylation enhances its transactivation capacity, thereby promoting cell cycle progression, cellular proliferation, and tumorigenic processes [23]. In this study, we observed enhanced acetylation of FoxM1 in activated pulmonary fibroblasts. We demonstrated that decreased SIRT3 expression impaired the deacetylation of FoxM1 in activated pulmonary fibroblasts. Upregulation of SIRT3 could induce the deacetylation of FoxM1, and effectively suppressed TGF-β1-induced activation of pulmonary fibroblasts.

It has been reported that SIRT3, a mitochondrial deacetylase predominantly expressed in tissues with high metabolic demands, can also function within the nucleus to regulate stress-related gene expression [53], which could promote the resolution of lung fibrosis. Except for mitochondrial proteins, SIRT3 could regulate the acetylation of various transcription factors, including FoxO3a, p53 [54,55]. Growing evidences demonstrated the putative anti-fibrotic roles of SIRT3 in multiple organs systems [56]. Exogenous restitution of SIRT3 levels in aged mice could restore capacity for fibrosis resolution via recovering the susceptibility of fibroblasts to apoptosis [22]. SIRT3 ablation could lead to accelerated aging and age-related diseases [57]. In IPF patients, low levels of SIRT3 were observed in alveolar epithelial cells, which exerted anti-fibrotic effects via regulation of mitochondrial DNA damage and epithelial cell apoptosis, as well as inhibition of macrophages recruitment from monocytes into the alveoli [58]. In our study, we demonstrated that knockdown of SIRT3 could aggravate BLM-induced pulmonary fibrosis via enhancing the activation of pulmonary fibroblasts. Restoration of SIRT3 via nicotinamide effectively suppressed TGF-β1-induced fibroblast activation and impaired the progression of pulmonary fibrosis. In conclusion, our work provides new molecular insight into the pathogenesis of pulmonary fibrosis, which support a critical role for SIRT3 and FoxM1 in pulmonary fibroblast activation to orchestrate the progression of pulmonary fibrosis. Therapeutic approaches aimed at restoring these integrated longevity pathways to potentially mitigate or reverse organ fibrosis warrant further investigation.

Ethical approval and consent to participate

The study was approved by the Ethics Committee of Nanjing university (IACUC-2003135, Date: 2020-3-24). All patients provided written informed consent, and approval was provided by the ethics committee of Drum Tower Hospital of Nanjing (2020-176-13, Date: 2020-3-10).

Availability of data and materials

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

CRediT authorship contribution statement

Jian Dong: Conceptualization, Formal analysis, Investigation, Writing – original draft. Lulu Wang: Data curation, Formal analysis, Project administration. Ai Wei: Conceptualization, Funding acquisition. Jiajia Lin: Formal analysis, Methodology, Software. Yulong Xuan: Formal analysis. Zichen Jiao: Resources. Yongkang Bai: Conceptualization, Resources. Xiaoming Shi: Supervision. Zirui Zhang: Methodology. Wei Sun: Formal analysis, Funding acquisition, Validation, Visualization. Tao Wang: Conceptualization, Funding acquisition, Supervision, Validation, Visualization, Writing – review & editing. Xiang Chen: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare no conflict of interest in this work.