Inhibition of myocardin-related transcription factor/serum response factor signaling decreases lung fibrosis and promotes mesenchymal cell apoptosis

Abstract

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Figure 1

CCG-203971 (CCG) inhibits myocardin-related transcription factor A (MRTF-A) nuclear localization. IMR-90 fibroblasts were cultured on glass coverslips and treated with/without 2 ng/mL transforming growth factor (TGF)-β1 and/or 30 μmol/L CCG-203971 for 24 hours. A: The distribution of MRTF-A was assessed by immunofluorescence staining. B: The nuclear-to-cytoplasmic ratio of MRTF-A was determined for each condition as described in Materials and Methods. Data are expressed as means ± SEM. ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus untreated controls; ^†††P < 0.001 versus TGF-β1treated fibroblasts. Original magnification, ×20 (A).

Figure 2

Inhibition of serum response factor/myocardin-related transcription factor with CCG-203971 prevents myofibroblast differentiation but not Smad phosphorylation after treatment with transforming growth factor (TGF)-β1. Normal adult human lung fibroblasts (CCL-210) were cultured to 60% confluence in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum and growth arrested in serum-free DMEM for 24 hours. Cells were treated with 0, 0.3, 3.0, or 30 μmol/L CCG-203971 for 30 minutes before treatment with/without 2 ng/mL TGF-β1 or with 30 μmol/L CCG-203970 in the absence of TGF-β1. A: Whole-cell lysates were obtained after 24 hours, and α-smooth muscle actin (SMA) expression was determined by Western blot analysis. Blots were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: For each Western blot, the density of the α-SMA band and the corresponding GAPDH band were measured with ImageJ version 1.45s (NIH, Bethesda, MD). The ratio of α-SMA to GAPDH was determined for each condition, and then normalized so that the ratio in untreated cells was equivalent to 1.0. Pooled analysis from at least three independent experiments with each concentration of CCG-203971 is shown. C: CCL-210 fibroblasts were treated with or without 2 ng/mL TGF-β1 in the presence or absence of 30 μmol/L CCG-203971 for 30 and 60 minutes. Whole-cell lysates were assessed for Smad3 phosphorylation by Western blot analysis, and blots were stripped and reprobed for total Smad3. Data are expressed as means ± SEM. ^∗∗P < 0.01 versus untreated controls; ^†P < 0.05 versus TGF- β1 treatment in the absence of CCG-203971.

Figure 3

CCG-203971 blocks basal and transforming growth factor (TGF)-β1 induced fibronectin expression with or without 2 ng/mL TGF-β1 in the presence or absence of 30 μmol/L CCG-203971 for 30 and 60 minutes. A: Total fibronectin expression was determined by Western blot analysis. Each blot was stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Fibronectin-to-GAPDH ratios were determined and normalized to the ratio observed in untreated cells. Data presented are a pooled analysis that includes a minimum of three experiments with each concentration of CCG-203971. Data are expressed as means ± SEM. ^∗∗∗P < 0.001 versus untreated controls; ^†††P < 0.001 versus cells treated with TGF-β1 alone.

Figure 4

CCG-203971 (CCG) reduces lung fibrosis after intratracheal bleomycin (bleo). Wild-type C57BL/6 mice were treated with 1.2 U/kg intratracheal bleomycin or phosphate-buffered saline (PBS) as previously described. Starting on day 11 after bleomycin administration, the mice received CCG-203971 (100 mg/kg b.i.d. via i.p. injection) or an equal volume of vehicle control (dimethyl sulfoxide; DMSO). A: Weight changes over time. Weights were obtained on days 4, 6, and 8 before the administration of CCG-203971 and then daily through day 20 after the initiation of CCG-203971 administration. B: Hydroxyproline concentrations of both lungs at day 21 after bleomycin administration. Two independent experiments were completed with similar results, and the figure shown represents one of the experimental replicates. C: Representative histological examination. D: Representative picrosirius red staining. Data are expressed as means ± SEM. n = 7 mice per group (A and B). ^∗P < 0.05, ^∗∗P < 0.01, bleomycin/CCG-203971 and bleomycin/DMSO versus PBS/CCG-203971 and PBS/DMSO; ^††P < 0.01, PBS/CCG-203971 and PBS/DMSO versus bleomycin/DMSO; ^‡P < 0.05, bleomycin/CCG-203971 versus bleomycin/DMSO; ^§§§§P < 0.0001 versus untreated controls and CCG-203971 alone; ^¶¶¶¶P < 0.0001 versus bleomycin-treated mice.

Figure 5

CCG-203971 (CCG) attenuates lung fibrosis after type II alveolar epithelial injury. Surfactant protein C promoter–diphtheria toxin receptor (SPC-DTR⁺) and DTR⁻ transgenic mice were injected with/without 10.0 mg/kg diphtheria toxin (DT) i.p. once daily for 14 days. CCG-203971 100 mg/kg i.p. b.i.d. was administered from day 11 to 21 in the indicated groups. A: Weights were obtained at the indicated time points. CCG-203971 treatment alone did not lead to any weight loss in the DTR⁺ mice that did not receive DT (data not shown). B: At day 21, both lungs from at least five mice per group were assessed for hydroxyproline. The experiment was performed twice, with similar results. C: Representative histological examination and picrosirius red staining from additional mice at day 21. Insets represent digital enlargements of the indicated areas of the corresponding 40× image. Data are expressed as means ± SEM (A and B). n = 5 to 7 per group (A and B). ^∗P < 0.05, ^∗∗P < 0.01 versus DTR mice treated with DT alone; ^†P < 0.05 versus DTR controls without DT treatment; ^‡‡P < 0.01 versus DTR mice treated with DT and CCG-203971. H&E, hematoxylin and eosin. DMSO, dimethyl sulfoxide.

Figure 6

Fas-mediated apoptosis is enhanced by CCG-203971 (CCG) in IMR-90 fibroblasts. IMR-90 fibroblasts were treated with 0.3, 3.0, or 30 μmol/L CCG-203971 alone or in combination with 250 ng/mL Fas-activating antibody (Fas-Ab). A: Apoptosis was assessed by the detection of a green-fluorescent signal indicating cleavage by caspase-3/7 and quantified at the indicated time points using IncuCyte software version 2011A as described in Materials and Methods. B: Cells were treated with 250 ng/mL Fas-Ab, 30 μmol/L CCG-203971, or the combination, and apoptosis was similarly assessed at baseline and 16 hours. C and D: IMR-90 fibroblasts were treated with 250 ng/mL Fas-Ab and/or 0, 3.0, or 30 μmol/L CCG-203971 for 8 hours (C) or 16 hours (D). Apoptosis was assessed by Western blot analysis for cleaved poly-(ADP-ribose) polymerase (PARP); blots were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). E: Similarly, IMR-90 fibroblasts were treated with Fas-Ab and/or 30 μmol/L CCG-203971 for 8 or 16 hours, and apoptosis was assessed by enzyme-linked immunosorbent assay detection of histone-associated DNA fragments. Data are expressed as means ± SEM. Each data point represents three replicates, with each replicate comprised of nine different images from each well at each time point (A and B). n = 3 at each time point for each condition (E). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 versus control; ^†P < 0.05 versus baseline controls; ^‡‡‡P < 0.001 versus Fas-Ab alone and CCG-203971 at 16 hours; ^§§P < 0.01 versus Fas-Ab + CCG-203971 at 8 hours.

Figure 7

Inhibition of serum response factor/myocardin-related transcription factor A (MRTF-A) signaling by CCG-203971 and MRTF-A deficiency promotes Fas-mediated apoptosis in fibroblasts. CCL-210 fibroblasts were exposed to 250 ng/mL Fas-activating antibody (Fas-Ab) with or without 0, 3.0, or 30 μmol/L CCG-203971 for 8 hours. A: Apoptosis was assessed by Western blot analysis for cleaved poly-(ADP-ribose) polymerase (PARP). Blots were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Relative densitometry was based on the ratio of cleaved PARP to GAPDH for each condition. C: Apoptosis in CCL-210 fibroblasts was assessed by enzyme-linked immunosorbent assay (ELISA) detection of histone-associated DNA fragments. D: Lung fibroblasts from wild-type (WT) mice and transgenic mice deficient in MRTF-A (Mkl1) were treated with 250 ng/mL Fas-activating antibody (Fas-Ab), and apoptosis was assessed at 8 hours by detection of histone-associated DNA fragments. E: Normal primary adult lung fibroblasts (left graph) and fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis (IPF; right graph) were treated with/without 250 ng/mL Fas-Ab and/or 30 μmol/L CCG-203971, and apoptosis was assessed after 8 hours by ELISA detection of histone-associated DNA fragments. Data are expressed as means ± SEM. n = 3 or more replicates per condition (B); n = 3 per group (C); n = 3, WT, and n = 6, Mkl^−/− (D); n = 4 normal and 4 IPF cell lines (E). ^∗∗P < 0.01 versus untreated control; ^†P < 0.05 versus Fas-Ab alone, CCG-203971 alone, and CCG-203971 3 μmol/L + Fas-Ab; ^‡P < 0.05, ^‡‡P < 0.01 versus untreated control; ^§P < 0.05 versus WT and Mkl1^−/− control and WT fibroblasts treated with Fas-Ab.

Figure 8

Transforming growth factor (TGF)-β1 fails to reduce Fas-mediated apoptosis in IMR-90 fibroblasts treated with CCG-203971. IMR-90 fibroblasts were cotreated with 250 ng/mL Fas-activating antibody (Fas-Ab) with/without 30 μmol/L CCG-203971 and/or 2 ng/mL TGF-β1 for 8 hours. Apoptosis was assessed by Western blot analysis for cleaved poly-(ADP-ribose) polymerase (PARP) (A) with densitometry (B) and by enzyme-linked immunosorbent assay for histone associated DNA fragments (C). Data are expressed as means ± SEM. n = 2 per condition (B and C). ^∗P < 0.05 versus controls, TGF-β1 alone, Fas-Ab alone, and Fas-Ab + TGF-β1; ^†P < 0.05 versus Fas-Ab + CCG-203971, with or without TGF- β1; ^‡P < 0.05 versus all groups that did not receive Fas-Ab + CCG-203971. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 9

Transforming growth factor (TGF)-β1 does not rescue CCL-210 fibroblasts from Fas-mediated apoptosis in the presence of CCG-203971 (CCG). CCL-210 fibroblasts were treated with 250 ng/mL Fas-activating antibody (Fas-Ab) with/without 30 μmol/L CCG-203971 and/or 2 ng/mL TGF-β1 for 8 hours. Apoptosis was assessed by Western blot analysis for cleaved poly-(ADP-ribose) polymerase (PARP) (A) with quantification by densitometry (B), enzyme-linked immunosorbent assay for histone associated DNA fragments (C), and cleavage of a fluorescent substrate by activated caspase-3/7 with image analysis at 2-hour intervals and quantification of the fluorescent signal using the IncuCyte software version 2011A, ×20 magnification (D). E: Representative images of each condition at baseline and after 10 hours. Data are expressed as means ± SEM. n = 2 per group (B); n = 3 per group (C); n = 2 with 16 independent images per replicate analyzed at each time point (D) (the detail of this analysis is given in Materials and Methods). ^∗∗∗P < 0.001 versus control, TGF-β1 alone, Fas-Ab alone, and Fas-Ab + CCG-203971; ^†P < 0.05 versus Fas-Ab + CCG-203971 with or without TGF-β1; ^‡P < 0.05 versus control, Fas-Ab alone, CCG-203971 alone, and Fas-Ab + TGF-β1; ^§P < 0.05, ^§§P < 0.01 versus control and CCG-203971 alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 10

CCG-203971 sensitizes differentiated myofibroblasts to Fas-mediated apoptosis. CCL-210 fibroblasts were treated with/without 2 ng/mL transforming growth factor (TGF)-β1 for 24 hours and then treated with/without 250 ng/mL Fas-activating antibody (Fas-Ab) and/or 30 μmol/L CCG-203971. Apoptosis was assessed by Western blot analysis for poly-(ADP-ribose) polymerase (PARP) (A) and cleavage of a fluorescence substrate by activated caspase-3/7 with image analysis at 8 hours (B). Data are expressed as means ± SEM. n = 3 replicates of each condition with 16 separate images of each replicate analyzed. ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control; ^†††P < 0.001 versus Fas-Ab + CCG-203971. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 11

Inhibition of serum response factor/myocardin-related transcription factor with CCG-203971 prevents transforming growth factor (TGF)-β1 induction of X-linked inhibitor of apoptosis (XIAP) and secretion of plasminogen activator inhibitor (PAI)-1. CCL-210 fibroblasts were treated with 2 ng/mL TGF-β1 in the presence/absence of CCG-203971 (indicated doses) for 24 hours. Whole-cell lysates were assessed for XIAP expression by Western blot analysis (A) with densitometric analysis (B). Cell culture supernatants were assessed for total (C) and active (D) PAI-1. Data are expressed as means ± SEM. n = at least 3 replicates per condition (B); n = 3 for CCG-203971 alone and 12 for all other conditions (C and D). ^∗P < 0.05, ^∗∗∗∗P < 0.0001 versus control; ^††P < 0.01, ^††††P < 0.0001 versus TGF-β1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 12

Myofibroblast apoptosis is increased in vivo after the administration of CCG-203971 during the fibroproliferative phase of lung injury. Lung sections were analyzed for cell death by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (red) 2 weeks after the instillation of intratracheal phosphate-buffered saline (A) or bleomycin (Bleo) (B and C) and 3 days after the initiation of dimethyl sulfoxide (B) or CCG-203971 (CCG971) (C). D: TUNEL-positive (pos) cells were quantified in 10 random 400× fields by a blinded observer (K.K.K.). E and F: Lungs from bleomycin-injured mice treated with CCG-203971 were co-stained for TUNEL (E) and α-smooth muscle actin (SMA; green; F). G: Merged images demonstrate colocalization of TUNEL and α-SMA. Data are expressed as means ± SEM. n = 3 per group (D). ^∗P < 0.05 versus bleomycin alone.

Figure 13

CCG-203971 treatment decreases alveolar plasminogen activator inhibitor (PAI)-1 in murine models of lung injury and fibrosis. Mice were injured with bleomycin (A) or targeted type II alveolar epithelial cell (AEC) injury (B), and CCG-203971 treatment was initiated on day 11. Bronchoalveolar lavage fluid was collected on day 21 after bleomycin injury and on day 15 in the AEC-injury model. Levels of active PAI-1 were quantified by using a carboxylated microsphere–based enzyme-linked immunosorbent assay. Data are expressed as means ± SEM. n = 5, control group (A); n = 9, bleomycin-treated groups (A); n = 5, all groups (B). ^∗P < 0.05, ^∗∗P < 0.01 versus controls; ^†P < 0.05 versus bleomycin or diphtheria toxin receptor (DTR) mice treated with diphtheria toxin (DT). WT, wild type.