STL1267 Inhibits Myofibroblast Differentiation in a TGFβ1-Driven Human Lung Fibroblast Model

Abstract

Pulmonary fibrosis is a progressive interstitial lung disease characterized by excessive fibroblast-to-myofibroblast transition (FMT) and extracellular matrix (ECM) deposition, largely driven by transforming growth factor-beta 1 (TGFβ1). Existing therapies offer limited efficacy, particularly in advanced disease. Circadian rhythms have recently emerged as key modulators of lung inflammation and fibrosis. In this study, we developed an in vitro model of chronic fibrotic signaling using adenovirus-mediated TGFβ1 overexpression (Ad-TGFβ1) or human recombinant protein TGFβ1 in primary human lung fibroblasts. Using this model, we investigated the antifibrotic potential of STL1267, a next-generation Rev-erbα agonist with improved potency, specificity, and pharmacokinetic properties. RNA sequencing and pathway analysis revealed that STL1267 significantly reversed Ad-TGFβ1-induced expression of genes associated with ECM remodeling, collagen biosynthesis, and immune suppression. STL1267 also upregulated pathways related to IL-10, IL-4, and IL-13 signaling, which are known to counteract fibrotic responses. Quantitative PCR and immunoblotting confirmed STL1267’s ability to downregulate key pro-fibrotic markers, including COL1A1, αSMA, FN1, and FAP, at both gene and protein levels. Comparative studies with other Rev-erbα agonists (GSK4112, SR9009), Saracatinib, and FDA-approved antifibrotic drugs (Pirfenidone, Nintedanib) demonstrated superior efficacy of STL1267 in inhibiting both preventive and post-fibrotic induction models. Moreover, lentiviral overexpression of Rev-erbα suppressed TGFβ1-induced αSMA expression, supporting a direct antifibrotic role. These findings highlight Rev-erbα as a key regulator of myofibroblast differentiation and support both STL1267 and GSK4112 as promising candidates for circadian-based antifibrotic therapy. Future in vivo studies are warranted to evaluate its translational potential in idiopathic pulmonary fibrosis.

NEW & NOTEWORTHY:

This study introduces a novel in vitro adenovirus-based model of persistent TGFβ1 signaling in human lung fibroblasts to mimic chronic fibrosis. Using this model, we show that the next-generation Rev-erbα agonist STL1267 robustly inhibits myofibroblast differentiation and ECM gene expression. STL1267 outperforms FDA-approved antifibrotic drugs and modulates immune and circadian signaling pathways, supporting its potential as a promising circadian-based therapeutic strategy for pulmonary fibrosis.

Graphical Abstract

INTRODUCTION

Circadian rhythms are endogenous 24-hour cycles that regulate diverse physiological functions such as sleep-wake patterns, metabolism, and hormone secretion (1, 2). These rhythms are orchestrated by a master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which synchronizes peripheral clocks across various tissues. At the molecular level, core clock proteins CLOCK and BMAL1 form a heterodimer that drives the expression of period (PER) and cryptochrome (CRY) genes (1, 2). The resulting PER and CRY proteins inhibit CLOCK:BMAL1 activity, forming self-regulating transcriptional feedback loops. Additional regulation is provided by nuclear receptors REV-ERBα/β and RORα/γ, which competitively bind to ROR response elements (ROREs) to modulate BMAL1 transcription, thus maintaining circadian stability (1, 2). Emerging evidence suggests that disruption of circadian gene regulation contributes to the pathogenesis of fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF) (3–7).

IPF is a chronic, progressive interstitial lung disease of unknown etiology, characterized by excessive scarring, architectural distortion, and impaired gas exchange, ultimately leading to respiratory failure and death (8). Although FDA-approved antifibrotic agents such as pirfenidone and nintedanib can slow disease progression, their therapeutic efficacy is limited in advanced disease stages (9, 10). A hallmark feature of IPF pathogenesis is the fibroblast-to-myofibroblast transition (FMT), driven primarily by transforming growth factor-beta 1 (TGFβ1), a key pro-fibrotic cytokine (11). While myofibroblasts are essential for normal wound repair, persistent epithelial injury and dysregulated healing lead to sustained myofibroblast activation, excessive extracellular matrix (ECM) deposition, and irreversible fibrosis (12, 13).

Pharmacological modulation of circadian clock regulators has emerged as a novel strategy for targeting fibrotic pathways. Rev-erbα agonists such as GSK4112 and SR9009 have been shown to reduce fibrosis in vitro, in ex vivo human lung explants, and in vivo mouse models (4–6). In contrast, Rev-erbα antagonist SR8278 treatment promotes pro-fibrotic phenotypes (5). Additionally, arrhythmic Clock^Δ19 mutant mice exhibited reduced NRF2 levels and glutathione availability, resulting in elevated oxidative damage and an exaggerated spontaneous pro-fibrotic phenotype, with heightened susceptibility to bleomycin-induced lung fibrosis (14). While early Rev-erbα agonists such as SR9009 and SR9011 have demonstrated promising antifibrotic effects in preclinical models, their translational potential is limited by off-target activity and poor pharmacokinetic properties (15, 16). STL1267 is a next-generation Rev-erbα agonist that exhibits superior potency, binding affinity, selectivity, and pharmacokinetics, with minimal off-target effects (17). This makes STL1267 a more precise and reliable probe for investigating Rev-erbα signaling in fibrotic remodeling.

In this study, we aimed to comprehensively evaluate the effects of Rev-erbα agonists in modulating adenoviral (Ad-TGFβ1) or human recombinant protein TGFβ1-induced fibrotic responses in human lung fibroblasts. Using a panel of compounds: STL1267, GSK4112, SR9009, Saracatinib, Pirfenidone, and Nintedanib, we assessed their ability to suppress TGFβ1-induced FMT phenotypes and compared their antifibrotic efficacy, uncovering novel therapeutic avenues for circadian-based intervention in lung fibrosis.

MATERIALS AND METHODS

Cell Culture and Treatment

WI-38 human embryonic lung fibroblasts were obtained from ATCC (Manassas, VA, USA). Adult normal human lung fibroblasts (NHLFs; Cat. No. CC-2512) were purchased from Lonza (Walkersville, MD, USA). Additional adult NHLFs and diseased human lung fibroblasts (DHLFs; IPF) were generously provided by Dr. Steven K. Huang (University of Michigan, Ann Arbor, MI, USA). These primary fibroblasts were isolated from non-fibrotic (healthy) or IPF lungs of deceased donors supplied by Gift of Life, Michigan. The donor demographics and characteristics for NHLF and DHLF are provided in Table S1. NHLF cells were cultured in a humidified incubator maintained at 37°C with 5% CO₂ and 75–80% relative humidity. The complete growth medium consisted of DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. For treatment experiments, the medium was modified to contain 1% FBS while maintaining 1% antibiotic-antimycotic. WI-38 and NHLF cells were subcultured in T75 flasks and passaged either by trypsinization or gentle scraping, followed by centrifugation at 2000 × g for 5 minutes. Cell viability was assessed prior to each passage and seeding using acridine orange/propidium iodide (AO/PI) staining. Primary normal human lung fibroblasts from donor 1 were used for all experiments involving adenoviral-mediated TGF-β1 stimulation using the human TGF-β1–encoding construct (Ad-TGF-β1). Fibroblasts from the remaining three donors were primarily used for experiments involving stimulation with recombinant human TGF-β1 protein.

Cell Viability and Cytotoxicity

To evaluate the cytotoxic effects of REV-ERBα agonists, two normal human lung fibroblast (NHLF) donor cell lines were used. Cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well and cultured until they reached 70–80% confluence. The culture medium was then replaced with complete medium containing 1% FBS and incubated for 16 hours prior to treatment.

The REV-ERBα agonist STL1267 was synthesized in the Burris laboratoy using standard medicinal chemistry techniques, as previously reported (17). STL1267, GSK4112, and SR9009 were prepared as 2.5 mM stock solutions in DMSO and diluted to final working concentrations of 5, 10, and 20 μM in complete medium supplemented with 1% FBS. A vehicle control containing 0.8% (v/v) DMSO, corresponding to the highest concentration used in treated wells, was included for comparison. After 48 hours of exposure, cellular morphology and detachment were monitored by phase-contrast microscopy, and representative images were captured from one donor. Cells were then collected for viability assessment using acridine orange/propidium iodide (AO/PI) staining and quantified with a Countess automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).

In parallel, culture supernatants were collected to assess cytotoxicity via lactate dehydrogenase (LDH) release, using the CyQUANT LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, 50 μL of each sample supernatant or LDH positive control was added to a black 96-well clear-bottom plate, followed by 50 μL of LDH reaction mixture per well. After incubation for 10 minutes at room temperature in the dark, 50 μL of stop solution was added, and fluorescence was recorded at 560 nm excitation and 590 nm emission using a BioTek fluorescence plate reader. Relative LDH fluorescence values across treatment groups were analyzed and compared. For quantitative presentation of LDH and AO/PI viability data, results from two replicates per donor were pooled and presented.

Adenovirus-Mediated TGFβ1 (Ad-TGFβ1) and Antifibrotic Compound Treatment

To evaluate the effects of sustained TGFβ1 expression and continuous signaling in human lung fibroblasts (NHLF: Donor 1), primary cells were transduced with an adenoviral DNA construct encoding the human TGFβ1 gene (Ad-TGFβ1). Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and cultured until reaching 70–80% confluency. Cells were then treated with Ad-TGFβ1 at a concentration of 1 × 10⁸ plaque-forming units (PFU) per well in complete medium containing 1% FBS for 16 hours. Following transduction, fresh complete medium containing 1% FBS was replaced, and cells were incubated for an additional 24 hours to allow for the induction of a myofibroblast-like phenotype. After this incubation period, cells were treated with one of the following antifibrotic compounds: STL1267 [10 μM], GSK4112 [20 μM], SR9009 [10 μM], Saracatinib [0.3 μM], Nintedanib [1 μM], or Pirfenidone [100 μM]. The concentrations of compounds used were selected based on previously published reports (5, 17, 18). For SR9009 (10 μM), cells were pretreated for 4 hours in complete medium containing 1% FBS, after which the medium was replaced with fresh complete medium, as previously described (5).

Human Recombinant TGFβ1 Protein and Antifibrotic Compound Treatment

NHLF (donors 2 and 3) and idiopathic pulmonary fibrosis (IPF) donor (DHLF; donor 7) were stimulated with recombinant human TGF-β1 as previously described (5). Briefly, cells were seeded in 6-well plates and cultured until reaching 70–80% confluence. The following day, cells were serum-starved in complete medium containing 1% FBS for 16 hours. Cells were then pre-treated with antifibrotic compounds at the concentrations specified above for 1 hour, followed by co-treatment with TGF-β1 (10 ng/mL) for 48 hours. For SR9009 (10 μM), cells were pre-treated for 4 hours in 1% FBS-containing medium, after which the medium was replaced with fresh treatment medium containing TGF-β1 (10 ng/mL) and incubated for an additional 48 hours, as previously reported (5). After treatment, cells were harvested and stored for downstream RNA expression analysis by quantitative real-time PCR (qRT-PCR).

Slot-Blot Analysis of Secretory COL1A1

Secreted collagen I (COL1A1) levels in conditioned media from three NHLF donors were quantified using a slot-blot assay as previously described (5). Conditioned media were thawed on ice and loaded onto a 0.2 μm nitrocellulose membrane mounted in a slot-blot manifold. Each well was pre-equilibrated with 200 μL PBS, allowed to drain under vacuum, and then loaded with 60 μL of conditioned medium. After sample application, an additional 60 μL PBS was drawn through each slot for 5 min under vacuum to ensure uniform loading. Membranes were blocked in 5% BSA/TBST for 1 hr, followed by incubation with anti-COL1A1 primary antibody (1:10,000) for 2 hrs. After two 5-min washes in TBST, membranes were incubated with HRP-conjugated anti-mouse secondary antibody (1:20,000) for 1 hr and washed again (4 × 10 min). Signal was detected using chemiluminescent substrate and imaged on an Odyssey Fc (LI-COR) system. Densitometric quantification was performed using ImageJ (NIH).

Lentiviral-Mediated Rev-erbα Overexpression

To generate a Rev-erbα overexpressing lung fibroblast model, WI-38 cells were transduced with a lentiviral construct designed for Rev-erbα overexpression. Lentiviral constructs, including both the vector control and Rev-erbα overexpression constructs, were obtained from VectorBuilder (Chicago, IL, USA). Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and cultured until they reached 70–80% confluency. Once the desired confluency was achieved, cells were treated with polybrene (5 μg/mL, VectorBuilder, USA) to enhance transduction efficiency. After 5–6 hours, the medium was replaced with complete medium containing lentivirus at a concentration of 5 × 10⁵ transducing units (TU)/mL, and the cells were incubated for 48 hours. Transduced cells were continuously monitored for EGFP expression, and those exhibiting 80–90% EGFP-positive cells (for both vector control and Rev-erbα overexpression) were selected for expansion. These cells were then used for downstream experiments and analyses.

Total RNA Isolation

Frozen cell pellets from treated samples (donors 1–3) stored at −80 °C were thawed on ice and lysed with 700 μL of lysis buffer, followed by vortexing to ensure complete disruption. Total RNA was extracted using the RNeasy^® Mini Kit (Cat#: 74106, Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. To remove potential genomic DNA contamination, samples were treated with RNase-free DNase I enzyme (Cat#: 79256, Qiagen, USA) for on-column digestion, as recommended in the protocol. After DNA digestion, the columns were washed, and pure RNA was eluted from the column using RNase-free water. Isolated RNA samples were used immediately or stored at −80°C until used for downstream applications.

RNA Sequencing Analysis

The quality and integrity of total RNA isolated from donor 1 were assessed using the Agilent Bioanalyzer. Stranded mRNA sequencing was performed on the Illumina NovaSeq 6000 platform at the genomics core facility (University of Kansas Medical Center, KS, USA). Initial RNA quality control was conducted using the Agilent TapeStation 4200 with the RNA ScreenTape Assay kit (Agilent Technologies, Santa Clara, CA, USA; Cat#: 5067–5576). For library preparation, 500 ng of total RNA was processed using the Universal Plus mRNA-Seq with NuQuant stranded mRNA library preparation protocol (Tecan Genomics, Redwood City, CA, USA; Cat#: 0520-A01) on the Tecan DreamPrep NGS automation system. The RNA was enriched for mRNA using oligo-dT bead capture, followed by mRNA fragmentation, cDNA synthesis, end repair, adaptor ligation with Unique Dual Index (UDI) barcodes, and strand-specific library amplification by PCR (12 cycles) on a Bio-Rad S1000 thermal cycler. Library validation was performed using the D1000 ScreenTape Assay (Agilent Technologies, USA; Cat#: 5067–5582) on the TapeStation 4200. The library concentrations were quantified using the NuQuant module of the library prep kit with a Qubit 4 Fluorometer (Thermo Fisher/Invitrogen, Waltham, MA, USA), normalized to 1.9 nM, and pooled. The multiplexed library pool was quantified in triplicate using the LightCycler 96 (Roche, Basel, Switzerland) with FastStart Essential DNA Green Master (Roche, Cat#: 06402712001) and KAPA Library Quantification Standards (KAPA Biosystems, Cape Town, South Africa; Cat#: KK4903). Pooled libraries were denatured with 0.2 N NaOH (final concentration: 0.04 N) and neutralized with 400 mM Tris-HCl (pH 8.0). The final library pool was diluted to 380 pM before being subjected to onboard clonal clustering on the NovaSeq 6000 using the S1 Reagent Kit v1.5 (200 cycles; Illumina, San Diego, CA, USA; Cat#: 20028318). Sequencing was performed with a 2 × 101 cycle paired-end read configuration and dual-indexing: Read 1–101 cycles, Index Read 1–8 cycles, Index Read 2–8 cycles, and Read 2–101 cycles. Following sequencing, raw data in BCL format were converted to FASTQ format using bcl2fastq software and demultiplexed. FASTQ files were then securely transferred via FTP or uploaded to Illumina BaseSpace for downstream analysis. Differential gene expression analysis was performed using DESeq2 (19).

Bioinformatic Analysis

Gene set enrichment analysis (GSEA) was performed using the Enrichr tool (20–22) to identify significantly enriched biological pathways from the Reactome database for differentially expressed upregulated and downregulated genes within each pairwise comparison. Genes with an absolute log₂ fold change (|log₂FC|) ≥ 1.5 and an adjusted P-value < 0.05 were considered for downstream analysis. For each pairwise comparison, such as Ad-TGFβ1 vs. control and Ad-TGFβ1+STL vs. Ad-TGFβ1, genes meeting the above thresholds were subjected to GSEA using Enrichr. The top ten most statistically significant pathways from the Reactome database were highlighted. To assess the effect of STL1267 treatment, Venn diagram analysis was performed between the upregulated genes from the Ad-TGFβ1 vs. control comparison and the downregulated genes from the Ad-TGFβ1+STL1267 vs. Ad-TGFβ1 comparison. The common/shared genes were then subjected to GSEA. Similarly, the shared genes identified in the Venn diagram analysis between the downregulated genes of the Ad-TGFβ1 vs. control comparison and the upregulated genes of the Ad-TGFβ1+STL1267 vs. Ad-TGFβ1 comparison were also analyzed for GSEA using Enrichr.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis

Complementary DNA (cDNA) synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) using total RNA isolated from donors 1–3, as previously reported (5). Briefly, RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and 1 μg of purified RNA was used for cDNA synthesis. The resulting cDNA was diluted with DNase/RNase-free water prior to use in downstream qRT-PCR analysis. qRT-PCR was performed using PowerTrack^™ SYBR^™ Green Master Mix (Thermo Fisher Scientific) and gene-specific primers targeting pro-fibrotic markers, including Fn1 (Fibronectin 1), Col1a1 (Collagen type I alpha 1 chain), Acta2 (α-smooth muscle actin), Col4a1 (Collagen type IV alpha 1 chain), Itgb1 (Integrin β1), Tgfβ1 (Transforming growth factor β1), Lox (Lysyl oxidase), and Tagln1 (Transgelin). Expression of these genes was analyzed in Ad-TGFβ1-treated NHLF (donor 1). For human recombinant TGFβ1-stimulated NHLF cells (donors 2 and 3), the expression of pro-fibrotic genes such as Fn1, Col1a1, Acta2, and Col4a1 was evaluated. Amplification was performed on the CFX Opus 96 Real-Time PCR System (Bio-Rad, USA) as described previously (5, 23). Gene expression was determined using quantification cycle (Cq) values, and relative expression was calculated using the 2^−ΔΔCt method, with Gapdh or Hprt1 serving as the internal reference gene (23, 24) (Table S2).

Protein Isolation and Western Blot Analysis

Total protein was extracted from cells and quantified using the Pierce BCA assay kit (Thermo Scientific, USA). Equal amounts of protein (30 μg) were loaded per lane onto a 7.5% SDS-polyacrylamide gel for electrophoresis (SDS-PAGE). After separation, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 2 hours at room temperature. Following blocking, the membranes were incubated overnight at 4°C with primary antibodies, diluted 1:1000, against the following targets: COL1A1, FN1, αSMA, FAP, MYOD, cleaved PARP, total PARP, cleaved caspase-3, total caspase-3, CLOCK, BMAL1, and REV-ERBα. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 in 5% BSA; goat anti-mouse IgG [H+L] or goat anti-rabbit IgG [H+L], Invitrogen, USA) for 2 hours at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL, Thermo Fisher Scientific, USA) and imaged with a ChemiDoc Imaging System (Bio-Rad, USA). For detection of multiple targets from the same blot, membranes were stripped using a commercial stripping buffer (Thermo Fisher Scientific, USA), washed, and reprobed with a different primary antibody. Band intensities were quantified via densitometric analysis using ImageJ software (NIH), and target protein expression levels were normalized to GAPDH as a loading control to calculate relative fold changes.

Immunofluorescence Staining and Confocal Microscopy

Cells were seeded and treated in 8-well chambered slides at a density of 50,000 cells per well. NHLF donor 1 was treated with Ad-TGFβ1 in combination with test compounds as described previously. NHLF donors 2, 3, and 4 were treated with human recombinant TGFβ1 (10 ng/mL) together with the same test compounds, following the protocol described above. Following treatment, cells were rinsed with ice-cold 1× PBS and fixed with 4% formalin for 10 minutes at room temperature (RT). Permeabilization was performed using 0.2% Triton X-100 in 1× PBS for 10 minutes at RT, followed by washing with PBS. To block nonspecific binding, cells were incubated with 5% BSA in PBST (1× PBS containing 0.05% Tween 20) for 1 hour at RT. Primary antibody incubation was performed overnight at 4°C in blocking buffer using the following dilutions: α-SMA (1:200), FN1 (1:500), and COL1A1 (1:500). The next day, cells were washed five times with PBST and incubated with species-specific secondary antibodies (1:1000 dilution; goat anti-mouse or goat anti-rabbit IgG [H+L], labeled with Alexa Fluor^® 555 or Alexa Fluor^® 488; Invitrogen, USA) for 1 hour at RT. Following secondary antibody incubation, cells were washed again five times with PBST. Coverslips were mounted using ProLong^™ Gold Antifade Reagent containing DAPI (Thermo Fisher Scientific) and imaged using a confocal microscope (Nikon, USA) equipped with immersion oil at 60× magnification. For each treatment condition, 6–8 images were captured from different regions of each well. Pixel fluorescence intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA) to assess relative protein expression levels. These data were plotted as column graphs, and appropriate statistical analyses were performed. Detailed information on qRT-PCR primer sequences, antibodies, kits and reagents, and primary NHLF/DHLF donor demographics used in this study is provided in Tables S1–S3.

Statistical Analysis

All data are presented as mean ± standard deviation (SD). For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied. Statistical significance was defined as P < 0.05, with P < 0.01 and P < 0.001 indicating progressively higher levels of significance. All statistical analyses were performed using GraphPad Prism 9 (La Jolla, CA).

RESULTS

REV-ERBα Agonists do not Affect Viability or Morphology of Primary Normal Human Lung Fibroblasts

Cytotoxicity of the REV-ERBα agonists STL1267, GSK4112, and SR9009 was evaluated in NHLFs at concentrations of 5, 10, and 20 μM, ranges used in this study and previously reported (5). Bright-field imaging revealed no noticeable changes in cell morphology or evidence of cytotoxic detachment in DMSO control or drug-treated cells (Figure 1A). Cell viability, assessed by acridine orange/propidium iodide (AO/PI) staining, indicated >95% viability across all treatment groups, including the vehicle control (Figure 1B). Consistently, lactate dehydrogenase (LDH) release assays demonstrated comparable fluorescence levels between DMSO-treated and drug-treated samples, indicating similar membrane integrity and confirming the absence of cytotoxicity (Figure 1C). Collectively, these results demonstrate that the REV-ERBα agonists tested were non-toxic to primary normal human lung fibroblasts at the concentrations used.

Figure 1. Cellular cytotoxicity of REV-ERBα agonists (STL1267, GSK4112, SR9009) in primary normal human lung fibroblasts.

Cytotoxicity of REV-ERBα agonists was evaluated in primary normal human lung fibroblasts (NHLF; two independent donors). Cells were treated with STL1267 or GSK4112 (5, 10, or 20 μM) for 48 hours. For SR9009 (5, 10, or 20 μM), cells were treated for 4 hours, then washed and incubated in fresh culture medium for an additional 48 hours. (A) Representative phase-contrast images showing NHLF morphology after treatment. (B) Cell viability quantified by acridine orange/propidium iodide (AO/PI) staining. (C) Cytotoxicity assessed by lactate dehydrogenase (LDH) release in culture supernatants using the CyQUANT LDH Cytotoxicity Assay Kit. Data represent pooled results from two technical replicates across two donors.

Inhibition of αSMA and FN1 Protein Expression by Pre-treatment with Rev-erbα Agonists in TGFβ1-Stimulated Lung Fibroblasts

To evaluate the effect of REV-ERBα agonists on αSMA and FN1 expression, NHLF (Donor 1) were pretreated with REV-ERBα agonists, saracatinib, or established antifibrotic drugs (nintedanib or pirfenidone) prior to stimulation with TGFβ1 (10 ng/mL). Cells were then immunostained and analyzed by confocal microscopy. TGFβ1 markedly increased αSMA and FN1 levels, whereas pre-treatment with STL1267, GSK4112, SR9009, saracatinib, pirfenidone, or nintedanib significantly reduced TGFβ1-induced αSMA and FN1 expression (Figure 2A). Treatment with any of these compounds in the absence of TGFβ1 did not induce a pro-fibrotic response (Figure S1). A schematic overview of the experimental design is shown in Figure 2B.

Figure 2. Rev-erbα agonist treatment strongly inhibits TGFβ1-induced αSMA and FN1 protein expression in primary normal human lung fibroblasts compared to other antifibrotic drugs.

Primary normal human lung fibroblasts (NHLF: Donor 1) were grown in 8-well chamber slides, serum-starved overnight, and pretreated with or without Rev-erbα agonists or antifibrotic drugs before TGFβ1 stimulation. Cells were pretreated for 1 hour with GSK4112 (20 μM), STL1267 (10 μM), Saracatinib (0.3 μM), Nintedanib (1 μM), or Pirfenidone (100 μM), followed by co-treatment with TGFβ1 (10 ng/ml) for 48 hours. Cells were pretreated with SR9009 (10 μM) for 4 hours, washed, and then incubated in medium containing TGFβ1 (10 ng/ml) for 48 hours. (A) Representative confocal microscopy images (60× magnification; scale bar: 100 μm) showing αSMA (α-smooth muscle actin, red), FN1 (fibronectin 1, green), and nuclei counterstained with DAPI (blue). (B) Schematic overview of the experimental design. (C-D) Quantification of αSMA and FN1 fluorescence intensity expressed as pixel intensity. Data are shown as mean ± SD (n = 5–9/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001 versus TGFβ1; ^#P < 0.05, ^{# #}P < 0.01, ^{# # #}P < 0.001 for the other remaining pairwise comparisons.

Comparative analysis revealed that STL1267 more effectively reduced αSMA than pirfenidone and nintedanib, with effects comparable to saracatinib (Figure 2C). GSK4112 significantly suppressed αSMA relative to nintedanib and showed a trend toward greater suppression than pirfenidone, while its effects were similar to saracatinib, SR9009, and STL1267. For FN1, STL1267, GSK4112, pirfenidone, and nintedanib demonstrated comparable inhibition, whereas saracatinib was slightly less effective but still significant (Figure 2D). SR9009 showed a non-significant downward trend in FN1 expression. Overall, STL1267, GSK4112, pirfenidone, and nintedanib significantly reduced TGFβ1-induced FN1 relative to SR9009 and saracatinib.

These findings were confirmed across three additional independent NHLF donors (donors 2–4). Fluorescence microscopy and quantitative analysis demonstrated that all tested compounds significantly reduced αSMA and FN1 compared with TGFβ1 alone. In donor 2, STL1267, GSK4112, SR9009, and saracatinib were more effective than pirfenidone and nintedanib in suppressing both markers (Figure 3). In donor 3, all compounds except saracatinib significantly reduced αSMA, and all except nintedanib significantly reduced FN1; saracatinib’s effect on FN1 was less pronounced than STL1267, GSK4112, SR9009, and pirfenidone (Figure S2A). In donor 4, GSK4112 and saracatinib more strongly reduced αSMA compared with pirfenidone, nintedanib, and SR9009, while all compounds significantly decreased FN1, with STL1267 and GSK4112 showing superior efficacy (Figure S2B). Among REV-ERBα agonists, STL1267 and GSK4112 consistently outperformed SR9009. Collectively, immunofluorescence analysis indicates that STL1267 and GSK4112 produce the most pronounced reduction in pro-fibrotic markers (αSMA and FN1) compared with SR9009 and FDA-approved antifibrotic drugs.

Figure 3. REV-ERBα agonist treatment strongly inhibits TGFβ1-induced αSMA and FN1 protein expression in normal human lung fibroblasts compared to other antifibrotic drugs.

Primary normal human lung fibroblasts (NHLF: Donor 2) were cultured in 8-well chamber slides, serum-starved for 16 hours, and pretreated with or without REV-ERBα agonists or other antifibrotic drugs prior to TGFβ1 stimulation. Cells were pretreated for 1 hour with GSK4112 (20 μM), STL1267 (10 μM), saracatinib (0.3 μM), nintedanib (1 μM), or pirfenidone (100 μM), followed by co-treatment with TGFβ1 (10 ng/mL) for 48 hours. For SR9009, cells were pretreated with 10 μM for 4 hours, washed, and then incubated in fresh medium containing TGFβ1 (10 ng/mL) for 48 hours. Representative confocal microscopy images (60× magnification; scale bar: 60 μm) show αSMA (α-smooth muscle actin, red), FN1 (fibronectin 1, green), and nuclei (DAPI, blue). Quantification of αSMA and FN1 fluorescence intensity is shown as pixel intensity. Data are shown as mean ± SD (n = 5–9/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 versus TGFβ1; ^#P < 0.05, ^{# #}P < 0.01, ^{# # #}P < 0.001 for the other remaining pairwise comparisons.

Ad-TGF β1-Induced Myofibroblast Transition and Differential Expression of Genes Post STL1267 Treatment

To assess the effect of sustained TGFβ1 expression and continuous signaling, we developed a novel in vitro model of human lung fibroblast-to-myofibroblast transition using adenovirus-mediated expression of human TGFβ1, as illustrated in Figure 4A. Ad-TGFβ1 treatment enhanced the protein expression of pro-fibrotic markers FN1, COL1A1, and αSMA but reduced the protein level of REV-ERBα (Figure S3A-B). Ad-GFP does not induce the pro-fibrotic response in normal human lung fibroblasts (Figure S3A). To investigate the effects of the Rev-erbα agonist STL1267 on Ad-TGFβ1-induced gene expression changes, we performed RNA sequencing (RNA-seq) of total RNA from primary human lung fibroblasts treated with Ad-TGFβ1 followed by STL1267.

Figure 4. Differential gene expression and enriched molecular pathways in the Ad-TGFβ1 primary normal human lung fibroblast model with and without Rev-erbα agonist (STL1267) treatment.

(A) Schematic overview of the experimental design. Primary normal human lung fibroblasts (NHLF: Donor 1) were transduced with Ad-TGFβ1 (1 × 10⁸ PFU) for 16 hours, followed by replacement with complete medium. After 24 hours, the cells transitioned into a myofibroblast-like phenotype before treatment with the Rev-erbα agonist, STL1267 (10 μM), for 48 hours. Total RNA was extracted, and RNA sequencing (RNA-seq) was performed. (B-C) Differently expressed genes (DEGs) with a |log₂FC| ≥ 1.5 are shown in volcano plots for two different comparisons: Ad-TGFβ1 vs. control (B) and Ad-TGFβ1+STL1267 vs. Ad-TGFβ1 (C). Upregulated genes are shown in red, and downregulated genes are shown in blue. Pathway enrichment analysis reveals distinct biological pathways associated with these DEGs in both comparisons.

A volcano plot analysis of differentially expressed genes (Ad-TGFβ1 vs. control) revealed a significant number (867) of upregulated genes, including extracellular matrix (ECM) and their remodeling genes such as Acta2, Col1a1, Col5a1, Col20a1, Tgfβ1, Mmp11, and other genes such as Spp1 and Sparc (Figure 4B). We found 1,439 genes downregulated in the Ad-TGFβ1 vs. control analysis, which includes some peculiar chemokine and cytokine response genes such as Ccl2, Ccl8, Ccl11, Ccl20, Cxcl6, Cxcl12, Il33 and Tnfsf18. Pathway enrichment analysis of upregulated genes revealed that they are mainly associated with ECM organization, ECM proteoglycans, smooth muscle contraction, integrin cell surface interaction, and collagen chain trimerization, along with collagen biosynthesis and modifying enzymes (Figure 4B), whereas the pathway enrichment analysis of the downregulated genes in the same pairwise comparison reflected pathways involved in chemokine signaling, IL-10 signaling, ABC transporter in lipid homeostasis, TNF binding to their physiological receptors, and GPCR ligand binding signaling as the most affected pathways post-Ad-TGFβ1 treatment of primary human lung fibroblasts.

Ad-TGFβ1 + STL1267 vs. TGFβ1 pairwise comparisons revealed that STL1267 treatment upregulated 518 genes (Figure 4C), including Bcl2 (apoptotic signaling), Ccl20, Cxcl2, Cxcl3, Il1a, Itga6 (integrins), Mmp10, Myc, Nr1d1 (also known as Rev-erbα), and Nfil3, while downregulating 446 genes, including pro-fibrotic Col20a1, Col11a1, Col8a2, Eln, and Wnt2 (Wnt signaling), as well as mitochondrial genes Mt-atp8, Mt-nd3, and Mt-nd1. Pathway enrichment analysis of upregulated genes in this pairwise comparison showed significant enrichment of pathways related to the response of EIF2AK1 to heme deficiency, IL-4 and IL-13 signaling, IL-10 signaling and cytokine signaling in the immune system (Figure 4C). Whereas downregulated genes showed enrichment of FASTK family proteins regulating mitochondrial RNA, tRNA metabolism in mitochondria, muscle contraction and acetylcholine neurotransmitter signaling.

We also performed the Venn diagram analysis between upregulated genes of pairwise comparison Ad-TGFβ1 vs. control and downregulated genes of pairwise comparison Ad-TGFβ1+ STL1267 vs. Ad-TGFβ1. We identified 84 common genes that were upregulated by Ad-TGFβ1 and subsequently downregulated following STL1267 treatment (Figure 5A). Pathway enrichment analysis of these 84 genes revealed significant enrichment of collagen metabolism and ECM organization proteins, along with their associated pathways. We then curated a subset of significantly affected ECM remodeling genes, including collagens, integrins, and matrix metalloproteinases (MMPs), from the downregulated gene set in the Ad-TGFβ1 + STL1267 vs. Ad-TGFβ1 pairwise comparison. The normalized counts of the selected genes are highlighted in the form of a heatmap (Ad-TGFβ1 vs. Ad-TGFβ1+STL), as shown in Figure 5B. The heatmap showed that these upregulated myofibroblast phenotype-promoting genes are blocked by STL1267 treatment. Hierarchical clustering of the heatmap confirmed that these genes exhibited a distinct expression profile depending on the treatment group.

Figure 5. Venn diagram and heatmap illustrating differentially expressed genes in STL1267-treated primary normal human lung fibroblasts in the Ad-TGFβ1 model.

Primary normal human lung fibroblasts (NHLF: Donor 1) were transduced with adenoviral Ad-TGFβ1 (1 × 10⁸ PFU) for 16 hours, followed by replacement with complete medium. After 24 hours, cells transitioned into a myofibroblast-like phenotype before treatment with the Rev-erbα agonist STL1267 (10 μM) for 48 hours. Total RNA was extracted and analyzed via RNA sequencing (RNA-seq). (A) Venn diagram showing the overlap of significantly upregulated genes (Log₂FC > 1.5) in the Ad-TGFβ1 vs. Control comparison and significantly downregulated genes (Log₂FC < −1.5) in the Ad-TGFβ1+STL1267 vs. Ad-TGFβ1 comparison. A subset of 84 shared genes, significantly downregulated in the Ad-TGFβ1+STL1267 vs. Ad-TGFβ1 comparison, was further analyzed for pathway enrichment using Enrichr, revealing key pathways such as collagen biosynthesis and extracellular matrix (ECM) organization. (B) Heatmap depicting differentially expressed ECM-metabolizing genes, including integrins and matrix metalloproteinases, which were upregulated by Ad-TGFβ1 but downregulated following Ad-TGFβ1+STL1267 treatment. Hierarchical clustering and heatmap generation were performed using one minus Pearson correlation (linkage method: average; clustering: column) in Morpheus (Broad Institute).

We also performed the Venn diagram analysis between downregulated genes of pairwise comparison Ad-TGFβ1 vs. control and upregulated genes of pairwise comparison Ad-TGFβ1 + STL1267 vs. Ad-TGFβ1 reflected 167 common genes (Figure S4A). Pathway enrichment analysis of these 167 genes showed significant enrichment of IL-10, IL-4, IL-13, Senescence Associated Secretory Phenotype (SASP), and NOTCH 2 and 3 signaling as top pathways whose associated genes were downregulated by Ad-TGFβ1 treatment but brought up by STL1267. Heatmap analysis of the normalized count of some key signaling molecules is highlighted in Figure S4B. Overall, RNA-Seq analysis of lung fibroblasts revealed that Ad-TGFβ1 upregulated pro-fibrotic genes associated with myofibroblast transition, while STL1267 reversed these effects by downregulating ECM and collagen metabolism genes and modifying immune and mitochondrial pathways. We also found that treatment with Ad-TGFβ1 significantly affected the expression of core clock genes. Specifically, it led to a significant reduction in the normalized counts of Arntl (Bmal1) and Nr1d2 (Rev-erbβ), while increasing the expression of Nr1d1 (Rev-erbα), Nfil3, Cry1, Per1, and Dbp compared to the control (Figure S4C).

Inhibition of Pro-Fibrotic Gene Expression by Rev-erbα Agonist in TGFβ1-Stimulated Lung Fibroblasts

To evaluate the impact of the REV-ERBα agonist STL1267 on TGFβ1-induced fibrotic activation, primary normal human lung fibroblasts (NHLF donors 2 and 3) were pretreated with REV-ERBα agonists, saracatinib, or established antifibrotic drugs (nintedanib or pirfenidone) prior to TGFβ1 stimulation. qRT-PCR analysis was performed to assess the expression of selected pro-fibrotic genes: Fn1 (Fibronectin 1), Col1a1 (Collagen type I alpha 1 chain), Acta2 (α-smooth muscle actin), and Col4a1 (Collagen type IV alpha 1 chain) (Figure 6A–B). As expected, TGFβ1 markedly increased the expression of all genes, confirming successful induction of a myofibroblast-like phenotype.

Figure 6. REV-ERBα agonist pre-treatment strongly inhibits TGFβ1-induced pro-fibrotic gene expression in normal human lung fibroblasts.

REV-ERBα agonists (STL1267, GSK4112, and SR9009) were evaluated for their ability to suppress TGFβ1-induced pro-fibrotic gene expression in primary normal human lung fibroblasts (NHLF) derived from two independent donors (Donors 2 and 3). (A–B) Cells were pretreated for 1 hour with STL1267 (10 μM), GSK4112 (20 μM), Saracatinib (0.3 μM), Nintedanib (1 μM), or Pirfenidone (100 μM), followed by co-treatment with TGFβ1 (10 ng/mL) for 48 hours. For SR9009, cells were pretreated with 10 μM for 4 hours, washed, and subsequently incubated in fresh medium containing TGFβ1 (10 ng/mL) for 48 hours. Total RNA was extracted, cDNA synthesized, and expression of pro-fibrotic genes, Fn1 (fibronectin 1), Col1a1 (collagen type I alpha 1 chain), Acta2 (α-smooth muscle actin), and Col4a1 (collagen type IV alpha 1 chain), was quantified by qRT-PCR. Hprt1 was used as the housekeeping gene, and relative expression was calculated using the 2^−ΔΔCt method. Data are shown as mean ± SD (n = 5–6/group). *P < 0.05, **P < 0.01, ***P < 0.001 versus TGFβ1; ^#P < 0.05, ^{# #}P < 0.01, ^{# # #}P < 0.001 for the other remaining pairwise comparisons.

In donor 2, Fn1 expression was significantly inhibited only by nintedanib, while all other compounds had minimal effect. For Col1a1, GSK4112, SR9009, and nintedanib significantly reduced transcript levels, whereas STL1267 and saracatinib showed a non-significant downward trend; pirfenidone had no effect. Acta2 expression was significantly suppressed by all REV-ERBα agonists, saracatinib, and SR9009, with STL1267 being less effective than GSK4112 but more effective than pirfenidone and nintedanib. Finally, Col4a1 expression was comparably reduced by all tested compounds (Figure 6A).

In donor 3, Fn1 transcript levels were comparably reduced by all tested compounds. For Col1a1, STL1267 and GSK4112 showed stronger inhibition than SR9009, saracatinib, pirfenidone, and nintedanib. Acta2 was significantly suppressed by all REV-ERBα agonists, saracatinib, and nintedanib, while pirfenidone had no effect. In contrast, Col4a1 expression was not significantly affected by any REV-ERBα agonist, whereas saracatinib, pirfenidone, and nintedanib effectively reduced its expression, with nintedanib showing the most pronounced inhibition (Figure 6B). Overall, these results indicate that pre-treatment with REV-ERBα agonists effectively blocks TGFβ1-driven fibroblast-to-myofibroblast transition and, in several instances, demonstrates greater antifibrotic efficacy than currently FDA-approved drugs across multiple NHLF donors.

Inhibition of Ad-TGFβ1-induced Pro-Fibrotic Gene Expression by Rev-erbα Agonist

To assess the functional efficacy of the Rev-erbα agonist STL1267 on Ad-TGFβ1-induced lung fibrosis in NHLF, we performed qRT-PCR to measure the expression of key pro-fibrotic genes, including Fn1 (Fibronectin 1), Col1a1 (Collagen type I alpha 1 chain), Acta2 (α smooth muscle actin), Col4a1 (Collagen type 4 alpha 1 chain), Itgb1 (Integrin subunit β 1), Tgfβ1 (Transforming growth factor β1), Lox (Lysyl oxidase), and Tagln1 (Transgelin) (Figure 7). The results showed that Ad-TGFβ1 treatment significantly upregulated the expression of all pro-fibrotic genes, consistent with the induction of a myofibroblast-like phenotype. However, treatment with STL1267 significantly inhibited the expression of multiple pro-fibrotic genes, including Col1a1, Acta2, Col4a1, Itgb1, Tgfb1, Lox, and Tagln. Although the Fn1 expression was also reduced, it did not reach statistical significance. These data indicate that STL1267 effectively blocks Ad-TGFβ1-induced myofibroblast differentiation and fibrotic gene expression, which validates our RNA-seq analysis finding shown in Figure 5B, as most of the Ad-TGFβ1-induced upregulated genes associated with pro-fibrotic response were significantly downregulated by STL1267 treatment. We also analyzed the effect of another Rev-erbα agonist (GSK4112) on the Ad-TGFβ1-induced expression profile of these key pro-fibrotic genes and found that the treatment with GSK4112 reduced the expression of a few genes (Acta2, Col4a1, Itgb1, and Tgfβ1), while others remained unchanged (Figure S5). Overall, STL1267 effectively blocked Ad-TGFβ1-induced fibrosis by downregulating expression of the key pro-fibrotic genes, with GSK4112 showing a less pronounced effect.

Figure 7. REV-ERBα agonist (STL1267) inhibits Ad-TGFβ1-induced pro-fibrotic gene expression in normal human lung fibroblasts.

Primary normal human lung fibroblasts (NHLF Donor 1) were transduced with adenoviral Ad-TGFβ1 (1 × 10⁸ PFU) for 16 hours, followed by replacement with complete medium. After 24 hours, cells transitioned into a myofibroblast-like phenotype before treatment with the Rev-erbα agonist STL1267 (10 μM) for an additional 48 hours. Total RNA was isolated, cDNA was synthesized, and gene expression levels of pro-fibrotic markers, including Fn1 (fibronectin 1), Col1a1 (collagen type I alpha 1 chain), Acta2 (α smooth muscle actin), Col4a1 (collagen type 4 alpha 1 chain), Itgb1 (integrin subunit β 1), Tgfβ1 (transforming growth factor β1), Lox (lysyl oxidase), and Tagln1 (transgelin), were quantified by qRT-PCR. Gapdh was used as the housekeeping gene, and relative expression was calculated using the 2^−ΔΔCt method. Data are shown as mean ± SD (n = 5–6/group). ** P < 0.01, *** P < 0.001 versus control group; ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001 versus Ad-TGFβ1 group.

Inhibition of Pro-fibrotic Protein Markers by Rev-erbα Agonist Treatment

To further validate the effects of Rev-erbα agonists on key pro-fibrotic markers, we analyzed protein expression of COL1A1, FN1, αSMA, and FAP (Fibroblast Activation Protein) using Western blot analysis. Consistent with the gene expression results, Ad-TGFβ1 treatment significantly increased the expression of these pro-fibrotic proteins (COL1A1, FN1, αSMA, FAP). However, treatment with STL1267 significantly reduced the protein levels of COL1A1, αSMA and FAP. There was also a trend toward decreasing FN1 that was not statistically significant. Treatment with GSK4112 also significantly reduced the protein levels of the pro-fibrotic markers COL1A1 and αSMA, with a trend towards decreased FN1 and FAP (Figure 8A).

Figure 8. REV-ERBα agonist inhibits Ad-TGFβ1-induced protein expression of pro-fibrotic markers and promotes apoptosis in normal human lung fibroblasts.

(A) Primary normal human lung fibroblasts (NHLF: Donor 1) were transduced with adenoviral Ad-TGFβ1 (1 × 10⁸ PFU) for 16 hours, followed by replacement with complete medium. After 24 hours, cells transitioned into a myofibroblast-like phenotype before treatment with the Rev-erbα agonists STL1267 (10 μM) or GSK4112 (20 μM) for an additional 48 hours. Whole-cell lysates were prepared, and protein abundance of pro-fibrotic markers, including COL1A1, FN1, αSMA, and FAP, was measured by immunoblot analysis. GAPDH served as the housekeeping control. (B) Whole-cell lysates were also used to assess protein levels of cleaved PARP, total PARP, cleaved caspase 3, and total caspase 3 via immunoblot analysis, with GAPDH as the housekeeping control. Representative Western blot images are shown, and densitometry analysis of the bands was performed using ImageJ. Protein abundance is expressed as relative intensity compared to the control group. (C) Representative images of primary human lung fibroblasts after immunocytochemistry, showing αSMA (α smooth muscle actin, red) and the nucleus (DAPI, blue). Images were captured using a confocal microscope (60× magnification; scale bar: 100 μm). Quantification of αSMA staining is shown as pixel intensity. Data are shown as mean ± SD (n = 3–4/group for WB; n = 5–6/group for ICC). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. ** P < 0.01, *** P < 0.001 versus control group; ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001 versus Ad-TGFβ1 group.

The assessment of pro-apoptotic proteins revealed that Ad-TGFβ1 treatment decreased the expression of cleaved PARP, which was significantly restored by GSK4112 treatment. Although STL1267 treatment also increased cleaved PARP levels, the effect was marginal compared to GSK4112. The total PARP protein levels remained largely unchanged across the studied groups, except in the Ad-TGFβ1+GSK group, where the reduction was likely due to increased cleavage into cleaved PARP (Figure 8B). Similarly, cleaved caspase-3 levels were elevated in both the Ad-TGFβ1+GSK and Ad-TGFβ1+STL groups compared to the Ad-TGFβ1 group. Total caspase-3 levels remained consistent across most groups, except for Ad-TGFβ1+GSK, where its expression was significantly higher than in the Ad-TGFβ1, Ad-TGFβ1+GSK, and Ad-TGFβ1+STL groups. Overall, Rev-erbα agonists modulated protein expression of pro-fibrotic markers, with STL1267 and GSK4112 significantly reducing COL1A1, αSMA, and FAP and showing a decreasing trend for FN1. Additionally, GSK4112 strongly restored cleaved PARP levels, while both agonists increased cleaved caspase-3, indicating their role in apoptosis regulation. We also analyzed the protein levels of the core-clock protein and found that the TGFβ1 treatment significantly increased the CLOCK, BMAL1 and REV-ERBα compared to the control, which was brought down by the GSK4112 treatment (Figure S6). GSK4112 sole treatment increased the expression of these core-clock proteins compared to controls.

We evaluated TGFβ1-induced pro-fibrotic protein expression in diseased human lung fibroblasts (DHLF; one IPF donor). TGFβ1 robustly increased COL1A1, αSMA, and FN1 abundance, whereas co-treatment with the REV-ERBα agonist GSK4112 markedly attenuated this induction (Figure S7A). To compare the anti-fibrotic efficacy of REV-ERBα agonists with FDA-approved therapeutics, we assessed their effects on TGFβ1-driven fibroblast activation. GSK4112 co-treatment produced a pronounced reduction in COL1A1, αSMA, and FN1 protein levels. In contrast, pirfenidone, nintedanib, and saracatinib did not suppress COL1A1 or αSMA, although they each reduced FN1 expression relative to TGFβ1 alone (Figure S7B).

We next quantified extracellular collagen deposition by measuring secreted COL1A1 via slot-blot analysis. TGFβ1 stimulation increased COL1A1 secretion in conditioned media collected from three independent NHLF donors. Treatment with REV-ERBα agonists (GSK4112 and SR9009) as well as FDA-approved anti-fibrotic agents reduced TGFβ1-induced COL1A1 release; however, the magnitude and consistency of these effects varied across donors (Figure S8).

Inhibition of Pro-fibrotic Protein Expression by Post-treatment of Rev-erbα Agonists in Ad-TGFβ1 Transduced Normal Human Lung Fibroblasts

To assess the impact of Rev-erbα agonists on fibrotic gene expression, normal human lung fibroblasts were first transduced with Ad-TGFβ1 to induce myofibroblast differentiation and subsequently treated with either Rev-erbα agonists (STL1267, GSK4112, SR9009) or saracatinib or established antifibrotic agents (nintedanib, pirfenidone) (Figure 9A). Following treatment, cells were subjected to immunostaining and analyzed by confocal microscopy. Ad-TGFβ1 transduced cells markedly increased the expression of αSMA, FN1, and COL1A1 proteins, indicative of myofibroblast transition. However, treatment with Rev-erbα agonists STL1267 and GSK4112 significantly attenuated the Ad-TGFβ1-induced expression of αSMA, FN1, and COL1A1 (Figure 9B–D). All three Rev-erbα agonists (STL1267, GSK4112, and SR9009) effectively suppressed αSMA expression to levels comparable to saracatinib and greater than those achieved by pirfenidone or nintedanib. Notably, STL1267 and GSK4112 more effectively inhibited FN1 and COL1A1 expression compared to saracatinib, pirfenidone, and nintedanib. While SR9009 significantly reduced αSMA and FN1 expression, its ability to suppress COL1A1 was less potent than STL1267, GSK4112, or the established antifibrotic agents (Figure 9B–D). These findings suggest that Rev-erbα agonists, particularly STL1267 and GSK4112, exert robust antifibrotic effects in vitro in primary normal human lung fibroblasts, with greater suppression of certain fibrotic markers compared to current FDA-approved antifibrotic drugs in this model.

Figure 9. Rev-erbα agonist treatment strongly inhibits Ad-TGFβ1-induced pro-fibrotic protein expression in normal human lung fibroblasts compared to other antifibrotic drugs.

(A) Primary normal human lung fibroblasts (NHLF: Donor 1) were transduced with Ad-TGFβ1 (0.5 × 10⁸ PFU) for 16 hours, followed by replacement with complete medium. After 24 hours, the cells transitioned into a myofibroblast-like phenotype before treatment with STL1267 (10 μM), GSK4112 (20 μM), saracatinib (0.3 μM), nintedanib (1 μM), or pirfenidone (100 μM) for 48 hours. Cells were pretreated with SR9009 (10 μM) for 4 hours, washed, and then incubated in complete medium for 48 hours. Representative confocal microscopy images (60× magnification; scale bar: 100 μm) show αSMA (α-smooth muscle actin, green), FN1 (fibronectin 1, red), COL1A1 (red) and nuclei stained with DAPI (blue). (C-D) Quantification of αSMA, FN1, and COL1A1 staining is shown as pixel intensity. αSMA, FN1 and COL1A1 expression inhibition efficiency of Rev-erbα agonists was quantified in terms of pixel intensity and analyzed compared to saracatinib, nintedanib, or pirfenidone. Data are shown as mean ± SD (n = 5–9/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001 versus Ad-TGFβ1; ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001 for the other remaining pairwise comparisons.

Overexpression of Rev-erbα Reduces TGFβ1-Induced αSMA Expression in Lung Fibroblasts

To establish the role of the Rev-erbα in regulating expression of αSMA, WI-38 human fetal lung fibroblasts were first transduced with lentivirus expressing human Rev-erbα and enhanced green fluorescent protein (EGFP) and then treated with TGFβ1. Both groups, vector control (VC) and overexpression (OE), were stained for REV-ERBα and αSMA protein. Confocal microscopy analysis of vector control and Rev-erbα-overexpressing cells revealed that nearly all cells were positive for EGFP, indicating efficient transduction with the lentiviral construct. Quantitative analysis showed significantly higher nuclear REV-ERBα expression in the OE group compared to the VC. Notably, TGFβ1 treatment appeared to marginally increase the REV-ERBα expression in VC but not in the OE group (Figure 10). However, upon TGFβ1 stimulation, αSMA expression was significantly upregulated in VC cells, while Rev-erbα-overexpressing cells maintained αSMA levels at the basal state, indicating a suppressive effect of REV-ERBα overexpression on TGFβ1-induced αSMA expression.

Figure 10. Rev-erbα overexpression suppresses TGFβ1-induced αSMA expression in lung fibroblasts.

Lentiviral constructs expressing human Rev-erbα genes and EGFP were transduced in WI-38 cells to generate overexpression (OE) cell populations. Vector control (VC) cells were transduced with viral constructs with EGFP but without Rev-erbα genes using the same plasmid backbone. Both VC and OE cells were treated with TGFβ1 (10 ng/ml) for 48 hours and then immunostained for REV-ERBα and αSMA. The representative confocal image showed inherently expressing EGFP (green), REV-ERBα (red) and αSMA (red). For quantitative representation of the REV-ERBα, the mean pixel intensity of around 50 nuclei was determined using Image J, and then data were analyzed using a scatter plot in GraphPad Prism. For quantitative representation of the αSMA, pixel intensity of images (n=5–6) was determined from different areas, and then they were plotted as a scatter plot using GraphPad Prism. Data are shown as mean ± SD (n = 5–9/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, ***P < 0.001 versus VC; ^{# # #} P < 0.001 versus VC or VC + TGFβ1 groups.

DISCUSSION

In this study, we developed an in vitro model of persistent pro-fibrotic signaling using adenoviral-mediated TGFβ1 overexpression (Ad-TGFβ1) in primary normal human lung fibroblasts. Previous in vivo studies have utilized Ad-TGFβ1 to model pulmonary fibrosis (25–27). To the best of our knowledge, this is the first application of this approach in an in vitro system to recapitulate key phenotypic and transcriptomic features of pulmonary fibrosis. This model induced robust myofibroblast differentiation, marked by upregulation of ECM genes and suppression of immune-related pathways, along with elevated classical fibrotic markers at both mRNA and protein levels. This sustained fibrotic state enabled us to assess not only the preventive but also the therapeutic effects of novel Rev-erbα agonist STL1267 and comparative testing of GSK4112, SR9009, Saracatinib, Pirfenidone, and Nintedanib.

Previous studies have demonstrated context-dependent roles of several pathways in pulmonary fibrosis, including chemokine signaling (28), IL-10 signaling (24), ABC transporter in lipid homeostasis (29, 30), Class A/1-Rhodopsin-like receptor (LPARs) (31), TNF receptor signaling (32), and GPCR ligand binding (33). In our transcriptomic analysis, gene set enrichment of downregulated genes in the Ad-TGFβ1 group revealed these pathways among the top 10 significantly enriched, underscoring their suppression during sustained fibrotic signaling. Consistent with the known role of TGFβ1 as a driver of FMT and ECM gene expression (11), our data showed strong enrichment of ECM-related pathways, including ECM organization, ECM proteoglycans, smooth muscle contraction, integrin-mediated cell surface interaction, collagen chain trimerization, and collagen biosynthesis/modifying enzyme pathways in the upregulated gene set of the Ad-TGFβ1 group.

Although prior studies have implicated Rev-erbα role in the regulation of mitochondrial metabolism (34–36). Notably, we observed significant enrichment of mitochondrial RNA metabolism pathways among downregulated genes in the Ad-TGFβ1+STL vs. Ad-TGFβ1 comparison. Similarly, among the same comparison, pathways such as EIF2AK1 response to heme deficiency, IL-4 and IL-13 signaling, IL-10 signaling, and broader cytokine signaling in the immune system were significantly enriched among upregulated genes. These results suggest that myofibroblast transition is not solely a fibrotic process but may involve complex interactions between immune signaling and structural cell behavior in chronic fibrotic environments.

Analysis of genes upregulated in the Ad-TGFβ1 group but significantly downregulated after STL1267 treatment revealed strong enrichment in pathways related to muscle contraction, collagen metabolism, and ECM organization. This indicates that STL1267 treatment is associated with reduced expression of genes involved in ECM remodeling. Notably, STL1267 significantly reduced the expression of key ECM components and remodeling genes involved in collagen synthesis, integrins, and MMPs, which are known to drive pulmonary fibrosis progression through myofibroblast transition (11). These transcriptomic findings were further corroborated by qRT-PCR, confirming that STL1267 suppressed Ad-TGFβ1-induced expression of pro-fibrotic genes, including Fn1, Col1a1, Acta2, Col4a1, Itgb1, Tgfb1, Lox, and Tagln. Additional validation of gene expression analysis revealed that the Rev-erbα agonists, particularly STL1267 and GSK4112, consistently suppressed hallmark pro-fibrotic genes (Fn1, Col1a1, Acta2, and Col4a1) more effectively than the FDA-approved antifibrotic drugs pirfenidone and nintedanib in at least two primary NHLF donors, as determined by qRT-PCR. However, some donor-specific variability was observed. For instance, the Fn1 expression in donor 2 was not inhibited by Rev-erbα agonists, whereas in donor 1 (Ad-TGFβ1–stimulated) and donor 3 (TGFβ1–stimulated), Fn1 was significantly downregulated by STL1267. Similarly, Col4a1 expression in donor 3 was unaffected by Rev-erbα agonists, while a significant reduction was observed in donors 1 and 2. These findings underscore the importance of personalized therapeutic approaches, as inter-individual variability in drug response is increasingly recognized across multiple disease models and highlights the need to account for donor-specific differences when designing antifibrotic strategies (37).

Ingenuity Pathway Analysis of genes that were downregulated in the Ad-TGFβ1 group but restored upon STL1267 treatment showed enrichment in IL-10, IL-4, IL-13 signaling, senescence-associated secretory phenotype (SASP), and NOTCH2/3 signaling pathways. These findings align with prior studies indicating that IL-10 delivery attenuates bleomycin-induced TGFβ1 production and fibrosis in murine lungs (24). Thus, STL1267-induced upregulation of IL-10 signaling may contribute to the suppression of the pro-fibrotic phenotype in lung fibroblasts observed in this study. Furthermore, the data suggest that STL1267 may modulate additional pathways implicated in FMT-driven pulmonary fibrosis, including IL-4, IL-13, SASP, and NOTCH signaling (38, 39), further underscoring its therapeutic potential.

Recent studies have linked circadian rhythm regulation to collagen turnover and secretion. Specifically, SR9009 treatment in fibroblasts shows reduced collagen fiber content per cell in Clock mutants, while CRY protein stabilization had variable effects depending on the circadian context (40). Consistent with these findings, we observed that Rev-erbα agonists STL1267 and GSK4112 significantly downregulated key pro-fibrotic proteins, including COL1A1, FN1, and αSMA. Fibroblast activation protein (FAP), previously reported to be elevated in IPF lungs (41), was also significantly reduced by STL1267 treatment in the Ad-TGFβ1 group. In the IPF donor, TGFβ1 robustly induced the pro-fibrotic markers COL1A1, αSMA, and FN1, consistent with activation of a myofibroblast-like phenotype. Notably, co-treatment with the REV-ERBα agonist GSK4112 effectively suppressed this induction, demonstrating a stronger inhibitory effect than the currently FDA-approved anti-fibrotic therapies tested. These findings suggest that targeting REV-ERBα may offer a more potent strategy for attenuating TGFβ1-driven fibroblast activation and remodeling in IPF.

TGFβ1 is well known to promote apoptosis resistance in fibroblasts, contributing to persistent myofibroblast activation and excessive ECM production (42). In this study, the Ad-TGFβ1 group shows reduced cleaved PARP levels, a marker of apoptosis. GSK4112 restored cleaved PARP expression, suggesting a reversal of apoptosis resistance. Compared to Ad-TGFβ1 alone, STL1267 treatment also showed an upward trend in cleaved PARP levels. This suggests that STL1267 may help myofibroblasts become more susceptible to apoptosis.

While FDA-approved therapies such as pirfenidone and nintedanib offer modest benefits by slowing lung function decline, they do not fully halt disease progression, particularly at later stages (9, 10). This underscores the need for more effective therapies targeting the underlying molecular mechanisms of fibrosis progression. Our study reflects this limitation: both FDA-approved drugs demonstrated only partial efficacy when applied after myofibroblast differentiation. In contrast, the Rev-erbα agonists STL1267 and GSK4112 exhibited stronger antifibrotic effects, effectively reversing established fibrotic markers (FN1 and αSMA) in both Ad-TGFβ1-induced and TGFβ1-induced models across three primary normal human lung fibroblast donors, as confirmed by immunostaining and fluorescence microscopy. Moreover,

However, SR9009 was less effective in reducing Ad-TGFβ1-induced COL1A1 expression; STL1267 and GSK4112 showed significantly greater efficacy, outperforming Saracatinib and performing comparably to Pirfenidone and Nintedanib. Saracatinib, a selective Src kinase inhibitor, has been shown to attenuate bleomycin-induced lung fibrosis in mice and is currently designated as an orphan drug for IPF treatment (18). These findings support the potential of circadian clock-targeting compounds as antifibrotic agents. Notably, STL1267 and GSK4112 reduced fibrotic marker expression (FN1, COL1A1, and αSMA) when treated after pro-fibrotic induction in vitro.

Interestingly, Ad-TGFβ1 groups showed reduced Rev-erbα expression that aligns with previously reported downregulation of Rev-erbα in aged lung fibroblasts following bleomycin-induced lung fibrosis (43). WI-38 cells provide a genetically defined and highly reproducible system that ensures experimental consistency and have been extensively utilized by our group and others to study TGFβ1-induced fibroblast-to-myofibroblast transition (5, 44–46). The genetic reconstitution of the Rev-erbα in human lung fibroblasts (WI-38) suppressed TGFβ1-induced αSMA and maintained the basal levels even after 48 hrs post-TGFβ1 treatment, further supporting its antifibrotic role in the in vitro model.

GSK4112 demonstrated comparatively stronger anti-fibrotic activity than STL1267 in some assays; however, its poor in vivo pharmacokinetics and low bioavailability severely limit its therapeutic potential. SR9009, an optimized derivative of GSK4112, exhibited improved potency and pharmacokinetic/pharmacodynamic (PK/PD) properties (47), but its in vivo application remains constrained by significant cytotoxicity, which also necessitated a short 4-hour exposure in our in vitro experiments.

STL1267 is a newer, rationally engineered REV-ERBα agonist developed to overcome these limitations. It exhibits superior binding affinity for REV-ERBα (Ki = 253 ± 30 nM vs. 692 ± 209 nM) and REV-ERBβ (Ki = 98 ± 14 nM vs. 2546 ± 127 nM) relative to SR9009 (48), along with enhanced NCoR recruitment (17), improved bioavailability, broader tissue distribution, and reduced cytotoxicity in C2C12 and HepG2 cells (17). Collectively, these pharmacological and drug-like properties highlight STL1267’s enhanced therapeutic potential compared with earlier REV-ERBα agonists.

Existing in vivo studies demonstrate systemic distribution of STL1267 to plasma, liver, brain, skeletal muscle, and white adipose tissue, as well as modulation of hepatic Bmal1 activity (17). However, its biodistribution and toxicity profile in key organs, particularly the lungs, remain insufficiently characterized and warrant further investigation.

In summary, our findings suggest that activation of Rev-erbα with STL1267 and GSK4112 inhibits myofibroblast differentiation and fibrotic marker expression in an in vitro model of primary human lung fibroblasts. STL1267 mediates these effects by downregulating genes involved in ECM remodeling and fibrosis. Although this study provides important insights, several limitations should be acknowledged. All experiments were performed in vitro and thus require validation in physiologically relevant preclinical models. The use of normal human lung fibroblasts (NHLFs) from three independent donors strengthens the robustness of our findings; however, the limited donor number may not fully represent inter-individual variability. While prior studies have reported systemic distribution and hepatic activity of STL1267 (17), its pharmacokinetic behavior, tissue-specific accumulation, and safety profile, particularly within the lungs, remain to be established. Further in vivo investigations are warranted to assess pulmonary exposure, long-term safety, and therapeutic efficacy in models that more accurately recapitulate fibrotic disease pathology. These investigations will be critical in defining the full therapeutic potential of circadian rhythm pathway modulation in IPF and may pave the way for novel circadian-based interventions, either as standalone treatments or in combination with existing antifibrotic therapies.

Supplementary Material

Supplemental Tables S1-S3; https://doi.org/10.6084/m9.figshare.29546342

Supplemental Figures S1-S8; https://doi.org/10.6084/m9.figshare.29546342

DATA AVAILABILITY

All data necessary to evaluate the conclusions of this manuscript are provided in the main text and online supplemental (Tables S1-S3 and Figures S1-S5). The raw datasets supporting this study will be available in the NCBI Gene Expression Omnibus repository, GEO Series accession number: GSE293962. Source data for this study will be openly available after the publication at GEO accession GSE293962.

For Review: Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE293962

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