TGF-β1 promotes SCD1 expression via the PI3K-Akt-mTOR-SREBP1 signaling pathway in lung fibroblasts

House dust mite (HDM) extract was purchased from Greer laboratories (Lenoir, NC, USA). Enzyme-linked immunosorbent assay (ELISA) kits for immunoglobulin E (IgE) were purchased from RayBiotech (Norcross, GA, USA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). The SCD1 inhibitor A939572 and the PI3K inhibitor LY294002 were purchased from MedChemExpress (Shanghai, China). The SREBP1 inhibitor Fatostatin HBr and the mTOR inhibitor Torin1 were purchased from Selleck (Shanghai, China). OA-bovine serum albumin (BSA) was purchased from Sigma‒Aldrich (St. Louis, MO, USA). Small interfering RNA targeting SCD1 (siSCD1) and negative control small interfering RNA (siNC) were obtained from GenePharma (Suzhou, China). The siRNA sequence information is listed in Additional file 1: Table S1. The primers used in this study were synthesized by Synbio Technologies (Suzhou, China). Cytosolic, nuclear and membrane proteins were extracted using a Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Beijing, China) according to the manufacturer’s instructions. The antibodies used are listed in Additional file 1: Table S2.

The human fetal lung fibroblast cell line (HFL1) was obtained from our laboratory as previously described [14]. HFL1 cells were cultured in F12K medium supplemented with 10% fetal bovine serum (Gibco, New Zealand) at 37 °C in an atmosphere with 5% CO₂. Once the HFL1 cells reached 80–90% confluence, they were placed in serum-free medium for 24 h and then treated with inhibitors and TGF-β1 for the indicated times. siRNA transfection was performed with a transfection reagent (Lipofectamine 3000 from Invitrogen) according to the manufacturer’s protocol.

Female C57BL/6 mice aged 6–8 weeks were obtained from Southern Medical University and housed under pathogen-free conditions with 12 h light/dark cycles. The mice were acclimatized for one week before the start of the experiments. All animal studies were conducted in accordance with the animal use guidelines of the Southern Medical University, and the protocols were approved by the Animal Ethics Committee of Southern Medical University. A total of 40 mice were used (n = 10 in each experimental group). The mice were randomly divided into four groups: (1) control group, phosphate-buffered saline (PBS)-sensitized/PBS-challenged mice treated with the solvent; (2) HDM group, HDM-sensitized/HDM-challenged mice treated with the solvent; (3) A93 group, PBS-sensitized/PBS-challenged mice treated with A939572; and (4) HDM + A93 group, HDM-sensitized/HDM-challenged mice treated with A939572.

Briefly, the mice were sensitized with 25 μg of HDM extract diluted in 100 μl of PBS on Days 1 and 8 via intraperitoneal injections. Beginning on Day 15, the mice were exposed intranasally to 25 μg of HDM extract (in 20 μl of PBS) for 5 consecutive days and then allowed to rest for 2 days, and this cycle was repeated for 5 weeks. The control mice were sensitized or challenged with the same volume of PBS. The mice were gavaged with A939572 (5 mg/kg) 2 h before challenge, and the control mice received the same volume of solvent for comparison. The mice were sacrificed 24 h after the last HDM or PBS challenge.

The lung resistance index of anesthetized and mechanically ventilated (Buxco Electronics, Wilmington, NC, USA) mice was determined in response to increasing doses of methacholine (0, 3.125, 6.25, 12.5, 25, and 50 mg/ml) administered by ultrasonic nebulization. Measurements of lung resistance were performed every 5 min following each nebulization step until a plateau phase was reached.

Blood samples were collected from the retro-orbital plexus, incubated at room temperature for 1 h, and then centrifuged at 3000 rpm for 10 min. The supernatants were harvested, and the total IgE level was measured using an ELISA kit according to the manufacturer’s instructions.

The left lung lobes were fixed in 4% neutral paraformaldehyde and then embedded in paraffin according to standard procedures. The right lung lobes were immediately snap-frozen in liquid nitrogen and stored at −80 °C for subsequent protein or RNA analysis. Lung sections (4 μm) were used for hematoxylin and eosin (HE) staining. The results were scored by three observers in a random blinded manner and semiquantified as previously described [15]. Periodic acid-Schiff (PAS) staining was performed to quantify the percentages of goblet cells among airway epithelial cells as previously described [16]. Peribronchial collagen deposition was assessed by Masson trichrome staining, and quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) as previously described [17].

Sections of lung tissue were treated with 0.3% H₂O₂ for 10 min to quench endogenous peroxidase activity and then blocked in PBS containing 5% BSA for 30 min. After incubation with rabbit anti-collagen I antibody (ab34710, Abcam, USA) at a dilution of 1:200 overnight at 4 °C, the sections were incubated with biotinylated anti-rabbit IgG secondary antibody (Zsbio, Beijing, China) for 1 h and exposed to a substrate chromogen mixture for 2 min (Zsbio, Beijing, China). The staining intensity of collagen I per micrometer length of the basement membrane of bronchioles was calculated using Image-Pro Plus 6.0 software as previously described [18].

After treatments, lung tissue or cells were lysed in lysis buffer containing PMSF, protease and phosphatase inhibitors (Keygen Biotech, Nanjing, China). The lysates were separated on SDS‒PAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then immunoblotted with primary antibodies overnight at 4 °C. All antibodies were diluted 1:1000, with the exception of Lamin B1, which was diluted 1:10,000. After incubation with secondary antibodies conjugated with IRDye® 680 (LI-COR Biosciences, Lincoln, NE, USA) at a dilution of 1:10,000 for 1 h at room temperature, immunoreactive bands were detected using an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA). The quantitative analysis was performed using ImageJ 1.8.0 software (NIH, Bethesda, MD, USA).

RNA was isolated from cells or lung tissue using an RNAiso Plus Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Reverse transcription was performed with reverse transcription reagents (Takara, Dalian, China). The levels of mRNA were measured with a Bio-Rad CFX96 Real-Time system using Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotech, Shanghai, China). The relative changes in mRNA expression were quantified using the 2^−ΔΔCq method. The primers used in the present study are listed in Additional file 1: Table S3.

Lung tissue was fixed in 4% neutral paraformaldehyde at 4 °C for 24 h, treated with 30% sucrose at 4 °C for 24 h, embedded in O.C.T. compound (Sakura Finetek, Torrance, CA, USA) and used to prepare lung sectsions (6 μm). HFL1 cells were seeded on glass-cover dishes. HFL1 cells were washed with PBS after being treated and then fixed in 4% neutral paraformaldehyde. Subsequently, the lung sections or cells were treated with 0.5% Triton-X-100 (Sigma‒Aldrich) in PBS for 20 min and blocked with 5% BSA in PBS for 30 min. The samples were then incubated with primary antibodies at a dilution of 1:200 overnight at 4 °C and then with secondary antibodies conjugated with Alexa Fluor® 488 or Alexa Fluor® 594 (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:100 for 1 h at room temperature. The cell nuclei were labeled with DAPI (Beyotime Biotechnology, Shanghai, China) for 5 min. Fluorescent images were captured with a laser scanning confocal microscope (Olympus, Tokyo, Japan).

To investigate the expression of SCD1 during fibroblast activation, we examined the effects of TGF-β1 on the HFL1 cell line in vitro. We observed that TGF-β1 treatment increased SCD1 expression in a concentration-dependent manner, and the expression of α-SMA was also increased (Fig. 1A–C). In addition, western blotting demonstrated that TGF-β1 induced fibronectin, COL1A1, α-SMA and SCD1 protein expression in HFL1 cells in a time-dependent manner (Fig. 1D–H). Similarly, TGF-β1 increased the mRNA expression levels of fibronectin 1 (FN1), COL1A1, collagen type III alpha 1 (COL3A1), ACTA2 (α-SMA) and SCD1 in HFL1 cells (Fig. 1I–M). These findings suggest that TGF-β1-induced fibroblast activation is associated with increased levels of SCD1 in HFL1 cells.

To determine the effects of SCD1 on airway remodeling and airway inflammation in HDM-induced asthmatic mice, we administered A939572, a small molecule that specifically inhibits SCD1 enzymatic activity, by gavage (Fig. 2A). As expected, HDM-induced asthmatic mice exhibited a significantly increased AHR. Treatment with A939572 partially reduced AHR induced by methacholine (Fig. 2B). Overproduction of mucus and goblet cell hyperplasia were also observed after sensitization and challenge with HDM, and these effects were significantly decreased by A939572, as shown by PAS staining (Fig. 2C, D). Similarly, Masson trichrome staining showed a significant increase in collagen deposition in the interstitium of the airways and vessels of mice exposed to HDM extract, and this increase was attenuated in mice treated with A939572 (Fig. 2E, F). Moreover, the accumulation of collagen I in the peribronchiolar region induced by HDM was also reduced by treatment with A939572 (Fig. 2G, H).

One central pathway in airway remodeling involves TGF-β1-induced fibroblast activation, which leads to increased production of ECM proteins, airway wall thickening and airflow obstruction [19]. HDM exposure induced a marked elevation of TGF-β1 in BALF, and this effect was significantly decreased by treatment with A939572 (Additional file 1: Fig. S1A). Western blotting showed similar increases in the levels of the ECM proteins fibronectin and collagen type I alpha 1 (COL1A1) and the myofibroblast differentiation marker α-SMA in lung homogenates from the HDM group, and these effects were decreased by treatment with A939572 (Additional file 1: Fig. S1B). In addition, treatment with A939572 suppressed fibroblast activation, as shown by immunofluorescence colocalization of S100A4, a specific marker of fibroblasts, and fibronectin in peripheral airways (Additional file 1: Fig. S1C).

We also investigated the effects of SCD1 on airway inflammation. A histology analysis showed a typical feature of peribronchiolar and perivascular inflammatory cell infiltration in the lungs of the HDM group, but the degree of infiltration was not reduced in the mice treated with A939572 (Fig. 2I, J). Consistently, higher amounts of total cells and increased numbers of macrophages, lymphocytes, neutrophils and eosinophils in BALF were found in the mice exposed to HDM extract. We did not find a reduction in inflammatory cells in BALF of mice treated with A939572 (Fig. 2K–O). Moreover, HDM challenge increased the level of serum IgE, but treatment with A939572 did not reduce this level (Fig. 2P).

Above all, these data indicate that A939572 could relieve airway remodeling and inhibit fibroblast activation but fails to reduce airway inflammation in the HDM-induced mouse model of chronic asthma.

We then further investigated whether SCD1 regulates TGF-β1-induced fibroblast activation. Western blotting showed that the inhibition of SCD1 partially decreased the upregulation of fibronectin, COL1A1 and α-SMA induced by TGF-β1 in HFL1 cells (Fig. 3A–D). Immunofluorescence staining of fibronectin, collagen I and α-SMA revealed a similar reduction in HFL1 cells cotreated with TGF-β1 and A939572 (Fig. 3E–H). Consistent with the results obtained with A939572 treatment, SCD1 knockdown decreased the protein expression of fibronectin, COL1A1, and α-SMA in response to TGF-β1 in HFL1 cells (Fig. 3I–M). Immunofluorescence staining showed that the fibronectin, collagen I and α-SMA levels were also reduced in HFL1 cells after SCD1 knockdown (Fig. 3N–Q).

OA is the main MUFA generated by SCD1 and is subsequently incorporated into membrane phospholipids [20]. Therefore, we used OA-BSA, a complex containing BSA that is suitable for cell culture, to investigate whether the addition of exogenous OA might rescue the inhibition of fibroblast activation induced by A939572 and SCD1 knockdown. We observed that the A939572-induced inhibition of protein production was obviously alleviated in the presence of exogenous OA (Fig. 3R–U). Moreover, treatment with exogenous OA increased the protein levels of fibronectin, COL1A1 and α-SMA in SCD1-knockdown HFL1 cells (Additional file 1: Fig. S2A–D). Collectively, these results suggest that SCD1 is needed for TGF-β1-induced fibroblast activation and ECM protein production.

SREBP1 plays a transcriptional activation role in enzymes associated with fatty acid synthesis [21]. To determine whether SREBP1 regulates TGF-β1-induced SCD1 expression in HFL1 cells, we used Fatostatin HBr, a specific inhibitor of SREBP1 activation. Interestingly, treatment with Fatostatin HBr decreased the expression of SREBP1 and the downstream fatty acid synthesis enzymes FASN and SCD1 in HFL1 cells (Fig. 4A–D). Similarly, a significant reduction in the transcript levels of the SREBP1 target genes ACACA, FASN, and SCD1 was observed in HFL1 cells treated with TGF-β1 and Fatostatin HBr (Fig. 4E–G). Immunofluorescence staining also showed reduced expression of SREBP1 after treatment with Fatostatin HBr (Fig. 4H). The fibroblast activation markers fibronectin, COL1A1 and α-SMA were also decreased by treatment with Fatostatin HBr (Fig. 4I–L). These results indicate that SREBP1 contributes to SCD1 expression and fibroblast activation downstream of TGF-β1.

Previous studies have shown that the PI3K-Akt-mTOR signaling pathway is activated by TGF-β and promotes protein synthesis and cell metabolism, which are required for cell growth, proliferation, and differentiation [22]. Thus, we treated HFL1 cells with TGF-β1 and the PI3K inhibitor LY294002. We found that treatment with LY294002 significantly inhibited the phosphorylation of Akt and its downstream effector molecule mTOR (Fig. 5A–D). Moreover, the inhibition of PI3K notably decreased the TGF-β1-induced upregulation of SREBP1, FASN, and SCD1 in HFL1 cells (Fig. 5E–H). Similarly, the transcript levels of the SREBP1 target genes ACACA, FASN, and SCD1 were reduced in HFL1 cells treated with TGF-β1 and LY294002 (Fig. 5I–K). In addition, the inhibition of PI3K decreased the TGF-β1-induced upregulation of fibronectin, COL1A1 and α-SMA in HFL1 cells (Fig. 5L–O). The mRNA levels of FN1, COL1A1, COL3A1, and ACTA2 in HFL1 cells were also significantly decreased by LY294002 (Fig. 5P–S). Taken together, these results demonstrate that TGF-β1 promotes SCD1 expression and fibroblast activation through PI3K-Akt-mTOR signaling.

mTOR, a downstream effector of Akt activation, impacts SREBP1 transcription, processing and nuclear localization [23]. To further establish the role of mTOR in regulating fatty acid synthesis and fibroblast activation downstream of TGF-β1, we treated HFL1 cells with TGF-β1 and Torin1, an inhibitor of mTOR complexes. Treatment with Torin1 reduced the protein levels of SREBP1, FASN and SCD1 in HFL1 cells (Fig. 6A–D). Furthermore, treatment with Torin1 inhibited the nuclear localization of SREBP1 (Fig. 6E, F). The TGF-β1-induced expression of fibronectin, COL1A1, and α-SMA was also partially reduced by treatment with Torin1 (Fig. 6G–J). Immunofluorescence staining showed similar results: the protein expression of fibronectin, collagen I, and α-SMA in HFL1 cells was reduced by treatment with Torin1 (Fig. 6K–N). The above data demonstrate that mTOR regulates the TGF-β1-induced nuclear localization of SREBP1, expression of fatty acid synthesis enzymes, and fibroblast activation.