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American Journal of Physiology - Cell Physiology logoLink to American Journal of Physiology - Cell Physiology
. 2015 Sep 2;309(9):C616–C626. doi: 10.1152/ajpcell.00086.2015

TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts

Wendy A Neveu 1, Stephen T Mills 1, Bashar S Staitieh 1, Viranuj Sueblinvong 1,✉
PMCID: PMC4628935  PMID: 26333597

Abstract

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-β1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-β1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-β1. TGF-β1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-β1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-β1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-β1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-β1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.

Keywords: pulmonary fibrosis, thymocyte differentiation antigen 1, transforming growth factor-β1, deoxyribonucleic acid methyltransferase, 5-aza-2′-deoxycytidine

transforming growth factor (TGF)-β1 is a pleiotropic cytokine that is strongly associated with pulmonary fibrosis. Within the lung it is produced by a variety of cell types, including alveolar macrophages, epithelial cells, endothelial cells, fibroblasts, and myofibroblasts (30). TGF-β1 levels are increased in the lungs of experimental animals with bleomycin-induced lung injury and in humans with idiopathic pulmonary fibrosis (IPF) (5, 28, 37). Abundant evidence implicates a critical role of TGF-β1 in promoting fibroblast differentiation into myofibroblasts leading to lung fibrosis (10, 36).

However, the exact mechanisms of TGF-β1-mediated myofibroblast transdifferentiation are not fully understood. Multiple actions of TGF-β1 have been implicated. For example, TGF-β1 induces myofibroblast transdifferentiation through activation of the integrin and focal adhesion kinase (FAK) pathway (31). Other cell signaling pathways have also been implicated, including activation of Smad and non-Smad pathways (such as canonical Wnt, Erk, JNK/p38, small GTPase, and PI3K/Akt)(1, 39).

TGF-β1 may also induce myofibroblast differentiation through alternative signaling pathways that involve epigenetic regulatory mechanisms for controlling cellular phenotype. For example, TGF-β1-induced fibroblast to myofibroblast transdifferentiation is regulated by histone deacetylation, particularly HDAC4 (8). Another potential epigenetic pathway for the regulation of fibroblast phenotype involves methylation of fibrotic suppressor genes by TGF-β1. Notably, TGF-β1 has been shown to upregulate the expression of DNA methyltransferase (DNMT), one of the key epigenetic regulators (17, 38). One candidate gene for TGF-β1-induced promoter methylation that might play a critical role in myofibroblast differentiation is thymocyte differentiation antigen 1 (Thy-1 or CD90), a glycophosphatidylinositol-linked glycoprotein that is localized to plasma membrane lipid rafts in multiple cell types, including fibroblasts. Decreased Thy-1 expression has been associated with pulmonary fibrosis (19). Both animal and human studies identify that loss of Thy-1 promotes myofibroblast transdifferentiation (9, 13, 18, 23, 28, 29). However, the mechanisms underlying Thy-1 gene regulation have yet to be fully elucidated. Interestingly, the Thy-1 promoter is located in a high G/C content region, making it susceptible to DNA methylation, and there is evidence of Thy-1 suppression by promoter methylation in lung fibroblasts (20, 24) and nasopharyngeal cancer cells (15).

Given the role of TGF-β1 in lung fibrosis and DNA methylation, we hypothesized that one of the mechanisms by which TGF-β1 promotes fibroblast to myofibroblast transdifferentiation is through Thy-1 promoter hypermethylation. To test this hypothesis, we evaluated the links between Thy-1 expression, promoter methylation, DNMT activation, and expression of collagen type 1 (Col1A1) and α-smooth muscle actin (α-SMA) in TGF-β1-treated mouse primary lung fibroblasts.

MATERIALS AND METHODS

Primary mouse lung fibroblast culture and treatment.

Primary lung fibroblasts (PLFs) were derived from 3-mo-old wild-type C57BL/6 mouse lung tissue as previously described (21). Cells from passages 4–8 were seeded in six-well plates at a density of 5,000 cells/cm2 and allowed to grow in high-glucose (4.5 g/l) Dulbecco's modified Eagle's medium (Cellgro, Manassas, VA) supplemented with 20% FBS, 100 U/ml penicillin G sodium, 100 U/ml streptomycin, and 2 mM l-glutamine for 24 h. Media were then removed, and cells were rendered quiescent for 16 h in serum-free media. Cells were then supplemented with TGF-β1 (2 or 5 ng/ml; R&D Systems, Minneapolis, MN) ± 5-aza-2′-deoxycytidine (10 μM 5-AZA; Sigma Aldrich, St. Louis, MO). Control cells were treated with either PBS or DMSO. No measurable difference was observed between control groups, and, thus, PBS was selected as the internal control for the remaining in vitro experiments. The media were changed every 24 h with freshly added cytokine and/or methylation inhibitor. Cells were harvested for RNA, protein isolation, flow cytometry, and nuclear protein isolation for DNMT activity analysis. All studies were approved by the Institutional Animal Care and Use Committee at Emory University and conformed to institutional standards for the humane treatment of laboratory animals.

DNMT1 silencing RNA transfection.

PLFs were seeded in six-well plates and transfected the next day with 10 nM mouse DNMT1 silencing RNA vector (Invitrogen, Carlsbad, CA) or the same concentration of scrambled control vector (Invitrogen) using Lipofectamine 3000 (Invitrogen) as described in the manufacturer protocol. At 24 h posttransfection, some groups were exposed to TGF-β1 (5 ng/ml). Cells were then harvested for mRNA, DNMT activity, and quantitative methyl PCR analysis at 24 h and for protein analyses at 72 h.

Quantitative PCR.

At 24 and 72 h of treatment, total RNA was extracted from lung fibroblasts using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) as recommended by the manufacturer. First-strand cDNA synthesis was performed as previously described (28). Quantitative PCR was performed on cDNA using the iCycler iQ Detection System and SYBR Green kit from Bio-Rad Laboratories (Hercules, CA) with primer sets designed for mouse 18S, Thy-1, α-SMA, and Col1A1. Expression of target genes in PLFs was normalized to 18S levels, and relative values were determined by the comparative cycle threshold (Ct) method.

Flow cytometry analyses.

At 96 h of treatment, cells were stained with 1:50 dilution of an antimouse antibody against Thy-1 conjugated with phycoerythrin (BioLegend, San Diego, CA). Cells were incubated for 45 min at 4°C, washed in PBS, fixed in 4% paraformaldehyde for 15 min, and then analyzed by FACS using a LSRII flow cytometer (Becton Dickinson, San Jose, CA) as previously described (28). Flow cytometry data were analyzed using FlowJo 10.0 software (Tree Star, San Carlos, CA).

Western blot analyses.

After 72 h of treatment, whole cell extracts were prepared from PLFs, and protein was analyzed by Western blot analysis as previously described (28). Blots were probed with rabbit anti-DNMT1 and anti-DNMT3a (Cell Signaling, Danvers, MA), rabbit anti-DNMT3b (Santa Cruz, Dallas, TX), and rabbit anti-GAPDH (Sigma Aldrich), rabbit anti-α-SMA (Abcam, Cambridge, MA), and rabbit anti-Col1A1 (Millipore, Billerica, MA) antibodies followed by an appropriate secondary antibody. The immunoreactive bands were visualized using enzyme-linked chemiluminescence and analyzed with the ChemiDoc XRS system (Bio-Rad Laboratories).

DNMT activity assay.

At 24 h of treatment, nuclear proteins were extracted using the Active Motif Nuclear Extraction Kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA). DNMT activity was assayed using the EpiQui DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek Group, Farmingdale, NY) according to the manufacturer's instructions.

Quantitative DNA methylation PCR.

After 72 h of treatment, DNA was isolated from PLFs by the phenol-chloroform extraction method as previously described (22). Thy-1 methylation was quantified by use of the OneStep qMethyl kit according to the manufacturer's instructions (Zymo Research, Irvine, CA). Primer sets were designed with at least two methylation-sensitive restriction enzyme sites within CpG-rich regions in the Thy-1 promoter (mThy1.F 5′-GTC CCG GAG AGA ACC ATG AG-3′ and mThy1.R 5′-CTT TGC GCC CTT TCC CTG AG-3′) (see Fig. 4A) (26). Samples were amplified on the iCycler iQ Detection System, and percent gene methylation was determined by the comparative Ct method.

Fig. 4.

Fig. 4.

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TGF-β1 includes Thy-1 promoter methylation in mouse PLFs. Thy-1 methylation status was evaluated in mouse PLFs treated for 72 h with PBS or TGF-β1 (2 ng/ml) by qMethyl PCR using primers specific to methylation-sensitive restriction enzyme (MSRE) sites in CpG-rich regions of the Thy-1 promoter. A: illustration of methylation-sensitive PCR primer location in relation to mouse Thy-1.2 gene and its promoter region. Gene reference no. M12379. B: Thy-1 promoter methylation status as analyzed by qMethyl PCR. Data are represented as means ± SE. *P < 0.05 changed compared with PBS treatment. Data are representative of 4 independent experiments.

Immunofluorescent staining.

Mouse lung fibroblasts were cultured on poly-d-lysine cover slips in the presence or absence of TGF-β1 and/or 5-AZA (at 0.5, 1, and 5 μM) for 96 h. Cells were fixed, permeabilized, and then stained for α-SMA overnight using rabbit antimouse α-SMA antibody at a 1:200 dilution at 4°C (Abcam, San Francisco, CA). Cover slips were incubated with goat anti-rabbit secondary antibody (Invitrogen) and examined using an Olympus BX-41 fluorescence microscope (Olympus, Center Valley, PA) at ×40 magnification. Cells count was performed from two random fields per sample (×20 magnification) from triplicate experiments. Data were expressed as percent of cells with stress fiber formation compared with number of total cells.

Statistical analyses.

The data are presented as means ± SE. The significance of difference for single and multiple comparisons was determined by the unpaired two-tailed Student's t-test and one-way ANOVA, respectively. P value <0.05 was considered significant. GraphPad Prism version 4.03 (La Jolla, CA) was used for statistical analysis.

RESULTS

TGF-β1-mediated Thy-1 suppression can be mitigated by the DNMT inhibitor 5-AZA in mouse lung fibroblasts.

Thy-1 expression is reduced in fibroblastic foci in contrast to increased levels of TGF-β1 in IPF and in murine models of lung fibrosis (5, 9, 18, 24, 37). To date, the relationship between TGF-β1 and Thy-1 has not been elucidated. To determine the effect of TGF-β1 on the expression of Thy-1, mouse PLFs were stimulated with TGF-β1 for 72 h, and Thy-1 expression was evaluated by real-time RT-PCR. There was a significant reduction of Thy-1 gene expression (P < 0.05) in PLFs after 72 h of TGF-β1 treatment (Fig. 1A). In parallel, TGF-β1 treatment significantly decreased Thy-1 protein expression in fibroblasts as determined by FACS analysis (P < 0.05; Fig. 1B). Previous studies in pulmonary fibrosis have shown that Thy-1 expression in lung fibroblasts is epigenetically regulated by promoter hypermethylation (20, 24). We therefore hypothesized that inhibition of methylation would prevent TGF-β1-mediated Thy-1 suppression in lung fibroblasts. We found a significant increase in Thy-1 gene (∼200% increase, P < 0.05) and protein (∼50% increase, P < 0.05) expression in lung fibroblasts exposed to 5-AZA alone (Fig. 1, A and B, respectively). Cells exposed to 5-AZA and TGF-β1 showed a significant increase of Thy-1 gene and protein expression compared with cells treated with TGF-β1 alone (Fig. 1). These data suggested that TGF-β1 might mediate Thy-1 expression through epigenetic mechanisms (e.g., induction of gene methylation).

Fig. 1.

Fig. 1.

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Transforming growth factor (TGF)-β1 decreases thymocyte differentiation antigen 1 (Thy-1) expression, and treatment with a DNA methyltransferase (DNMT) inhibitor attenuates TGF-β1-mediated Thy-1 suppression in mouse primary lung fibroblasts (PLFs). Mouse lung fibroblasts were treated with TGF-β1 (2 ng/ml) ± 5-aza-2′-deoxycytidine (5-AZA, 5 μM). Cells were harvested and analyzed at 24 and 72 h for Thy-1 gene expression and normalized to 18S by real-time RT-PCR (A) and protein levels by FACS analysis (B), respectively. MFI, mean fluorescence intensity. Data are represented as means ± SE. *P < 0.05, decreased compared with the PBS-treated group. **P < 0.05, increased compared with the PBS-treated group. Data are representative of 4 independent experiments.

TGF-β1 induces epigenetic changes through the induction of DNMT activity in mouse lung fibroblasts.

As shown above, an addition of 5-AZA to TGF-β1 attenuated the deleterious effect of TGF-β1 on Thy-1 expression. However, the increase in Thy-1 gene expression may also reflect DNA methylation-independent effects of 5-AZA, which have been previously reported (40). Accordingly, to assess the role of TGF-β1 on DNA methylation, we analyzed epigenetic changes in lung fibroblasts treated with TGF-β1 with a focus on the DNMT pathway. As shown in Fig. 2, TGF-β1 treatment increased DNMT activity by approximately threefold compared with PBS-treated lung fibroblasts (P = 0.05). To further elucidate the specificity of TGF-β1 on DNMT activity, the DNMT inhibitor 5-AZA was used. The addition of 5-AZA to TGF-β1 resulted in attenuation of DNMT activity (Fig. 2). Taken together, these findings suggest that TGF-β1 is sufficient to induce the methylation activity of DNMT enzymes in lung fibroblasts.

Fig. 2.

Fig. 2.

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TGF-β1 induces DNMT activation in mouse PLFs. DNMT activity levels from nuclear extracts prepared from lung fibroblasts treated with TGF-β1 (2 ng/ml) ± 5-AZA (5 μM) were measured by the EpiQuick DNMT Activity Assay. Data are represented as means ± SE. One-way ANOVA showed P = 0.038. Bonferroni's multiple-comparisons posttest was performed. *P = 0.05, changed compared with the PBS group. #P = 0.08, decreased from the TGF-β1 group. Data are representative of 3 independent experiments.

5-AZA attenuates TGF-β1-induced DNMT1 protein expression in lung fibroblasts.

To further elucidate the mechanism by which TGF-β1 influences DNMT activity, we evaluated the effect of TGF-β1 on DNMT1, DNMT3a, and DNMT3b protein expression by Western blot analyses. As shown in Fig. 3, lung fibroblasts exposed to TGF-β1 for 72 h showed an ∼50% increase in DNMT1 protein expression (P < 0.05) compared with PBS treatment (Fig. 3A) but did not have an effect on DNMT3a (Fig. 3B) or DNMT3b (Fig. 3C). As expected, exposure to 5-AZA resulted in a significant decrease in DNMT1 expression (Fig. 3A) and a trend toward a decrease in DNMT3a (Fig. 3B) and DNMT3b (Fig. 3C). Interestingly, addition of TGF-β1 to 5-AZA did not induce DNMT1 protein expression (Fig. 3A), suggesting that 5-AZA is a potent inhibitor of TGF-β1-induced DNMT1 expression. These results suggest that TGF-β1 selectively regulates DNMT1 protein expression.

Fig. 3.

Fig. 3.

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TGF-β1-induced DNMT1 protein expression in mouse PLFs. Mouse PLFs were treated with TGF-β1 (2 ng/ml) for 72 h and were harvested and analyzed for DNMT1 (A), DNMT3a (B), and DNMT3b (C) protein expression by Western blot analysis. Total protein levels were normalized to GAPDH. A representative immunoblot of DNMT1 protein expression in response to treatment is shown in A. Representative bands were selected, cropped, and placed into a panel with Photoshop CS5 without any adjustment of the exposure. Immunoblots for DNMT3a and DNMT3b were not included due to lack of a significant change with treatment. Data are represented as means ± SE. *P < 0.05, changed from the PBS group. Data are representative of 3 independent experiments.

TGF-β1 inhibits Thy-1 gene expression by inducing Thy-1 promoter hypermethylation.

As shown above, TGF-β1 attenuates Thy-1 expression and induces hypermethylation in lung fibroblasts associated with an increase in DNMT1 expression, but not DNMT3a or DNMT3b. Therefore, we hypothesized that TGF-β1 attenuates Thy-1 expression by inducing hypermethylation of the Thy-1 promoter. Using qMethyl PCR targeting the promoter region described in Fig. 4A, we found that lung fibroblasts treated with TGF-β1 had a threefold induction in Thy-1 promoter methylation compared with control cells (P < 0.05; Fig. 4B). This result suggests that methylation of the Thy-1 promoter in lung fibroblasts is regulated by TGF-β1.

Silencing of DNMT1 attenuates TGF-β1-induced DNMT activity and Thy-1 promoter methylation.

As shown in Fig. 3, we found that TGF-β1 induced DNMT1 protein expression, suggesting that TGF-β1 might modify PLF phenotype through induction of DNMT1. With the use of silencing RNA specific to DNMT1 (Fig. 5A), DNMT activity and Thy-1 promoter hypermethylation were analyzed. As shown in Fig. 5B, specific inhibition of DNMT1 using silencing RNA attenuated TGF-β1-induced DNMT activity (P < 0.05). In parallel, silencing of DNMT1 expression showed a trend toward decreased Thy-1 promoter methylation in the presence of TGF-β1 (P = 0.08; Fig. 5C).

Fig. 5.

Fig. 5.

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Silencing of DNMT1 attenuates TGF-β1-mediated DNMT activity and Thy-1 promoter methylation. Mouse PLFs were transfected with DNMT1 silencing RNA or scramble RNA and then treated with TGF-β1 (5 ng/ml). A: at 24 h, DNMT1 silencing RNA showed specific inhibition of DNMT1 mRNA expression but not DNMT3a or DNMT3b mRNA expression by quantitative PCR (inset). *P < 0.05 compared with scrambled RNA group. PLFs were transfected with DNMT1 silencing RNA (DNMT1 RNAi 1), treated with TGF-β1, and analyzed for DNMT activity (B) and Thy-1 promoter methylation (C) by quantitative methyl PCR, at 24 and 72 h, respectively. *P < 0.05 changed compared with scrambled RNA + TGF-β1 group. #P = 0.08 changed compared with scrambled RNA + TGF-β1 group. Data are representative of 3 independent experiments.

Silencing of DNMT1 attenuates TGF-β1-mediated Thy-1 gene and protein expression.

To assess the specific role of DNMT1 on Thy-1 expression, PLFs transfected with DNMT1 silencing RNA or appropriate controls were treated with TGF-β1 and then analyzed for Thy-1 mRNA and protein expression. We found that cells transfected with DNMT1 silencing RNA expressed higher levels of Thy-1 gene (∼60% increase, P < 0.05) and protein (∼10% increase, P < 0.05) as shown in Fig. 6, A and B, respectively.

Fig. 6.

Fig. 6.

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Silencing of DNMT1 attenuates TGF-β1-mediated Thy-1 gene and protein expression. Mouse PLFs were transfected with DNMT1 silencing RNA (DNMT1 RNAi 1) and then treated with TGF-β1 (5 ng/ml). A: at 24 h, cells were harvested and analyzed for Thy-1 gene expression. B: at 72 h, cells were harvested and analyzed for Thy-1 protein expression by flow cytometry. Data represent means ± SE. *P < 0.05 changed compared with scrambled RNA + TGF-β1 group. Data are representative of 4 independent experiments.

Silencing of DNMT1 attenuates TGF-β1-induced α-SMA and Col1A1 gene and Col1A1 protein expression but not α-SMA protein expression.

To assess the specific role of DNMT1 on TGF-β1-induced α-SMA and Col1A1 expression, cells transfected with DNMT1 silencing RNA treated with TGF-β1 were analyzed. As shown in Fig. 7, A and B, silencing of DNMT1 attenuated both α-SMA and Col1A1 gene expression (P < 0.05). In parallel, silencing of DNMT1 attenuated Col1A1 protein expression (P < 0.05; Fig. 7D) but not α-SMA protein expression (Fig. 7C).

Fig. 7.

Fig. 7.

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Silencing of DNMT1 inhibits TGF-β1-induced smooth muscle actin and collagen type 1 gene and protein expression. Mouse PLFs were transfected with DNMT1 silencing RNA (DNMT1 RNAi) and then treated with TGF-β1 (5 ng/ml). At 24 h, cells were harvested and analyzed for α-smooth muscle actin (α-SMA, A) and collagen type 1 (Col1A1, B) gene expression by real-time RT-PCR. At 72 h, cells were harvested and analyzed for α-SMA (C) and collagen type 1 (D) protein expression by Western blot. Data represent means ± SE. *P < 0.05 changed compared with scrambled RNA + TGF-β1 group. Data are representative of 3 independent experiments.

Treatment of lung fibroblasts with 5-AZA attenuates TGF-β1-induced smooth muscle actin stress fiber formation.

Previous studies have shown that fibroblastic foci in clinical specimens of patients with pulmonary fibrosis contain lung fibroblasts lacking Thy-1 expression (9, 10). Furthermore, it has been shown that Thy-1-negative lung fibroblasts are important in TGF-β1 activation and myofibroblast differentiation (18, 23, 42). Moreover, we show above that TGF-β1 inhibits Thy-1 gene and protein expression. Accordingly, we hypothesized that inhibition of DNMT1 could mitigate the effects of TGF-β1 on α-SMA and collagen type 1A1 and, finally, fibroblast to myofibroblast transdifferentiation. As expected, TGF-β1 increases α-SMA and collagen type 1A1 gene and protein expression compared with PBS control (P < 0.05; Fig. 8, A–D). Treatment with 5-AZA alone did not alter α-SMA and collagen type 1A1 gene and protein expression. Treatment with both 5-AZA and TGF-β1 showed a strong trend toward decreased α-SMA and collagen type 1A1 gene expression compared with TGF-β1 treatment (P between 0.05 and 0.06; Fig. 8, A and B). However, cotreatment with 5-AZA and TGF-β1 showed no change in α-SMA and collagen type 1 protein expression compared with TGF-β1 treatment alone by Western blot analyses (Fig. 8, C and D). Finally, to determine whether TGF-β1-mediated DNA methylation is sufficient for α-SMA stress fiber development and morphological changes consistent with the myofibroblast phenotype (11, 32), lung fibroblasts were treated with 5-AZA (0.5, 1, or 5 μM) ± TGF-β1 and analyzed by immunofluorescence microscopy (Fig. 9) and for percent of cells with stress fiber formation (Fig. 9J). As expected, cells treated with DMSO + TGF-β1 (Fig. 9, A–C) showed an increase in the number of α-SMA-expressing myofibroblasts compared with DMSO-treated control cells (data not shown). There was a significant decrease in cells with stress fiber formation in cells treated with 5-AZA + TGF-β1 compared with DMSO + TGF-β1 (P < 0.05; Fig. 9J). Interestingly, cells treated with 5-AZA + TGF-β1 showed disruption in stress fiber formation (Fig. 9, G–I) compared with DMSO + TGF-β1 groups. These findings suggested that epigenetic modification using 5-AZA inhibits TGF-β1-induced fibroblast to myofibroblast transdifferentiation through alteration in stress fiber formation.

Fig. 8.

Fig. 8.

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DNA methylation inhibitor attenuates TGF-β1-induced α-SMA and collagen gene expression but not protein expression. Mouse lung fibroblasts were treated with TGF-β1 (2 ng/ml) ± 5-AZA. At 24 h, cells were harvested and analyzed for α-SMA (A) and Col1A1 (B) gene expression by real-time RT-PCR. At 72 h, cells were harvested and analyzed for α-SMA (C) and collagen type 1 (D) protein expression by Western Blot. Data represent means ± SE. *P < 0.05, increased compared with the PBS-treated group. #P between 0.05 and 0.06, decreased compared with the TGF-β1 treatment group. Data are representative of 3 independent experiments.

Fig. 9.

Fig. 9.

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DNA methylation inhibitor 5-AZA attenuates TGF-β1-induced fibroblast to myofibroblast transdifferentiation. Mouse lung fibroblasts were treated with 5-AZA or DMSO ± TGF-β1 (5 ng/ml) for 96 h. Myofibroblast transdifferentiation was evaluated in lung fibroblasts using immunofluorescent staining for α-SMA (green). DAPI (blue) was used for nuclear staining. Representative slides are shown. A–C: cells treated with DMSO (0.5, 1, or 5 μM, respectively) + TGF-β1. D–F: cells treated with 5-AZA alone (0.5, 1, or 5 μM, respectively). G–I: cells treated with 5-AZA (0.5, 1, or 5 μM, respectively) + TGF-β1. Scale bar = 50 μm. J: 2 random fields from each sample were analyzed for percent of cells positive for stress fiber formation. Total cells counted = 276–478 from each treatment group. Data are representative of 3 independent experiments. *P < 0.05 changed compared with each corresponding DMSO + TGF-β1 treatment group. Data are representative of 3 independent experiments.

Inhibition of DNA methylation with 5-AZA restores Thy-1 expression in Thy-1-negative lung fibroblasts.

Loss of Thy-1 expression helps to promote fibroblast to myofibroblast transdifferentiation. Because studies have shown that this is secondary to epigenetic silencing of Thy-1 expression (9, 18, 23–25), we evaluated whether 5-AZA could restore protein expression in an enriched population of Thy-1-negative cells. As shown in Fig. 10, exposure to 5-AZA significantly upregulates Thy-1 protein expression compared with control (DMSO)-treated cells as analyzed by flow cytometry (P < 0.05). These findings suggest that loss of Thy-1 by methylation is a reversible process that could potentially influence the myofibroblast phenotype.

Fig. 10.

Fig. 10.

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Methylation inhibition restores Thy-1 expression in Thy-1low mouse lung fibroblasts. Lung fibroblasts were isolated from young mice and sorted by FACS on a BD FACSARIA cell sorter into Thy-1high and Thy-1low subpopulations. Thy-1low cells were incubated with DMSO or 5-AZA as described above. Cells were harvested and analyzed for Thy-1 cell surface expression by FACS analysis. Representative histograms are shown. Graph depicts the mean fluorescence intensity of Thy-1 expression as shown by percentage compared with untreated group (data not shown). Data represent means ± SE. *P < 0.05, increased compared with DMSO treatment. Data are representative of 4 independent experiments.

DISCUSSION

In this study we determined that TGF-β1 downregulates Thy-1 gene and protein expression in lung fibroblasts through induction of DNMT activity. Consistent with this conclusion, the effect of TGF-β1 on DNMT activity was attenuated by treatment with the DNMT inhibitor 5-AZA. We further showed that TGF-β1 upregulates DNMT1 expression and suppresses Thy-1 expression through induction of Thy-1 promoter hypermethylation. Inhibition of DNMT with 5-AZA attenuated TGF-β1-induced α-SMA and Col1A1 gene expression. In parallel, 5-AZA failed to attenuate TGF-β1-induced α-SMA and collagen type 1 protein expression, but it inhibited α-SMA stress fiber formation. Thy-1 protein expression was restored by treatment with 5-AZA. Finally, specific inhibition of DNMT1 using silencing RNA attenuated TGF-β1-mediated DNMT activity, Thy-1 gene and protein suppression, α-SMA and Col1A1 gene expression, and collagen type 1 protein expression. However, silencing of DNMT1 expression did not fully attenuate TGF-β1-mediated Thy-1 promoter methylation. Taken together, these results provide evidence that one of the mechanisms by which TGF-β1 regulates fibroblast phenotype is through epigenetic modifications to the Thy-1 promoter that lead to decreased Thy-1 expression and fibroblast to myofibroblast transdifferentiation.

These new findings build on previous studies showing that TGF-β1 contributes to the development and progression of fibrotic lung disease by regulating extracellular matrix (ECM) synthesis and cellular behavior (5, 30). TGF-β1 has been shown to induce fibroblast to myofibroblast transdifferentiation both in vitro and in vivo, in part due to adhesion signaling and FAK activation and possibly through epigenetic modification of Thy-1, a protein that is intimately involved in fibroblast phenotype determination (9, 23, 31, 41). It has been shown that fibroblasts lacking Thy-1 expression are important in activation of TGF-β1 (42) and development of fibrosis in patients with IPF and in animal models (9, 10). Furthermore, TGF-β1 has been shown to regulate cellular programming and epithelial to mesenchymal transition by DNA methylation in prostate cancer (14) and breast cancer (17) as well as in hepatic stellate cells in a liver fibrosis model (4). Accordingly, we hypothesized that TGF-β1 epigenetically inhibits Thy-1 expression in lung fibroblasts. We showed that TGF-β1 decreased Thy-1 gene and protein expression, and treatment with the DNMT inhibitor 5-AZA attenuated the effects of TGF-β1 on Thy-1 expression. Interestingly, 5-AZA exposure alone increased Thy-1 gene and protein expression, suggesting that the Thy-1 promoter might be partially hypermethylated at baseline. Whether this is an acquired epigenetic change in culture or a homeostatic regulation of Thy-1 gene expression is to be determined. However, the increase in Thy-1 gene expression may also reflect DNA methylation-independent effects of 5-AZA, which have been previously reported (40).

DNMT is a key factor in regulating the methylation of target genes within CpG dinucleotide-rich regions in the promoter. Several studies showed that TGF-β1 epigenetically regulates the development of fibrosis, either through DNMT suppression (16) or activation (4, 17, 38). In this study, we show that TGF-β1 induces DNMT activity in lung fibroblasts. In parallel, TGF-β1 selectively upregulates DNMT1 protein expression but not DNMT3a or DNMT3b expression in lung fibroblasts. These findings are further supported by our observation that TGF-β1 induces Thy-1 CpG promoter methylation around threefold, raising the possibility that DNMT1 may be the key methyltransferase acting downstream of TGF-β1 in the regulation of Thy-1 in lung fibroblasts. Collectively, these data show that TGF-β1 decreased Thy-1 expression via Thy-1 promoter hypermethylation. A recent study showed an increase in DNMT1 in the lung and lung fibroblasts of patients with IPF, and inhibition of DNMT1 activity through 5-AZA treatment decreased lung fibrosis in bleomycin-treated mice (6). In parallel, we found that specific inhibition of DNMT1 attenuated the suppression of overall DNMT activity and Thy-1 expression by TGF-β1 as well as its effects on α-SMA and Col1A1 gene, and collagen type 1 protein expression but not α-SMA expression. However, DNMT1 inhibition did not significantly inhibit Thy-1 promoter methylation as shown by qMethyl PCR, but did show a trend (P = 0.08). The modest level of attenuation may suggest that the persistent low level of DNMT activity that occurred after DNMT1 silencing may be sufficient to drive promoter hypermethylation. It may also indicate that other DNMT enzymes are involved in the process. Overall, though, our study provides further evidence that TGF-β1 expression and DNA methylation of Thy-1 influence lung fibroblast phenotype through DNMT1 in the pathogenesis of pulmonary fibrosis.

The mechanism by which TGF-β1 regulates fibroblast to myofibroblast transdifferentiation is not well understood. In the current study, we showed that addition of 5-AZA to TGF-β1 failed to suppress α-SMA protein expression, but morphological analyses showed that 5-AZA decreased actin stress fiber formation in a dose-dependent manner. These data suggest that TGF-β1 influences fibroblast to myofibroblast differentiation through DNA methylation during stress fiber formation (e.g., epigenetic modification of RhoA) but not through TGF-β1-induced α-SMA protein expression. Future studies to assess the relationship between TGF-β1 and Thy-1 expression on myofibroblast differentiation might allow us to develop therapeutic interventions for patients with IPF. One of the observations here that supports the therapeutic potential of 5-AZA is that treatment of Thy-1low lung fibroblasts with 5-AZA increases Thy-1 protein expression, which portends a less fibrogenic fibroblast phenotype (25, 42).

The present study has several limitations. The effects of TGF-β1 on Thy-1 expression were evaluated in vitro using a cell culture model. Therefore, the potential clinical relevance of these findings will require further investigation, including translating them to animal models of lung fibrosis and fibroblast cultures generated from IPF tissue. To fully address whether or not TGF-β1 induces fibroblast to myofibroblast transdifferentiation directly through Thy-1 methylation, future studies are needed with specific inhibitors of the TGF-β1 signaling pathway. Furthermore, although Thy-1 is one of the major methylation targets in lung fibrosis, other candidate genes shown to be hypermethylated in IPF such as phosphate and tensin homolog (35), caveolin-1 (34), CDKN2B, and CARD10 (12) should be evaluated as other potential TGF-β1 methylation targets. Additionally, future studies are necessary to investigate other transcriptional and posttranscriptional mechanisms of TGF-β1-mediated Thy-1 regulation, including histone deacetylation and cell surface receptor shedding. Although these mechanisms of Thy-1 regulation have been documented in the literature, their association with TGF-β1 is currently unknown (9, 25). Last, it is important to identify the specific pathway(s) within the TGF-β1 cascade that activates DNA methylation (e.g., MAPK, SMAD, PI3K/AKT, integrin-ECM pathways). Elucidation of these downstream events is important for the development of targeted fibrosis therapy, since long-term global DNMT inhibition by 5-AZA may have an unacceptable level of toxicity.

In summary, we determined that TGF-β1 inhibits Thy-1 expression in mouse lung fibroblasts by promoter hypermethylation and that TGF-β1-induced methylation promotes fibroblast to myofibroblast transdifferentiation (Fig. 11). These results are provocative since aberrant TGF-β1 expression in the lung has also been implicated in conditions such as alcohol-mediated susceptibility to acute lung injury (2, 3, 27) and chronic human immunodeficiency virus infection, both of which are characterized by increased cell senescence and TGF-β1 elevation in the lung (7, 33). Although it is known that TGF-β1 induces myofibroblast transdifferentiation through ECM-dependent signaling, adhesion-independent mechanisms of fibroblast cellular programming are not as well characterized. Our studies suggest that TGF-β1 may also induce myofibroblast transdifferentiation through epigenetic regulation of Thy-1. The reversibility of Thy-1 expression in lung fibroblasts with the methylation inhibitor 5-AZA, a current Food and Drug Administration-approved chemotherapy, could provide a therapeutic option for the amelioration of lung fibrosis. Therefore, future studies investigating DNA methylation in experimental models of lung fibrosis are warranted and could provide evidence supporting the design of novel clinical trials.

Fig. 11.

Fig. 11.

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Schematic representation of the hypothesized effect of TGF-β1 on Thy-1 regulation in mouse PLFs. Cellular stress (e.g., aging, reactive oxygen species, and alcohol exposure) triggers the release of latent TGF-β1 from PLFs. Upon activation, TGF-β1 induces Thy-1 promoter methylation (e.g., DNMT activation). Loss of Thy-1 expression primes PLFs for a profibrotic response by promoting extracellular matrix (ECM) remodeling, autocrine TGF-β1 production, and myofibroblast differentiation.

GRANTS

This study was supported by National Institutes of Health Grants K08-AA-021404-01 (V. Sueblinvong), P50-AA-013757 (V. Sueblinvong and DMG), T32-HL-116271 (W. A. Neveu) and T32-HL-076118 (B. S. Staitieh).

DISCLOSURES

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Author contributions: W.A.N. and V.S. conception and design of research; W.A.N., S.T.M., and V.S. performed experiments; W.A.N. and V.S. analyzed data; W.A.N., B.S.S., and V.S. interpreted results of experiments; W.A.N. and V.S. prepared figures; W.A.N. and V.S. drafted manuscript; W.A.N., B.S.S., and V.S. edited and revised manuscript; V.S. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank David Guidot for providing support and mentoring for W. A. Neveu. We thank Xian Fan and Michael Koval for helpful scientific discussions and suggestions during the preparation of this manuscript.

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