Histone modifications are responsible for decreased Fas expression and apoptosis resistance in fibrotic lung fibroblasts

Abstract

Although the recruitment of fibroblasts to areas of injury is critical for wound healing, their subsequent apoptosis is necessary in order to prevent excessive scarring. Fibroproliferative diseases, such as pulmonary fibrosis, are often characterized by fibroblast resistance to apoptosis, but the mechanism(s) for this resistance remains elusive. Here, we employed a murine model of pulmonary fibrosis and cells from patients with idiopathic pulmonary fibrosis (IPF) to explore epigenetic mechanisms that may be responsible for the decreased expression of Fas, a cell surface death receptor whose expression has been observed to be decreased in pulmonary fibrosis. Murine pulmonary fibrosis was elicited by intratracheal injection of bleomycin. Fibroblasts cultured from bleomycin-treated mice exhibited decreased Fas expression and resistance to Fas-mediated apoptosis compared with cells from saline-treated control mice. Although there were no differences in DNA methylation, the Fas promoter in fibroblasts from bleomycin-treated mice exhibited decreased histone acetylation and increased histone 3 lysine 9 trimethylation (H3K9Me3). This was associated with increased histone deacetylase (HDAC)-2 and HDAC4 expression. Treatment with HDAC inhibitors increased Fas expression and restored susceptibility to Fas-mediated apoptosis. Fibroblasts from patients with IPF likewise exhibited decreased histone acetylation and increased H3K9Me3 at the Fas promoter and increased their expression of Fas in the presence of an HDAC inhibitor. These findings demonstrate the critical role of histone modifications in the development of fibroblast resistance to apoptosis in both a murine model and in patients with pulmonary fibrosis and suggest novel approaches to therapy for progressive fibroproliferative disorders.

OPEN

Citation: Cell Death and Disease (2013) 4, e621; doi:10.1038/cddis.2013.146

& 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13 http://www.nature.com/cddis

Web End =www.nature.com/cddis

SK Huang*1, AM Scruggs1, J Donaghy1, JC Horowitz1, Z Zaslona1, S Przybranowski1, ES White1 and M Peters-Golden1

Although the recruitment of broblasts to areas of injury is critical for wound healing, their subsequent apoptosis is necessary in order to prevent excessive scarring. Fibroproliferative diseases, such as pulmonary brosis, are often characterized by broblast resistance to apoptosis, but the mechanism(s) for this resistance remains elusive. Here, we employed a murine model of pulmonary brosis and cells from patients with idiopathic pulmonary brosis (IPF) to explore epigenetic mechanisms that may be responsible for the decreased expression of Fas, a cell surface death receptor whose expression has been observed to be decreased in pulmonary brosis. Murine pulmonary brosis was elicited by intratracheal injection of bleomycin. Fibroblasts cultured from bleomycin-treated mice exhibited decreased Fas expression and resistance to Fas-mediated apoptosis compared with cells from saline-treated control mice. Although there were no differences in DNA methylation, the Fas promoter in broblasts from bleomycin-treated mice exhibited decreased histone acetylation and increased histone 3 lysine 9 trimethylation (H3K9Me3). This was associated with increased histone deacetylase (HDAC)-2 and HDAC4 expression. Treatment with HDAC inhibitors increased Fas expression and restored susceptibility to Fas-mediated apoptosis. Fibroblasts from patients with IPF likewise exhibited decreased histone acetylation and increased H3K9Me3 at the Fas promoter and increased their expression of Fas in the presence of an HDAC inhibitor. These ndings demonstrate the critical role of histone modications in the development of broblast resistance to apoptosis in both a murine model and in patients with pulmonary brosis and suggest novel approaches to therapy for progressive broproliferative disorders.

Cell Death and Disease (2013) 4, e621; doi:http://dx.doi.org/10.1038/cddis.2013.146

Web End =10.1038/cddis.2013.146 ; published online 2 May 2013

Subject Category: Experimental Medicine

Normal wound healing is characterized by the recruitment and activation of broblasts to the areas of injury, followed by the clearance of broblasts after tissue has been repaired.1 The apoptosis of broblasts during this latter stage is felt to be critical in the prevention of pathological scarring.2 Undesired brosis can occur when activated broblasts persist in tissue and are not cleared by usual apoptotic mechanisms. Indeed, an overabundance of broblasts3 and broblast resistance to apoptosis47 are hallmarks of many brotic disorders. Overcoming apoptosis resistance and inducing apoptosis in broblasts may serve as effective strategies for the treatment of many chronic broproliferative disease which, by some estimates, accounts for up to 45% of all deaths in the developed world.8

Idiopathic pulmonary brosis (IPF) is a brotic disorder of the lungs that exacts signicant morbidity and mortality.9,10

Lung broblasts and myobroblasts are the major effector cells responsible for the excessive extracellular matrix production and scarring associated with IPF.11 Several studies have shown that broblasts from IPF patients are resistant to apoptosis,12,13 and in particular Fas-mediated

Keywords: epigenetics; histone deacetylation; histone methylation; histone deacetylase

Abbreviations: IPF, idiopathic pulmonary brosis; FasL, Fas ligand; PARP, poly-ADP ribose polymerase; ChIP, chromatin immunoprecipitation; HDAC, histone deacetylase; 5-aza-dC, 5-aza-20-deoxycytidine; TSA, trichostatin A; SAHA, suberoylanilide hydroxamic acid; H3K9Me3, histone 3 lysine 9 trimethylation; H3K27Me3, histone 3 lysine 27 trimethylation; c-FLIP, cellular FLICE-like inhibitory protein; XIAP, X-linked inhibitor of apoptosis

Histone modications are responsible for decreased Fas expression and apoptosis resistance in brotic lung broblasts

apoptosis,4,6,7,14,15 through a number of potential mechan

isms, including decreased expression of the surface receptor

Fas.4,16 Activation of Fas (CD95, Apo-1, TNF receptor superfamily member 6) by either soluble or cell surface-expressed Fas ligand (FasL) results in the formation of the intracellular death complex domain and activation of caspase 8, which lead to apoptosis.17 The failure to activate Fas and induce broblast apoptosis has been shown to have a critical role in the pathogenesis of pulmonary brosis.18,19 The

mechanism for the resistance of IPF broblasts to Fas-mediated apoptosis and for the decreased expression of Fas is unknown.

Although epigenetic alterations, including DNA methylation and histone modications, have been implicated in the dysregulation of genes associated with pulmonary brosis,2023 there are no studies that have examined whether

epigenetic mechanisms are responsible for the apoptosis resistance of broblasts in IPF. Methylation of DNA is associated with decreased gene expression, whereas acetylation of histones results in relaxation of chromatin facilitating gene transcription. These and other histone modications

1Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI, USA*Corresponding author: SK Huang, Pulmonary and Critical Care Medicine, University of Michigan, 6301 MSRB III, 1150 W Medical Center Dr., Ann Arbor, MI 48109, USA. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected]

Received 20.2.13; revised 28.3.13; accepted 3.4.13; Edited by G Raschell

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may function independently or in concert to regulate overall gene expression. Here, we investigated the mechanisms associated with the decreased expression of Fas in the lung broblasts of mice with bleomycin-induced pulmonary brosis and of patients with IPF and identied several histone modications associated with the FAS (TNFRSF6) gene promoter that are responsible for the decreased expression of Fas and resistance to Fas-mediated apoptosis.

Results

Fibroblasts from the lungs of mice with bleomycin-induced brosis exhibit diminished expression of Fas and resistance to Fas-mediated apoptosis. Administration of intratracheal bleomycin in C57Bl/6 mice induces pulmonary brosis, which is evident by days 1421,24 and is

a commonly used model of human brotic lung disease. Fibroblasts cultured from the brotic lungs of bleomycin-exposed mice have been shown to exhibit resistance to apoptosis,4,6,7,1214 but the mechanism(s) for apoptosis

resistance is unclear. Fas-mediated apoptosis can be triggered in vitro by the addition of Fas-activating antibody and enhanced by the addition of cycloheximide, which

prevents the synthesis of apoptosis inhibitor proteins.14

Fibroblasts cultured from the brotic lungs of mice 21 days after bleomycin exposure exhibited less apoptosis in response to Fas activation than did cells from saline-treated control mice (Figure 1). This resistance to Fas-mediated apoptosis was conrmed by assays of cleaved poly-ADP ribose polymerase (PARP) (Figure 1a) and cleaved caspase 8 (Figure 1b), both of which are diminished in broblasts from bleomycin-treated mice compared with saline-treated controls. To determine the mechanism for resistance to Fas-mediated apoptosis, we examined the expression of Fas and observed that cell surface expression of Fas (as assayed by ow cytometry), total protein expression of Fas from whole cell lysates (as determined by immunoblot), and Fas mRNA levels were all decreased in broblasts from bleomycin-treated mice compared with saline-treated controls (Figures 1ce). These ndings suggest that diminished expression of Fas may be responsible for the resistance to Fas-mediated apoptosis.

Diminished expression of Fas in broblasts from bleomycin-injured mice is attributed to histone modications. DNA methylation and histone modications have

Figure 1 Fibroblasts from the brotic lungs of bleomycin-injured mice are resistant to Fas-mediated apoptosis and exhibit decreased expression of Fas. Fibroblasts from mice treated with bleomycin or saline control were treated with the anti-Fas activating antibody (Fas Ab, 100 ng/ml) and cycloheximide (CHX, 0.5 mg/ml). Apoptosis was assayed by immunoblotting for (a) cleaved PARP and (b) cleaved caspase 8. Representative blot is shown, with densitometric ratio of cleaved to total PARP (n 3) and

cleaved caspase 8 to procaspase 8 (n 4) shown below each blot. (c) Expression of cell-surface Fas in broblasts from bleomycin- and saline-treated mice was assayed by

ow cytometry, with results expressed as mean uorescent intensity (n 3 for saline, n 4 for bleomycin). (d) Total Fas protein expression (n 4) and (e) Fas mRNA levels

(n 4 for saline, n 6 for bleomycin) were assayed as described in Methods. *Po0.05. NS, not signicant

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been shown to be responsible for the differential expression of certain genes in pulmonary brosis.20,22,23 To examine

whether such epigenetic mechanisms are responsible for the diminished expression of Fas in broblasts from bleomycin-injured mice, we rst examined levels of DNA methylation near the transcription start site of Fas. Performing bisulte sequencing, we observed no difference in DNA methylation between broblasts from brotic and nonbrotic lung at any of the 16 CpG loci analyzed (Supplementary Figure S1).

We next examined whether broblasts from bleomycin-treated mice exhibit differences in histone modications associated with the Fas promoter. We performed chromatin immunoprecipitation (ChIP) using antibodies to acetylated H3 and acetylated H4 and observed that the Fas promoter in broblasts from bleomycin-injured mice manifested decreased H3 (Figure 2a) and H4 (Figure 2b) acetylation. More than 11 isoforms of the classical histone deacetylase (HDAC) family, separated phylogenetically into two classes, are capable of deacetylating histones with varying specicity.25 Fibroblasts from the lungs of brotic mice were observed to exhibit increased levels of both HDAC2, a class I HDAC, and HDAC4, a class II HDAC (Figure 2c).

To determine whether the deacetylation of histones and the increase in HDACs in broblasts from bleomycin-injured mice are sufcient to cause diminished expression of Fas, we treated cells with two nonselective HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Treatment with either of the HDAC inhibitors, but not the DNA methylation inhibitor 5-aza-20-deoxycytidine (5-aza-dC), resulted in a signicant increase in Fas mRNA levels in cells from mice with bleomycin-induced brosis (Figure 3a). Neither TSA nor 5-aza-dC had any effect on Fas mRNA levels in broblasts from saline-control mice. Treatment of broblasts from bleomycin-treated mice with TSA or SAHA, but not 5-aza-dC, increased cell surface expression of Fas, as assayed by ow cytometry, to levels comparable with that observed in broblasts from saline-treated mice (Figure 3b). Total Fas protein expression in lung broblasts from bleomycin-treated mice was likewise increased in the presence of TSA or SAHA (data not shown). Treatment with SAHA furthermore resulted in a selective increase of acetylated histones at the Fas

promoter in cells from bleomycin-injured mice but not in those from saline controls (Figure 3c).

We next pretreated broblasts from saline-control and bleomycin-injured mice with TSA and examined the susceptibility of the cells to apoptosis by the Fas-activating antibody and cycloheximide. Fibroblasts from bleomycin-injured mice pretreated with TSA exhibited increased susceptibility to apoptosis by Fas-activating antibody and cycloheximide, as assayed by levels of cleaved caspase 8 (Figure 4a) and cleaved PARP (Figure 4b). TSA pretreatment of broblasts from saline-control mice did not alter the ability of Fas-activating antibody and cycloheximide to induce apoptosis in these already apoptosis-sensitive cells. Together, these results suggest that histone deacetylation is responsible for the decreased Fas expression and apoptosis resistance observed in brotic broblasts.

Histone acetylation/deacetylation is often associated with histone methylation changes at specic histone residues.26

These latter modications may be either the cause or effect of histone deacetylation. In particular, histone 3 lysine 9 trimethylation (H3K9Me3) and histone 3 lysine 27 trimethylation (H3K27Me3) are associated with decreased gene expression and the recruitment of HDACs.26 ChIP analysis using antibodies to H3K9Me3 and H3K27Me3 revealed that the Fas promoter is associated with increased H3K9Me3, but not H3K27Me3, in broblasts from bleomycin-injured mice compared with saline-control mice (Figure 5).

Histone modications are also responsible for the decreased expression of Fas in broblasts from IPF patients. Fas expression has been shown to be decreased in lung broblasts from patients with IPF, and diminished Fas expression in IPF broblasts is associated with increased resistance to Fas-mediated apoptosis4,16. The mechanisms

responsible for decreased Fas expression in IPF broblasts, however, are unknown. Based on our observations in the bleomycin mouse model of brosis, we sought to determine whether histone modications are also responsible for the decreased expression of Fas in cells from IPF patients. We performed ChIP utilizing antibodies to acetylated H3 and H3K9Me3 and assayed by real-time PCR two regions within

Figure 2 Fibroblasts from bleomycin-injured mice are associated with decreased histone acetylation. ChIP using antibodies against (a) acetylated H3 (n 6 for saline,

n 5 for bleomycin) and (b) acetylated H4 (n 6 for saline, n 4 for bleomycin) was performed in broblasts from saline- and bleomycin-treated mice. Relative enrichment of

the Fas promoter, normalized to saline control, was assayed by real-time PCR from immunoprecipitated DNA. (c) Expression of HDAC2 and HDAC4 were assayed by immunoblot in broblasts from bleomycin- and saline-treated mice, with mean densitometry values shown (n 4). *Po0.05

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Figure 3 Treatment of broblasts from bleomycin-injured mice with HDAC inhibitors resulted in increased expression of Fas. Fibroblasts from saline- and bleomycin-treated mice were treated for 72 h with the DNA methylation inhibitor 5-aza-dC (1 mM) or the HDAC inhibitors TSA (0.1 mM unless otherwise specied) and SAHA (1 mM) and (a) assayed for Fas mRNA levels by real-time RT-PCR (n 4 for saline, n 6 for bleomycin; normalized to untreated control) and (b) cell surface expression by ow cytometry

(n 2 for saline, n 4 for bleomycin). (c) ChIP for acetylated H3 was performed in cells pretreated with or without SAHA (n 3 for saline, n 4 for bleomycin). Enrichment for

the Fas promoter was assayed by PCR and normalized to untreated saline. *Po0.05 relative to untreated control

Figure 4 Pretreatment of broblasts from bleomycin-injured mice with TSA increased cell susceptibility to Fas-mediated apoptosis. Fibroblasts from the lungs of saline-and bleomycin-treated mice were pretreated with or without TSA (0.1 mM) for 72 h before being treated with the anti-Fas activating antibody (100 ng/ml) and cycloheximide (CHX, 0.5 mg/ml). (a) Cleaved caspase 8 and (b) cleaved PARP were assayed by immunoblot, with mean densitometric ratio of cleaved caspase 8 to procaspase 8 (n 4 for

both saline and bleomycin) and cleaved PARP to total PARP (n 2 for saline, n 3 for bleomycin) shown below each representative blot. *Po0.05

the FAS gene promoter. Fibroblasts from IPF patients exhibited decreased histone acetylation and increased H3K9Me3 at both regions interrogated along the gene promoter (Figure 6). Treatment of IPF broblasts with the

HDAC inhibitor TSA, but not the DNA methylation inhibitor 5-aza-dC, resulted in increased Fas mRNA (Figure 7a) and protein expression (Figure 7b). There was no difference in Fas promoter DNA methylation between IPF and nonbrotic

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Figure 5 Fibroblasts from bleomycin-treated mice were associated with increased H3K9Me3 at the Fas gene promoter. ChIP using antibodies against (a) H3K9Me3 (n 3) and (b) H327Me3 (n 6) was performed in broblasts from saline- and bleomycin-treated mice. Enrichment for the Fas promoter normalized to saline control was

assayed by real-time PCR. *Po0.05

Figure 6 The Fas gene promoter in IPF broblasts was also associated with decreased histone acetylation and increased H3K9 trimethylation. ChIP using antibodies against (b) acetylated H3 and (c) H3K9Me3 was performed in broblasts from IPF patients and nonbrotic controls. (a) Immunoprecipitated DNA was PCR amplied using primer pairs from two separate regions of the Fas gene promoter. *Po0.05

control broblasts (Supplementary Figure S1b). These data suggest that histone deacetylation and trimethylation of lysine 9 on H3 are responsible for the decreased Fas expression in IPF broblasts as also observed in bleomycin broblasts.

Discussion

Although the recruitment, expansion, and activation of broblasts at areas of tissue injury are considered integral to normal wound healing, apoptosis of broblasts is also critical for the eventual clearance of these cells after wound healing resolves.1,2 The persistence of activated broblasts beyond this period is felt to contribute to pathologic scarring, and broblast resistance to apoptosis is a key hallmark of many brotic disorders. The mechanism(s) for this resistance is, in most cases, unknown. Here, utilizing an animal model of

pulmonary brosis and examining primary cells from patients with IPF, we identied histone modications specically histone deacetylation and H3K9Me3 that are responsible for the decreased expression of the death receptor Fas and resistance to Fas-mediated apoptosis in broblasts from brotic lung. In both the mouse model and the human disease, treatment with HDAC inhibitors increased Fas expression and restored sensitivity of brotic cells to Fas-mediated apoptosis.

Changes in DNA methylation and histone modications have been shown to modulate the expression of several genes in pulmonary brosis;2023 however, this is the rst demonstration of histone modications being responsible for the specic downregulation of Fas and the resistance of brotic lung broblasts to Fas-mediated apoptosis. Although the function of Fas was originally described in studies of adaptive immunity,27,28 the importance of FasFasL interactions in other diseases, including pulmonary brosis, are well

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Figure 7 Treatment of IPF broblasts with TSA increased Fas expression. IPF broblasts were treated with the DNA methylation inhibitor 5-aza-dC or the HDAC inhibitor TSA at the respective concentrations and (a) assayed for Fas mRNA levels (n 3) and (b) protein expression by immunoblot

substantiated.18,19 It was originally suggested from indirect studies employing HDAC inhibitors in cancer that Fas expression may be regulated by histone acetylation.29,30

More recent studies have shown that diminished Fas expression is associated with histone deacetylation in models of colon and liver inammation.31,32 IPF is a disease of

unknown etiology but is thought to be inuenced by certain environmental exposures, some of which may affect epigenetic machinery.33 The persistence of apoptosis resistance in brotic lung broblasts through serial passages in culture further suggests that epigenetic mechanisms are responsible for these changes. Our studies presented here highlight the importance of histone modications in the resistance of brotic lung cells to apoptosis and in their role in the pathogenesis of pulmonary brosis.

Although we identied histone modications to be the mechanism for the diminished expression of Fas in pulmonary brosis, how these modications arise at the Fas promoter is unknown. This process is presumably acquired, as these modications were observed in murine models of pulmonary brosis in which brosis develops after bleomycin injury. The histone modications are furthermore heritable with cell division, as they are sustained in cell culture over several passages. It has been shown that Fas overexpression and increased expression of Fas by the cytokines tumor necrosis factor-a and interferon-g are sufcient to overcome the resistance of IPF broblasts to apoptosis.16,34 Further studies

would be needed to determine whether this cytokine-mediated increase in Fas expression is a result of modications to histones. The study by Wynes et al.16 did show that the cytokine-mediated increase in Fas depends on the transcription factor NF-kB, which recruits histone acetyltransferases to sites of transcription.35

In addition to histone deacetylation, we also observed that the Fas gene promoter in brotic cells exhibited an increase in H3K9Me3, but not H3K27Me3. Both H3K9Me3 and H3K27Me3 marks are associated with gene silencing and have been implicated as drivers of other chromatin changes, including histone deacetylation and DNA methylation.26

Interestingly, we did not observe any difference in DNA methylation at the Fas gene promoter in brotic lung broblasts from mice or IPF patients, and it is unclear in this case what the exact relationship is between H3K9Me3 and

histone deacetylation in regulating Fas expression in brotic lung broblasts. Others investigators have reported that H3K9Me3 marks may be a consequence, rather than a determinant, of histone deacetylation,36 and in our case, treatment of broblasts with HDAC inhibitors was sufcient to restore Fas expression and cell susceptibility to apoptosis. Fibrotic lung broblasts were also observed to exhibit increased expression of HDAC2 and HDAC4, suggesting that increased expression of HDACs may be sufcient for the deacetylation of histones and decreased expression of Fas. The presence of H3K9Me3, however, may reinforce gene silencing and promote the heritability of chromatin changes during cell division.

Apoptosis requires the complex interplay of many signaling molecules, and the deciency or overexpression of other apoptosis regulatory proteins besides Fas has also been implicated in the resistance of broblasts to apoptosis in the setting of pulmonary brosis.14,15 In fact, our nding that Fas

expression is diminished in broblasts from bleomycin-injured mice differs from that of Wallach-Dayan et al.,7 who did not observe a difference in Fas expression between broblasts from bleomycin-injured mice and controls. This discrepancy may be attributed to the fact that Wallach-Dayan et al.7 studied broblasts that were cultured from mice just 7 days after bleomycin-induced pulmonary injury. Because pulmonary brosis typically develops at days 1421 post-bleomycin exposure, the broblasts that we used in our experiments (taken from mice at day 21 post-bleomycin treatment) may be phenotypically different from those used in the study by Wallach-Dayan et al.7 and may illustrate the plasticity of histone modications in broblasts during the evolution of brosis. These as well as other investigators identied increased expression of cellular FLICE-like inhibitory protein (c-FLIP) as also contributing to the resistance of broblasts to apoptosis,15,37 although this was not consistently observed among broblasts in IPF tissue.38 Increased expression of inhibitors of apoptosis such as survivin14,39 and X-linked

inhibitor of apoptosis (XIAP)15 may also have a substantial role in apoptosis resistance. Indeed, the ability of cycloheximide to augment Fas-mediated apoptosis is thought to reect the importance of endogenous production of inhibitors of apoptosis. It would be informative to determine whether histone modications are also responsible for altered expression of c-FLIP, survivin, or XIAP in pulmonary brosis.

In our studies, the decreased Fas expression in brotic lung broblasts was observed at the transcriptional level that is congruent with its expression being regulated by epigenetic mechanisms. However, cell surface expression of Fas has also been reported to be regulated by post-translational mechanisms,4 and these mechanisms can be regulated by oxidative stress.40 Furthermore, broblast expression of FasL has been shown to promote epithelial cell apoptosis, which is also considered critical to the pathogenesis of IPF.41,42

Whether FasL expression is regulated by histone modications in pulmonary brosis is unknown.

Although bleomycin administration is a commonly used mouse model of pulmonary brosis, there are substantial limitations in its ability to approximate IPF. Nonetheless, in terms of Fas-mediated resistance to apoptosis, we observed similar mechanisms of histone modications in the bleomycin

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model as in the human disease. We found that the degree of histone deacetylation and H3K9 trimethylation as well as responsiveness to HDAC inhibitors varied among individual IPF patient lines. This is not surprising given the phenotypic heterogeneity of IPF broblasts observed in other contexts.39,43

IPF is a devastating lung disease with no clearly effective therapy aside from lung transplantation.10 Fibroblasts are the major effector cells responsible for the generation of extra-cellular matrix proteins that comprise scar tissue and are abundant within broblastic foci, the characteristic pathological lesion in IPF. Stimulation of broblast apoptosis is thus an appealing therapeutic approach, but broblast resistance to apoptosis poses a potential barrier to such therapy. The identication of histone modications as responsible for this resistance and the ability of HDAC inhibitors to overcome this resistance represent novel avenues for future therapy of this deadly disease.

Materials and MethodsCells. Bleomycin (0.0010 U/kg body weight) was administered intratracheally to C57Bl/6 mice (Charles River Laboratory, Wilmington, MA, USA) to induce pulmonary brosis, which develops by days 1421.24 Administration of intratracheal saline was used as control. At day 21, mice were euthanized and the lungs were minced and placed in Dulbeccos Modied Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and penicillin/streptomycin (Invitrogen). Outgrowths of broblasts from minced lungs were cultured, washed, and passaged for use in subsequent experiments.

Fibroblasts were also cultured from the outgrowths of lung tissue obtained by surgical lung biopsy of patients with IPF as previously described.43 All segments were conrmed histologically to show usual interstitial pneumonia. Nonbrotic control broblasts were cultured from histologically normal regions of the lung from patients undergoing resection for lung nodules. Fibroblasts were cultured in DMEM supplemented with 10% FBS and antibiotics.

Cells were treated with the DNA methylation inhibitor 5-aza-dC (15 mM, Sigma-Aldrich, St Louis, MO, USA) or the HDAC inhibitors TSA (0.1 mM1 mM, Sigma-

Aldrich) and SAHA (1 mM, Cayman Chemical, Ann Arbor, MI, USA) for 72 h before being harvested for expression of Fas. For apoptosis experiments, cells were pretreated with or without TSA 0.1 mM for 48 h, before the medium was removed and cells were treated with the anti-Fas-activating antibody (100 ng/ml; clone CH11

Millipore, Billerica, MA, USA for human cells and clone Jo2 No.554248 BD Biosciences, Sparks, MD, USA for mouse cells) and cycloheximide (0.5 mg/ml,

Sigma-Aldrich).

Bisulte sequencing. The DNA methylation of the Fas promoter was assayed by bisulte sequencing. DNA was isolated from cells using the DNeasy kit (Qiagen, Valencia, CA, USA) and subjected to bisulte conversion using the Zymo EZ DNA Methylation kit (Zymo Research, Irvine, CA, USA) as per the manufacturers protocol. Regions of the Fas promoter were PCR amplied using biotin-labeled primers obtained from EpigenDx, Inc (Worcester, MA, USA) specic for human and murine sequences. The PCR product was isolated by sepharose beads, washed, and sequenced using specic sequencing primers (EpigenDx, Inc) on the Q24 pyrosequencer (Qiagen).

ChIP. A total of 9 106 cells were cultured on three 100 mm plates. Cells were

incubated for 5 min in 1% formaldehyde to establish DNA-chromatin crosslinks, washed, and subsequently incubated in glycine to stop further xation. Nuclear material was isolated using nonionic detergent buffers, and chromatin was sheared to 200500 bp fragments by 15 min of sonication using the Covaris sonicator (Covaris, Inc, Woburn, MA, USA), as per the manufacturers protocol. Chromatin was incubated overnight with the following antibodies: anti-acetylated H3 (25 mg/ml, Nos.17615 Millipore), anti-acetylated H4 (25 mg/ml, Nos.06598

Millipore), anti-H3K9Me3 (20 mg/ml, Nos.17658 Millipore), and anti-H3K27Me3 (1 : 50, Nos.9733 Cell Signaling, Beverly, MA, USA). ChIP was performed using magnetic beads from the ActiveMotif ChIP-IT Express Kit (ActiveMotif, Carlsbad,

CA, USA) according to the manufacturers protocol. Immunoprecipitated chromatin was reverse cross-linked and DNA was cleaned using the DNA cleanup kit (Qiagen). Isolated DNA was PCR amplied by real time using primers predesigned to amplify a region 1 kilobases upstream of the transcription start site of the mouse Fas promoter (No.334001 GPM1034075( )01A, Qiagen). Primers for the human

Fas promoter include pair No.1: 50-ATAGCTGGGGCTATGCGATT-30 (forward primer), 50-GTTGTGGCTGCAACATGAGA-30 (reverse primer); and human Fas promoter primer pair No.2: 50-GTGAGCCTCTCATGTTGCAG-30 (forward primer), 50-GTTGGGGAGGTCTTGAAGGA-30 (reverse primer).

Real-time RT-PCR. Levels of Fas mRNA were assayed by real time RTPCR. RNA was isolated from cells using Trizol (Invitrogen) and quantitative mRNA levels were assayed by real-time RT-PCR using the Applied Biosystems 7300 Real-time PCR System (Carlsbad, CA, USA). Primers for human and murine Fas were obtained from Applied Biosystems. Primer and probes for human and murine b-actin were used as previously reported.44

Immunoblotting. Cell lysates were collected in lysis buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM orthovanadate, and protease cocktail inhibitor), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted using antibodies to the following: PARP (1 : 1000, No.9542 Cell Signaling), caspase 8 (1 : 1000, No.9429 Cell Signaling), murine Fas (1 : 1000, No.554258 BD Biosciences), human Fas (1 : 500, No.05201 Millipore), HDAC2 (1 : 2000, No.1710237 Millipore), HDAC4 (1 : 2000, No.7628 Cell Signaling) and a-tubulin (1 : 1000, Sigma-Aldrich). Bound primary antibodies were visualized with appropriate secondary antibody conjugated to horseradish peroxidase and developed with enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed on the visualized bands using Scion Image Software (NIH, Bethesda, MD, USA).

Flow cytometry. Cultured cells were scraped from plates, washed in PBS, and incubated with PE-labeled antibody to Fas (clone Jo2 No.554248 BD Biosciences) for 15 min at 4 1 C. Cells were subsequently washed three times and analyzed for uorescence by ow cytometry on the FACSCalibur (BD

Biosciences).

Statistical analysis. Data were analyzed on GraphPad Prism 5.0 (GraphPad Prism Software, San Diego, CA, USA) using ANOVA or Students t-test, as appropriate, with a Po0.05 dened as statistically signicant. Data are expressed as meanS.E.M.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. This work was supported by an American Thoracic Society Coalition for Pulmonary Fibrosis Foundation Research Grant and grants HL094657 (to SKH), HL105489 (to JCH), and HL094311 (to MP-G) from the National Heart, Lung, and Blood Institute.

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4. Buhling F, Wille A, Rocken C, Wiesner O, Baier A, Meinecke I et al. Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of brotic lung broblasts to FasL induced apoptosis. Respir Res 2005; 6: 37.

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