Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that is characterized by excessive proliferation of fibroblasts. The lipid mediator prostaglandin E 2 (PGE 2 ) has the capacity to limit fibrosis through its inhibition of numerous functions of these fibroblasts; however, in the setting of fibrosis, fibroblasts have been shown to be resistant to PGE 2 . We have linked such resistance to decreased expression levels of the E prostanoid 2 (EP2) receptor. In this study, in fibroblasts from both mice and humans with pulmonary fibrosis, we show that DNA hypermethylation is responsible for diminished EP2 expression levels and PGE 2 resistance. Bisulfite sequencing of the prostaglandin E receptor 2 gene (PTGER2) promoter revealed that fibrotic fibroblasts exhibit greater PTGER2 methylation than nonfibrotic control cells. Treatment with the DNA methylation inhibitors 5-aza-2-deoxycytidine and zebularine as well as DNA methyltransferase-specific siRNA decreased PTGER2 methylation, increased EP2 mRNA and protein expression levels, and restored PGE 2 responsiveness in fibrotic fibroblasts but not in nonfibrotic controls. PTGER2 promoter hypermethylation was driven by an increase in Akt signal transduction. In addition to results described for the PTGER2 promoter, fibrotic fibroblasts also exhibited increased global DNA methylation. These findings demonstrate that the down-regulation of PTGER2 and consequent PGE 2 resistance are both mediated by DNA hypermethylation; we identified increased Akt signal transduction as a novel mechanism that promotes DNA hypermethylation during fibrogenesis. (Am J
Differentiation of fibroblasts into a-smooth muscle actin (SMA)-expressing myofibroblasts represents a critical step in the pathogenesis of fibrotic disorders, and is generally regarded as irreversible. Prostaglandin E 2 (PGE 2 ) has been shown to prevent multiple aspects of fibroblast activation, including the differentiation of fibroblasts to myofibroblasts. Here, we investigated its ability to reverse this differentiated phenotype. Fetal and adult lung fibroblasts were induced to differentiate into myofibroblasts by 24-hour culture with transforming growth factor (TGF)-b1 or endothelin-1. Cells were then treated without or with PGE 2 for various intervals and assessed for a-SMA expression. In the absence of PGE 2 treatment, a-SMA expression induced by TGF-b1 was persistent and stable for up to 8 days. By contrast, PGE 2 treatment effected a dose-dependent decrease in a-SMA and collagen I expression that was observed 2 days after PGE 2 addition, peaked at 3 days, and persisted through 8 days in culture. This effect was not explained by an increase in myofibroblast apoptosis, and indeed, reintroduction of TGF-b1 2 days after addition of PGE 2 prompted dedifferentiated fibroblasts to re-express a-SMA, indicating redifferentiation to myofibroblasts. This effect of PGE 2 was associated with inhibition of focal adhesion kinase signaling, and a focal adhesion kinase inhibitor was also capable of reversing myofibroblast phenotype. These data unambiguously demonstrate reversal of established myofibroblast differentiation. Because many patients have established or even advanced fibrosis by the time they seek medical attention, this capacity of PGE 2 has the potential to be harnessed for therapy of late-stage fibrotic disorders.Keywords: E prostanoid receptor; transforming growth factor-b1; endothelin-1; a-smooth muscle actin; focal adhesion kinase Pathologic scarring or fibrosis results in impaired organ function in diseases such as cirrhosis, diabetes, end-stage renal disease, scleroderma, and pulmonary fibrosis (1, 2). The accumulation of myofibroblasts within pathologic lesions is a pivotal feature of many fibrotic disorders (1, 2). Fibroblasts possess the potential to differentiate into myofibroblasts, which are distinguished from fibroblasts by their expression of contractile proteins, such as a-smooth muscle actin (a-SMA), and their exuberant production of extracellular matrix proteins, such as collagen I. This expression of a-SMA and increased extracellular matrix production endow myofibroblasts with the ability to participate in wound contraction (3). Because the differentiation of fibroblasts to myofibroblasts is generally considered irreversible (4), resolution of normal wound repair is thought to require apoptosis of myofibroblasts (5). By contrast, pathologic fibrosis occurs when myofibroblasts fail to apoptose and instead accumulate and persist within tissues, contributing to progressive scarring. Indeed, idiopathic pulmonary fibrosis (IPF)-the most common and fatal type of lung fibrosis-is characterized b...
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.
Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n = 6) with those of nonfibrotic patient controls (n = 3) and commercially available normal lung fibroblast cell lines (n = 3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease.
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