Hypermethylation of PTGER2 Confers Prostaglandin E2 Resistance in Fibrotic Fibroblasts from Humans and Mice

Abstract

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

Reversal of Myofibroblast Differentiation by Prostaglandin E2

“…Variability in PGE 2 responsiveness is consistent with our previous studies on PGE 2 inhibition of collagen expression and fibroblast proliferation (26). In those studies involving fibroblasts, but not myofibroblasts, we identified several mechanisms that accounted for variable PGE 2 resistance, including decreased expression of EP2 (by DNA hypermethylation [44]) and decreased activation of the downstream signaling molecule, protein kinase A (26). Further studies would be needed to determine the extent and the mechanism(s) of resistance of IPF cells to PGE 2 reversal of myofibroblast differentiation.…”

Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

“…PGE2 has long been considered an antifibrotic molecule because it inhibits fibroblast proliferation, collagen synthesis, migration, and differentiation to myofibroblasts and induces fibroblasts apoptosis (45)(46)(47). Fibroblasts derived from IPF lungs exhibit a relative deficiency of PGE2 production as well as reduced responsiveness to PGE2 (48)(49)(50). Several studies suggested that bleomycin-induced fibrosis is enhanced when PTGS2-derived PGE2 is low or absent (51-53), whereas others did not observe the effects to the same extent (54) or suggested alternative pathways to PGE2 production (55).…”

Lung Pericytes and Resident Fibroblasts

“…15,24,91,92 However, fibroblasts from both bleomycin-injured and IPF lung become less responsive to this feedback from epithelial cells because promoter hypermethylation reduces expression of a key PGE 2 receptor, EP2. 26,28,93 In addition, myofibroblasts are a major source of plasminogen activator inhibitor 1; plasminogen activator inhibitor 1 inhibits the conversion of plasminogen to plasmin, and plasmin stimulates PGE 2 synthesis by lung epithelial cells. 15,94,95 Thus, the PGE 2 -dependent negative feedback loop may serve as an informative example of how regeneration occurs from a self-limited wound healing response, whereas an imbalance in feedback inhibition…”