Hypermethylation of PTGER2 confers prostaglandin E2 resistance in fibrotic fibroblasts from humans and mice.
Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that is characterized by excessive proliferation of fibroblasts. The lipid mediator prostaglandin E2 (PGE2) 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 PGE2. 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 PGE2 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 PGE2 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 PGE2 resistance are both mediated by DNA hypermethylation; we identified increased Akt signal transduction as a novel mechanism that promotes DNA hypermethylation during fibrogenesis.
Huang
Steven K
SK
Department of Internal Medicine, University of Michigan Medical School, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA. stehuang@umich.edu
Fisher
Aaron S
AS
Scruggs
Anne M
AM
White
Eric S
ES
Hogaboam
Cory M
CM
Richardson
Bruce C
BC
Peters-Golden
Marc
M
D000818:Animals; D002478:Cells, Cultured; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000473:pathology*; D005355:Fibrosis / Q000473:pathology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D058307:Receptors, Prostaglandin E, EP2 Subtype / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
C545644:PTGER2 protein, human; C545645:Ptger2 protein, mouse; D034741:RNA, Small Interfering; D058307:Receptors, Prostaglandin E, EP2 Subtype; D015254:DNA Modification Methylases; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; D015232:Dinoprostone