Thy-1 Promoter Hypermethylation

Abstract

Mechanisms regulating myofibroblastic differentiation of fibroblasts within fibroblastic foci in idiopathic pulmonary fibrosis (IPF) remain unclear. Epigenetic processes, including DNA methylation, produce heritable but potentially reversible changes in DNA or its associated proteins and are prominent in development and oncogenesis. We have shown that Thy-1 suppresses myofibroblastic differentiation of lung fibroblasts and that fibroblasts in fibroblastic foci are Thy-1(2). Epigenetic down-regulation of Thy-1 has been demonstrated in cellular transformation and clinical cancer. We hypothesized that epigenetic regulation of Thy-1 affects the lung fibroblast fibrogenic phenotype. RT-PCR, methylation-specific PCR (MSP), and bisulfite genomic sequencing were used to determine the methylation status of the Thy-1 promoter in Thy-1(1) and Thy-1(2) lung fibroblasts, and MSP-in situ hybridization (MSPISH) was performed on fibrotic tissue. Thy-1 gene expression is absent in Thy-1(2) human and rat fibroblasts despite intact Thy-1 genomic DNA. Cytosine-guanine islands in the Thy-1 gene promoter are hypermethylated in Thy-1(2), but not Thy-1(1), fibroblasts. RT-PCR and MSP demonstrate that, in IPF samples in which Thy-1 expression is absent, the Thy-1 promoter region is methylated, whereas in lung samples retaining Thy-1 expression, the promoter region is unmethylated. MSPISH confirms methylation of the Thy-1 promoter in fibroblastic foci in IPF. Treatment with DNA methyltransferase inhibitors restores Thy-1 expression in Thy-1(2) fibroblasts. Epigenetic regulation of Thy-1 is a novel and potentially reversible mechanism in fibrosis that may offer the possibility of new therapeutic options.

Lung Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis Exhibit Genome-Wide Differences in DNA Methylation Compared to Fibroblasts from Nonfibrotic Lung

“…HumanMethylation27 array) [16]. Studies of specific genes – namely, Thy-1 [19], PTGER2 [18], and p14 ARF [17] – have identified their differential methylation in IPF fibroblasts, but this is the first study of which we are aware that describes genome-wide differences in DNA methylation in these critical mesenchymal effector cells.…”

Epigenetics of idiopathic pulmonary fibrosis

“…Similarly, Thy-1 (CD90) is an important regulator of cell–cell and cell– matrix interactions that is expressed on normal lung fibroblasts but its expression is absent in myofibroblasts within fibroblastic foci (FF) in IPF. Thy-1 downregulation in rat lung fibroblasts is controlled by both promoter DNA hypermethylation [102] and histone modifications [103]. Thy-1 hypermethylation and dowregulation of expression have also been observed in human lung fibroblasts as a result of hypoxia [104].…”

Epigenetics in lung fibrosis

“…Several targeted studies have shown that epigenetic modulation (both DNA methylation and histone marks) regulates expression of genes and miRNAs involved in pathogenesis of IPF (Table 1), namely, cyclooxygenase-2 (COX2) (92, 93), chemokine IP-10 (94), Thy-1 (CD90) (95, 96), p14(ARF) (97), α-smooth muscle actin (α-SMA) (98, 99), and miR-17~92 cluster (100). Similarly, molecular processes of high relevance to pulmonary fibrosis are also epigenetically regulated; this has been demonstrated specifically for fibroblast apoptosis (101, 102), cell senescence (103), and innate immunity (104) in IPF.…”