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A2196 - Transforming Growth Factor Beta 1 (TGF- β1) Induced Profound Methylation Changes in Fibroblasts, and Revealed an Unexpected Role of Homer1 in Idiopathic Pulmonary Fibrosis
Author Block: M. Negreros1, J. S. Hagood2, C. Espinoza2, Y. I. Balderas-Martinez3, M. Selman4, A. Pardo1; 1Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico, Mexico, 2Pediatrics, Univ of California At San Diego, La Jolla, CA, United States, 3Research, Catedra CONACYT, Mexico, Mexico, 4Inst Nac De Enfermedades Respiratorias, Mexico City, Mexico.
RATIONALE
Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. There are few studies that explore global DNA methylation in IPF lungs fibroblast and none focused on Transforming Growth Factor beta-1 (TGF-β1) as a potential modifier of methylome. Here we analyzed normal and IPF methylome and transcriptome before and after TGF-β1 treatment, and one of the modified genes: Homer1. As far as we know this is the first study that explores the potential role of Homer1 in IPF.
METHODS One Primary culture of fibroblasts derived from normal human lung and one from IPF were treated with TGF-β1 (10ng/ml) for 24 h and 5 days. mRNA expression was analyzed with GeneChip PrimeView Human Gene Expression Array (Affymetrix) and DNA methylation status with the Infinium HumanMethylation450 BeadChip Kit (Illumina). Expression of Homer1 was validated with RT-PCR, and confirmed at the protein level with Western blot. Lung localization of Homer1 was examined by immunohistochemistry. Inverse transfection of siRNA against Homer1 in normal fibroblasts was performed. Apoptosis was analyzed with FLICA assay and TUNEL. Intracellular calcium release was examined with Fura-2 AM assay. Proliferation was measured with WST assay. RESULTS We analyzed at single nucleotide resolution the DNA methylome not only at promoters but also at other gene regions (gene body, 3’ and 5’ UTRs, 1st exon, between others) and also CpG Island reference: shore, shelf, open sea or island itself. We found that IPF fibroblasts with TGF-β1 treatment showed more methylation changes versus normal fibroblasts. Surprisingly we found that treatment with TGF-β1 in IPF fibroblasts resulted in hypermethylation but upregulation of Homer1, an scaffolding protein that has been implicated in Calcium modulation signaling. We confirmed the over expression of the gene and protein after TGF-β1 long treatment (5 days) in fibroblasts. Silencing of Homer1 significantly increased cell death, intracellular calcium release and deacresed proliferation. Interestingly Homer1 was immunolocalyzed in fibroblastic foci in IPF lungs. CONCLUSIONS Our findings demonstrate that TGF-β1 treatment modifies methylation patterns in IPF. Homer1 a hypermethylated and upregulated localized in fibroblastic foci in IPF lungs might play a role increasing resistance to apoptosis.