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CHRONIC HYPOXIA DECREASES THE EXPRESSION OF PKC EPSILON AND ALTERS METHYLATION STATUS OF PKC EPSILON PROMOTER IN H9c2 CELLS
Author(s) -
Patterson Andrew
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.629.2
Subject(s) - methylation , dna methylation , protein kinase c , microbiology and biotechnology , messenger rna , bisulfite sequencing , sodium bisulfite , hypoxia (environmental) , biology , chemistry , gene expression , andrology , kinase , dna , biochemistry , medicine , gene , oxygen , organic chemistry
Recent studies have demonstrated that chronic hypoxia during fetal development significantly decreased PKCε protein expression in the heart of adult offspring resulting in an increased heart susceptibility to ischemia and reperfusion injury. We tested the hypothesis that chronic hypoxia directly affects PKCε expression and alters methylation status of PKCε promoter in embryonic rat cardiac H9c2 cells. Cells were grown and divided into four groups, exposing to 21%, 10.5%, 3%, and 1% O 2 , respectively, for 24 hours. Protein and mRNA were isolated and Western immunoblotting and real‐time PCR were performed to determine the expression levels of PKCε. For methylation studies, DNA was isolated from H9c2 cells exposed to either 21% or 1% O 2 and treated with sodium bisulfite. Sodium bisulfite differentially converts all unmethylated, but not methylated cytosines to uracil. Methylation‐specific PCR was performed to assess the methylation status of transcription binding sites. We found cells treated with 1%, but not 10.5% and 3% O 2 , had significantly decreased PKCε protein and mRNA levels. Furthermore, 1% O 2 increased methylation of both SP1 bindings sites (−346, and −268) at PKCε promoter. ChIP assays demonstrated 1% O 2 significantly decreased SP1 binding to PKCε promoter, which was restored in the presence of 5‐aza‐2′‐deoxycytidine (DNA methylation inhibitor). Similarly, the addition of 5‐aza‐2′‐deoxycytidine blocked the hypoxia‐induced increase in methylation of both SP1 binding sites and restored PKCε mRNA and protein to the control levels. These findings suggest that chronic hypoxia decreases PKCε expression through increase methylation for SP1 binding sites at PKCε promoter in H9c2 cells.