METHYLATION STATUS OF NEUTRAL ENDOPEPTIDASE GENES DOWN‐REGULATED BY DIESEL EXHAUST PARTICULATES IN HUMAN AIRWAY EPITHELIUM

Abstract

Our previous study has reported that epithelial neutral endopeptidase (NEP, a 90–110 kDa zinc‐dependent, type II integral membrane protein) expression was down‐regulated by diesel exhaust particulates (DEP) in a concentration‐dependent manner. In addition, sequence analysisof the NEP promoter reveals a CpG island in the type II promoterregion containing five Sp1 elements and juxtaposed to a region containing multiple Alu repeats. To determine the possibility thathypermethylation of the NEP promoter involved a NEP downregulation following exposure to DEP, we conducted a series of experiments in BEAS‐2B cells, including concentration‐effect and time‐course design (7 days). Methylation‐specific PCR primers were designed to discriminate between methylated and unmethylated alleles after bisulfite treatment. Our data show that there is no evidence of methylation in DNA samples either from the dose‐effect (0, 5, 10, 20, 40, 60 μg/ml DEP for 24 hours or from the time‐course designs (2, 4, and 7 days at the concentrations of 40 μg/ml DEP), suggesting that methylationof the tested NEP 5′ CpG island region could not be associated with loss of NEP expressionin epithelial cells after DEP exposure. However, a more comprehensive analysisof epithelial specimens after long‐term DEP exposure is needed to fully rule out the effect of NEP promoter methylation (Supported by HEI).