Diesel exhaust particulates down‐regulate transcriptionally respiratory neutral endopeptidase

Abstract

Neutral endopeptidase (NEP) is a 90-110 kDa zinc-dependent, type II integral membrane protein abundantly expressed on airway epithelial cells. To determine the molecular effects of diesel exhaust particulates (DEP) on NEP expression, we analyzed temporal/concentrational patterns following exposure to 5-60 μg/ml standard DEP, SRM 2975 by using BEAS-2B cells. Data show that the NEP mRNA levels decreased in a time-dependent manner during 24 h DEP incubation. The significant change was detected as early as 8 h after DEP addition, persisting up to 24 h. To examine the sensitivity and specificity of NEP mRNA responses, we conducted a dose-effect experiment at concentrations of 0 (air controls), 5, 10, 20, 40, 60 μg/ml DEP. After 24 h exposure, NEP mRNA exhibits a concentration-dependent downregulation, with a maximum effect beginning at the exposure concentration of 40 μg/ml. Moreover, NEP protein and activity also decreased in a concentration-dependent manner after 24-h incubation. Todetermine whether the repression of NEP by DEP, occurs at the transcriptional or post-transcriptionallevel, we assessed the effect of DEP on NEP mRNA transcriptstability. BEAS-2B cells were treated with actinomycin D, exposed to DEP (40 μg/ml) or vehicle, and then assayed for NEPmRNA levels at various time points up to 24 h. Real-time PCRindicated no difference in the rate of NEP mRNA degradationbetween DEP and vehicle-treated samples, thusdemonstrating that DEP regulation of NEP gene expressionlikely occurs at the transcriptional level (Supported by HEI).