ToxSci Advance Access originally published online on June 2, 2005
Toxicological Sciences 2005 87(1):197-203; doi:10.1093/toxsci/kfi212
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Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Received March 25, 2005; accepted May 23, 2005
Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0–120 µg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80–120 µg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 µg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 µg CSC/ml. Moreover, 80–120 µg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.
Key Words: cigarette smoke condensate; lysyl oxidase relative transcriptional rate; lysyl oxidase mRNA stability; lysyl oxidase promoter activity; collagen type I; elastin.
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