Transcriptional and posttranscriptional inhibition of lysyl oxidase expression by cigarette smoke condensate in cultured rat fetal lung fibroblasts
Transcriptional and posttranscriptional inhibition of lysyl oxidase expression by cigarette smoke condensate in cultured rat fetal lung fibroblasts
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DOI：
10.1093/toxsci/kfi212
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发表时间：
2005-09-01
影响因子：
3.8
通讯作者：
Li, WD
中科院分区：
文献类型：
--
作者：
Gao, S;Chen, KY;Li, WD
赖氨酸氧化酶（LO）催化胶原蛋白和弹性蛋白的交联，对维持肺细胞外基质（ECM）的结构完整性至关重要。为了了解香烟烟雾（CS）诱导肺气肿的机制，我们研究了香烟烟雾凝聚物（CSC）对培养大鼠胎肺成纤维细胞（RFL6）中LO mRNA表达的影响。将RFL6细胞暴露于0-120 μ g CSC/ml中24小时，诱导了LO稳态mrna的剂量依赖性抑制，例如，在80-120 μ g CSC/ml中培养的细胞中，转录物水平降至对照的10%以下。核运行试验表明，在120 μ g CSC/ml处理的细胞中，LO相对转录率显著降低，为对照组的27.7%。放线菌素d -追逐实验显示CSC增强了LO转录本的不稳定性。在120 μ g CSC/ml处理的细胞中，LO mRNA的衰变t（1/2）由对照的24 h缩短至4.5 h。此外，80-120 μ g CSC/ml还可以抑制LO启动子活性，这是通过抑制LO启动子-荧光素酶载体转染细胞的报告基因表达而发现的。因此，抑制LO转录起始和增强LO mRNA不稳定性都导致了csc处理细胞中LO稳态mRNA的下调。值得注意的是，CSC对LO mRNA表达的抑制与LO的两种底物I型胶原和tropoelastin转录本水平的显著降低密切相关。因此，LO及其底物的转录扰动可能是cs诱导的肺气肿中ECM损伤的关键机制。
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 mu g 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 mu g 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 mu g CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t(1/2) for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 mu g CSC/ml. Moreover, 80-120 mu g 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.