title: Expression and regulation of the macrophage inflammatory protein-1 alpha gene by nicotine in rat alveolar macrophages.
abstract: Cigarette smoking causes inflammation mainly confined to the airway and lung. Nicotine is one of the primary constituents in cigarette smoke. Alveolar macrophages apparently play a pivotal role in mediating pulmonary inflammation via the production of chemokines. Macrophage inflammatory protein-1 alpha (MIP-1 alpha), a member of CC chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1 alpha mRNA expression in macrophages is induced by a variety of stimuli. In the present study, we further investigate whether nicotine can regulate the gene expression of MIP-1 alpha in macrophages and determine the mechanism leading to increased expression. A rat alveolar macrophage (RAM) cell line, NR8383, was treated with nicotine at a dose of 3.1, 31, 310 microM, or 3.1 mM. Northern blot analysis showed that the induction of MIP-1 alpha mRNA expression was dose-dependent. To define the time course of the inflammatory response, RAM cells were exposed to 31 microM nicotine, MIP-1 alpha mRNA was induced as early as 1 h after treatment, was maximally expressed at 4 and 6 hours, and reduced by 8 hours. Western blot analysis demonstrated a single band with an estimated molecular weight of 10 kD for MIP-1 alpha which was induced after nicotine treatment, suggesting that expression of MIP-1 alpha mRNA could reflect in protein synthesis. In addition. the increase in MIP-1 alpha mRNA expression induced by nicotine was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at doses of 10 and 20 mM, suggesting that the induction of MIP-1 alpha mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-1 alpha gene expression, RAM cells were exposed to nicotine. MIP-1 alpha mRNA levels were significantly increased in nuclear RNA preparations, indicating that transcriptional activation is involved in increased expression of MIP-1 alpha mRNA. Moreover, we performed RNA decay assay by measuring the half-life of MIP-1 alpha mRNA. Treatment of RAM cells with the transcriptional inhibitor actinomycin D following exposure to nicotine revealed that the half-life of MIP-1 alpha mRNA was markedly increased by nicotine treatment, supporting a role of post-transcriptional stabilization in MIP-1 alpha gene expression. These observations indicate that nicotine can induce MIP-1 alpha mRNA expression and protein synthesis in RAM cells, mediating, at least in part, via the generation of ROS. In addition, the increase in MIP-1 alpha mRNA level involves, both transcriptional activation and post-transcriptional stabilization.
authors.affiliation: Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. chong@cc.kmu.edu.tw
authors.forename: Inn-Wen
authors.initials: IW
authors.lastname: Chong
authors.forename: Shiu-Ru
authors.initials: SR
authors.lastname: Lin
authors.forename: Jhi-Jhu
authors.initials: JJ
authors.lastname: Hwang
authors.forename: Ming-Shyan
authors.initials: MS
authors.lastname: Huang
authors.forename: Tung-Heng
authors.initials: TH
authors.lastname: Wang
authors.forename: Jen-Yu
authors.initials: JY
authors.lastname: Hung
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Paulauskis
mesh_terms: D000818:Animals; D002460:Cell Line; D054407:Chemokine CCL4; D005786:Gene Expression Regulation / Q000187:drug effects; D006207:Half-Life; D007700:Kinetics; D019402:Macrophage Inflammatory Proteins / Q000235:genetics*; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology*; D008970:Molecular Weight; D009538:Nicotine / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D054407:Chemokine CCL4; D019402:Macrophage Inflammatory Proteins; D012333:RNA, Messenger; D009538:Nicotine