title: Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells.
abstract: Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H(2)O(2)) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H(2)O(2) increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells.
authors.affiliation: Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju, Republic of Korea.
authors.forename: Pham Ngoc
authors.initials: PN
authors.lastname: Khoi
authors.forename: Jung Sun
authors.initials: JS
authors.lastname: Park
authors.forename: Nam Ho
authors.initials: NH
authors.lastname: Kim
authors.forename: Young Do
authors.initials: YD
authors.lastname: Jung
mesh_terms: D000873:Anthracenes / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000378:metabolism
chemical_list: D000873:Anthracenes; D005419:Flavonoids; D007093:Imidazoles; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D055293:Receptors, Urokinase Plasminogen Activator; D018808:Transcription Factor AP-1; C432165:pyrazolanthrone; D009538:Nicotine; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one