title: Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-β1 in alveolar epithelial cells.
abstract: Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²⁺ chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²⁺-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²⁺/PKCα-β1 pathway independent of ATP signaling.
authors.affiliation: Centre de recherche, Centre hospitalier de l’Université de Montréal (CR-CHUM)-Hôtel-Dieu, Montreal, Quebec, Canada.
authors.forename: Émilie
authors.initials: É
authors.lastname: Boncoeur
authors.forename: Guillaume F
authors.initials: GF
authors.lastname: Bouvet
authors.forename: Francis
authors.initials: F
authors.lastname: Migneault
authors.forename: Valérie
authors.initials: V
authors.lastname: Tardif
authors.forename: Pasquale
authors.initials: P
authors.lastname: Ferraro
authors.forename: Danuta
authors.initials: D
authors.lastname: Radzioch
authors.forename: Juan B
authors.initials: JB
authors.lastname: de Sanctis
authors.forename: David
authors.initials: D
authors.lastname: Eidelman
authors.forename: Karuthapillai
authors.initials: K
authors.lastname: Govindaraju
authors.forename: André
authors.initials: A
authors.lastname: Dagenais
authors.forename: Yves
authors.initials: Y
authors.lastname: Berthiaume
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D011650:Pulmonary Alveoli / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000201:enzymology*
chemical_list: D008070:Lipopolysaccharides; D012333:RNA, Messenger; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D002118:Calcium
keywords: ATP; PKC, purinergic system; epithelial cells; iNOS