title: Involvement of phospholipases D1 and D2 in sphingosine 1-phosphate-induced ERK (extracellular-signal-regulated kinase) activation and interleukin-8 secretion in human bronchial epithelial cells.
abstract: Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin degradation, stimulates interleukin-8 (IL-8) secretion in human bronchial epithelial (Beas-2B) cells. The molecular mechanisms regulating S1P-mediated IL-8 secretion are yet to be completely defined. Here we provide evidence that activation of phospholipases D1 and D2 (PLD1 and PLD2) by S1P regulates the phosphorylation of extracellular-signal-regulated kinase (ERK) and IL-8 secretion in Beas-2B cells. S1P, in a time- and dose-dependent manner, enhanced the threonine/tyrosine phosphorylation of ERK. The inhibition of S1P-induced ERK phosphorylation by pertussis toxin and PD 98059 indicated coupling of S1P receptors to G(i) and the ERK signalling cascade respectively. Treatment of Beas-2B cells with butan-1-ol, but not butan-3-ol, abrogated the S1P-induced phosphorylation of Raf-1 and ERK, suggesting that PLD is involved in this activation. The roles of PLD1 and PLD2 in ERK activation and IL-8 secretion activated by S1P were investigated by infecting cells with adenoviral constructs of wild-type and catalytically inactive mutants of PLD1 and PLD2. Infection of Beas-2B cells with the wild-type constructs resulted in the activation of PLD1 and PLD2 by S1P and PMA. Also, the enhanced production of [(32)P]phosphatidic acid and [(32)P]phosphatidylbutanol in the presence of butan-1-ol and the increased phosphorylation of ERK by S1P were blocked by the catalytically inactive mutants hPLD1-K898R and mPLD2-K758R. Transient transfection of Beas-2B cells with human PLD1 and mouse PLD2 cDNAs potentiated S1P-mediated IL-8 secretion compared with vector controls. In addition, PD 98059 attenuated IL-8 secretion induced by S1P in a dose-dependent fashion. These results demonstrate that both PLD1 and PLD2 participate in S1P stimulation of ERK phosphorylation and IL-8 secretion in bronchial epithelial cells.
authors.affiliation: Department of Medicine, Division of Pulmonary and Critical Care, Johns Hopkins University, Baltimore, MD 21224, USA.
authors.forename: Lixin
authors.initials: L
authors.lastname: Wang
authors.forename: Rhett
authors.initials: R
authors.lastname: Cummings
authors.forename: Peter
authors.initials: P
authors.lastname: Usatyuk
authors.forename: Andrew
authors.initials: A
authors.lastname: Morris
authors.forename: Kaikobad
authors.initials: K
authors.lastname: Irani
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008246:Lysophospholipids; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D005419:Flavonoids; D016209:Interleukin-8; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D037342:Pertussis Toxin; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D013110:Sphingosine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one