title: Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: involvement of Src/EGFR/PI3-K/Akt pathway.
abstract: In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-kappaB in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.
authors.affiliation: Department of Physiology and Pharmacology, Chang Gung University, 259 Wen-Hua 1st Road, Kwei-San, Tao-Yuan, Taiwan.
authors.forename: Wei-Ning
authors.initials: WN
authors.lastname: Lin
authors.forename: Shue-Fen
authors.initials: SF
authors.lastname: Luo
authors.forename: Chou-Bing
authors.initials: CB
authors.lastname: Wu
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Lin
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D050881:E1A-Associated p300 Protein / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014132:Trachea / Q000166:cytology; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D000077191:Wortmannin; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000730:Androstadienes; D002867:Chromones; D008070:Lipopolysaccharides; D009025:Morpholines; D011799:Quinazolines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020032:Tyrphostins; D019010:Vascular Cell Adhesion Molecule-1; C101044:RTKI cpd; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin