Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: involvement of Src/EGFR/PI3-K/Akt pathway.
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.
Mesh Terms:
Androstadienes, Blotting, Western, Cell Adhesion, Cell Line, Chromones, Dose-Response Relationship, Drug, E1A-Associated p300 Protein, Humans, Lipopolysaccharides, Morpholines, Myocytes, Smooth Muscle, Neutrophils, Phosphatidylinositol 3-Kinases, Phosphorylation, Proto-Oncogene Proteins c-akt, RNA, Messenger, RNA, Small Interfering, Receptor, Epidermal Growth Factor, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Trachea, Transfection, Tyrphostins, Vascular Cell Adhesion Molecule-1, src-Family Kinases
Androstadienes, Blotting, Western, Cell Adhesion, Cell Line, Chromones, Dose-Response Relationship, Drug, E1A-Associated p300 Protein, Humans, Lipopolysaccharides, Morpholines, Myocytes, Smooth Muscle, Neutrophils, Phosphatidylinositol 3-Kinases, Phosphorylation, Proto-Oncogene Proteins c-akt, RNA, Messenger, RNA, Small Interfering, Receptor, Epidermal Growth Factor, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Trachea, Transfection, Tyrphostins, Vascular Cell Adhesion Molecule-1, src-Family Kinases
Toxicol. Appl. Pharmacol.
Date: Apr. 15, 2008
PubMed ID: 18182168
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