title: TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR.
abstract: BACKGROUND
  Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.
  
  
  METHODS
  18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10 mm cell culture dishes and were used from passages 10-14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 μM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors.
  
  
  RESULTS
  Exposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR.
  
  
  CONCLUSIONS
  Synergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.
authors.forename: James
authors.initials: J
authors.lastname: Yoo
authors.forename: Citlali Ekaterina
authors.initials: CE
authors.lastname: Rodriguez Perez
authors.forename: Wenxian
authors.initials: W
authors.lastname: Nie
authors.forename: James
authors.initials: J
authors.lastname: Sinnett-Smith
authors.forename: Enrique
authors.initials: E
authors.lastname: Rozengurt
mesh_terms: D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D004357:Drug Synergism; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D058628:Myofibroblasts / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology; D015854:Up-Regulation; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
chemical_list: C504821:GPR1 protein, human; D007211:Indoles; D008246:Lysophospholipids; D011799:Quinazolines; D043562:Receptors, G-Protein-Coupled; C416927:SU 6656; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C101044:RTKI cpd; D051546:Cyclooxygenase 2; C512478:EGFR protein, human; D066246:ErbB Receptors; D019061:src-Family Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020782:Matrix Metalloproteinases; C032881:lysophosphatidic acid