title: Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism.
abstract: BACKGROUND
  Cell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF.
  
  
  OBJECTIVE
  To determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration.
  
  
  METHODS
  Human coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, betaARK(CT) were also used.
  
  
  RESULTS
  ATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gbetagamma inhibitor betaARK(CT) inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect.
  
  
  CONCLUSIONS
  ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.
authors.affiliation: Vascular Biology and Therapeutics Program, Methodist DeBakey Heart and Vascular Center, Department of Cardiovascular Surgery, The Methodist Hospital, and The Methodist Hospital Research Institute, Houston, Tex. 77030, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Bakken
authors.forename: Clinton D
authors.initials: CD
authors.lastname: Protack
authors.forename: Elisa
authors.initials: E
authors.lastname: Roztocil
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Nicholl
authors.forename: Mark G
authors.initials: MG
authors.lastname: Davies
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D020032:Tyrphostins / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D006877:Hydroxamic Acids; D008565:Membrane Proteins; C092152:N-((2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl)-3-(2'-naphthyl)alanylalanine, 2-aminoethylamide; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010446:Peptide Fragments; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D034741:RNA, Small Interfering; D020032:Tyrphostins; C101044:RTKI cpd; D053829:Amyloid Precursor Protein Secretases; D014568:Urokinase-Type Plasminogen Activator; D051722:ADAM Proteins; C098339:ADAM9 protein, human; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human