Galardin (GM 6001), a broad-spectrum matrix metalloproteinase inhibitor, blocks bombesin- and LPA-induced EGF receptor transactivation and DNA synthesis in rat-1 cells.
Matrix metalloproteinases (MMPs) have been implicated in the transactivation of the epidermal growth factor receptor (EGFR) induced by G-protein coupled receptor (GPCR) agonists. Although EGFR phosphorylation and downstream signaling have been shown to be dependent on MMP activity in many systems, a role for MMPs in GPCR-induced DNA synthesis has not been studied in any detail. In this study we utilized the broad-spectrum matrix metalloproteinase inhibitor, galardin (Ilomastat, GM 6001), to study the mechanism of bombesin- or LPA-induced EGFR transactivation and the role of MMPs in early and late response mitogenic signaling in Rat-1 cells stably transfected with the bombesin/GRP receptor (BoR-15 cells). Addition of galardin to cells stimulated with bombesin or LPA specifically inhibited total EGFR phosphorylation, as well as site-specific phosphorylation of tyrosine 845, a putative Src phosphorylation site, and tyrosine 1068, a typical autophosphorylation site. Galardin treatment also inhibited extracellular signal-regulated kinase (ERK) activation induced by bombesin or LPA, but not by EGF. In addition, galardin inhibited bombesin- or LPA-induced DNA synthesis in a dose dependent manner, when stimulated by increasing concentrations of bombesin, and when added after bombesin stimulation. Furthermore, addition of galardin post-bombesin stimulation indicated that by 3 h sufficient accumulation of EGFR ligands had occurred to continue to induce transactivation despite an inhibition of MMP activity. Taken together, our results suggest that MMPs act as early as 5 min, and up to around 3 h, to mediate GPCR-induced EGFR transactivation, ERK activation, and stimulation of DNA synthesis.
Department of Medicine, School of Medicine, University of California, Los Angeles, CA 90095-1786, USA.
Chintda
C
Santiskulvong
Enrique
E
Rozengurt
D000818:Animals; D001839:Bombesin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004151:Dipeptides / Q000494:pharmacology*; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D008246:Lysophospholipids / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D018004:Receptors, Bombesin / Q000235:genetics; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D004151:Dipeptides; D008246:Lysophospholipids; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D018004:Receptors, Bombesin; D014443:Tyrosine; D004247:DNA; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D001839:Bombesin