Cell-matrix interactions induce tyrosine phosphorylation of MAP kinases ERK1 and ERK2 and PLCgamma-1 in two-dimensional and three-dimensional cultures of human fibroblasts.
Using immunoprecipitation and phosphotyrosine detection by Western blotting, intracellular signaling intermediates were analyzed in human primary dermal fibroblasts, either seeded as monolayers on collagen I coats (2D) or seeded within three-dimensional collagen I lattices (3D). Previous results demonstrated that integrin activation in these systems resulted in a cascade of protein tyrosine phosphorylation, including focal adhesion kinase (D. Roeckel and T. Krieg, 1994, Exp. Cell Res. 211, 42-48). Further downstream signaling events are now shown to include coordinate activation of ERK1 and ERK2 at 2 h after cell-collagen contact, irrespective of 2D or 3D culture conditions. Applying U-73122, an inhibitor of PLC, inhibits collagen lattice contraction in a dose-dependent fashion. Immunoprecipitation identified the isoform PLCgamma-1 as playing a role as signaling intermediate in fibroblast-collagen interactions. PLCgamma-1 becomes phosphorylated within 10 min after culture initiation and declines after 2 h. So far, no qualitative differences in signaling intermediates between 2D and 3D cultures have been identified.
Langholz
O
O
Department of Dermatology, University of Cologne, Cologne, Germany.
Roeckel
D
D
Petersohn
D
D
Broermann
E
E
Eckes
B
B
Krieg
T
T
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D003094:Collagen; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D012867:Skin / Q000166:cytology*; D012879:Skin Physiological Phenomena; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004963:Estrenes; D007527:Isoenzymes; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019000:Phosphotyrosine; D003094:Collagen; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma