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.