Vol. 6, 1009-1017, August 1995 Cell Growth & Differentiation 1009 Elucidation of a Signaling Pathway Induced by FGF-2 Leading to uPA Gene Expression in NIH 3T3 Fibroblasts’ Daniel Besser, Marco Presta, and Yoshikuni Nagamine2 Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland ED. B., Y. N.], and Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 1-25123 Brescia, Italy EM. P.] Abstract Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that the uPA gene is transcriptionally induced by FGF-2 as well as by 12-0- tetradecanoylphorbol-1 3-acetate involving a PEA3/AP1 element located 2.4 kb upstream of the transcription initiation site; neither induction requires ongoing protein synthesis. Unlike 1 2-O-tetradecanoylphorbol-1 3-acetate induction, FGF-2 induction was not impaired by protein kinase C down-regulation. Analyses of various signaling molecules by Western blotting, extracellular signal- regulated kinase (ERK) activity assays, and transient transfection assays (cotransfection of a uPA-reporter gene construct with expression vectors for wild-type or dominant negative type of these molecules or for ERK- specific protein phosphatase MKP-1) showed that a Ras/ Raf-1/MEK/ERK-2/JunD pathway is induced by FGF-2 and 1 2-O.tetradecanoylphorbol-1 3-acetate, leading to the activation of the uPA gene. Introduction FGFs3 are a family of closely related hepanin-binding pro- teins exhibiting various biological activities in a variety of cell types. They promote proliferation of cells of mesodem- mal and neuroectodenmal origin, influence differentiation either positively or negatively depending on cell type, and induce angiogenesis by acting on endothelial cells (me- viewed in Refs. 1 and 2). Thus fan, nine members ofthe FGF family and four types of high affinity cell surface FGFRs have been identified, each receptor responding to more than one type of FGF (reviewed in Ref. 3). It is not known how such a wide spectrum of cellular responses to FGF is Received 4/1 3/95; revised 5/1 5/95; accepted 5/26/95. 1 This work was partly supported by Gottlieb Daimler- and Karl Benz- Stiftung, Fellowship 2.91 .07 (to D. B.) and by an Associazione Italiana per Ia Ricerca sul Cancero grant (to M. P.). 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; PLCy, phospholipase C-y; uPA, urokinase-type plasminogen activator; TPA, 1 2-O-tetradecanoylphorbol-1 3-acetate; PKC, protein kinase C; CS, calf serum; MEK, MAP kinase/ERK kinase; ERK, extracellular signal-regulated kinase; CMV, cytomegalovirus; MKP-1 , MAP kinase phosphatase 1. induced through the interaction of different FGFs and me- ceptons in different cell types. All FGF receptors contain a split tymosine kinase domain in the cytoplasmic carboxyl terminal region, suggesting that tyrosine phosphorylation of cellular proteins is involved in FGF-induced signal trans- duction (1 , 2). Among various potential substrates for ty- mosine phosphomylation, only PLC’y has been shown to in- teract directly with one of the FGF receptors, FGFR-1 , and to be phosphorylated at a tyrosine residue, which is essen- tial for its activation (4). However, it is unlikely that all FGF activities are mediated by PLCy, because a mutant receptor unable to activate PLC-y is still capable of transducing mi- togenic- and differentiation-promoting signals (5). In many cases, cellular responses to extracellular signals accompany the induction of various genes. Therefore, the investigation ofthe regulatory mechanism ofthe induction should help to unravel cellular responses to extracellulan signals at the molecular level. Several genes have been shown to be induced by FGF (6-14), but the underlying molecular mechanisms remain largely unknown. Among the genes induced by FGF-2, the uPA is of interest because one of the major moles of FGF-2 in vivo is the induction of angiogenesis (1 5), which involves massive movement of cells using sev- enal extracellulan pmoteases including uPA (16). uPA is a secreted senine protease that converts the ubiq- uitous zymogen plasminogen to plasmin, a tnypsin-Iike senine protease with wide substrate specificity. Through interaction with a specific uPA receptor on the cell surface and subsequent generation of active plasmin, uPA plays an important role in cell-associated proteolytic processes such as tissue remodeling during development, cell migration during wound healing, angiogenesis, gametogenesis, and tumor metastasis (reviewed in Refs. 8 and 1 7-1 9). Reflect- ing its wide range of biological activity, uPA is expressed by a large number of cell types, and its expression is controlled by a variety of extracellulan signals, depending on the cell type (reviewed in Refs. 17 and 20). The regulation of uPA gene expression by TPA (21-23), cAMP (24-26), cytoskel- etal reorganization (23, 27), and the protein phosphatases 1/2A inhibitor okadaic acid (28, 29) has been extensively studied. In addition, the uPA gene is induced by UV (30), tumor necrosis factor-a and intenleukin 1 f3 (31), IFN-y (32), transforming growth factor 13 (33, 34), FGF-2 (8, 14), neti- noic acid (8), and oncogene products v-Src and v-Ras (35). In these cases, however, the mechanism of uPA gene acti- vation has not been elucidated. Sequence comparison of the 5’ flanking regions ofthe munine and porcine uPA genes showed extensive homology over several kilobases (36), suggesting that many of the regulation mechanisms of the uPA gene are conserved among different species. In the present work, we investigated a potential effect of FGF-2 on the uPA gene in NIH 3T3 fibroblast cells. Fibro- blasts are involved in the process of wound healing, in which uPA plays an important mole, and are responsive to FGF-2, at least in terms of cell proliferation, indicating that a signaling pathway can be elicited by FGF-2 in these cells.