Urokinase Expression and Binding Activity Associated With the Transforming Growth Factor  1 -Induced M igratory and Invasive Phenotype of M ouse Epidermal Keratinocytes Juan F. Santiba ´n ˜ ez, 1,2 Pilar Frontelo, 1 M aite Iglesias, 1 Jorge Martı ´nez, 2 and Miguel Quintanilla 1 * 1 Instituto de InvestigacionesBiome ´dicas CSIC-UAM, 28029-Madrid, Spain 2 Unidad de Biologı ´a Celular, INTA, Universidad de Chile, Santiago, Chile Abstract Transforming growth factor  1 (TGF- 1 ) is a stimulator of malignant progression in mouse skin carcino- genesis. TGF- 1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial-fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous-spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF- 1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI-1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF- 1 -induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF- 1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas. J. Cell. Biochem. 74:61–73, 1999.  1999 Wiley-Liss, Inc. Key words: uPA; TGF- 1 ; migration; invasiveness; keratinocytes; carcinogenesis During malignant progression the acquisi- tion of migratory and invasive properties are linked to changes in cell-cell and cell-extracellu- lar matrix (ECM) adhesiveness, the reorganiza- tion of cytoskeletal components, and the expres- sion and secretion of proteinases involved in ECM degradation [Stetler-Stevenson et al., 1993]. Sometimes, the alterations in the differ- entiation program are profound and associated to a drastic change of the cell phenotype. Thus, in natural and experimental carcinogenesis squamous cell carcinomas (SCCs) can progress to spindle carcinomas (SpCCs), a highly aggres- sive type of tumor formed by cells that have lost the epithelial phenotype and acquired fibro- blast-like characteristics [Portella et al., 1994– 95]. One of the best characterized animal mod- els for studying this transition is the mouse skin system, in which tumors are induced on the dorsal skin of mice by a single topical appli- cation of a chemical carcinogen followed by sequential treatment with a tumor promoter. A proportion of benign papillomas developed by chemical carcinogenesis progress spontane- ously to malignant SCCs. The later stages of Abbreviations used: ECM, extracellular matrix; EGF, epi- dermal growth factor; FBS, fetal bovine serum; HGF/SF, hepatocyte growth factor/scatter factor; PA, plasminogen activator; PAI, PAinhibitor; SCC, squamous cell carcinoma; SpCC, spindle cell carcinoma; TGF-, transforming growth factor ; uPA, urokinase-type PA; uPAR, uPA receptor. Grant sponsor: Comisio ´n Interministerial de Ciencia y Tec- nologı ´a; Grant number: SAF98–0085-C03–02; Grant spon- sor: Comunidad Auto ´noma de Madrid; Grant number: 8. 1/22/97. Grant sponsor: Fondo Nacional de Ciencia y Tecno- logia de Chile; Grant number: 890028. *Correspondence to: Miguel Quintanilla, Instituto de Inves- tigaciones Biome ´dicas CSIC-UAM, Arturo Duperier 4, 28029-Madrid, Spain. E-mail: mquintanilla@iib.uam.es Received 20 May 1998; Accepted 19 January 1999 Journal of Cellular Biochemistry 74:61–73 (1999)  1999 Wiley-Liss, Inc.