JOURNAL OF CELLULAR PHYSIOLOGY 199:284–292 (2004) Requirement of TGF-b Receptor-Dependent Activation of c-Jun N-Terminal Kinases (JNKs)/Stress-Activated Protein Kinases (Sapks) for TGF-b Up-Regulation of the Urokinase-Type Plasminogen Activator Receptor JIANBO YUE, BAODONG SUN, GUANGMING LIU, AND KATHLEEN M. MULDER* Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania We have previously demonstrated that activation of the Ras/Mapk pathways is required for transforming growth factor b (TGF-b) induction of TGF-b 1 expression. Here we examined the role of the Ras/Mapk pathways in TGF-b induction of urokinase-type plasminogen activator receptor (uPAR) expression in untrans- formed intestinal epithelial cells (IECs). TGF-b activated the stress-activated protein kinases (Sapk)/c-Jun N-terminal kinases (JNKs) within 5–10 min, an effect that preceeded TGF-b induction of uPAR expression in these cells. TGF-b induction of both JNK1 activity and JunD phosphorylation was blocked by expression of a dominant-negative mutant of the type II TGF-b receptor (DN TbRII), a dominant- negative mutant of MKK4 (DN MKK4), or a dominant-negative mutant of Ras (RasN17), or by the addition of the JNK inhibitor SP600125. TGF-b also induced AP- 1 complex formation at the distal AP-1 site (184 to 178) of the uPAR promoter within 2 h of TGF-b addition, consistent with the time-dependent up-regulation of uPAR expression. The primary components present in the TGF-b-stimulated AP-1 complex bound to the uPAR promoter were Jun D and Fra-2. Moreover, addition of SP600125, or expression of DN MKK4 or DN TbRII, blocked TGF-b up-regulation of uPAR in IECs. Accordingly, our results indicate that TGF-b activates the Ras/ MKK4/JNK1 signaling cascade, leading to induction of AP-1 activity, which, in turn, up-regulates uPAR expression. Our results also indicate that the type II TGF-b receptor (RII) is required for TGF-b activation of JNK1 and the resulting up-regula- tion of uPAR expression. J. Cell. Physiol. 199: 284 – 292, 2004. ß 2003 Wiley-Liss, Inc. Transforming growth factor b (TGF-b) is a pleiotropic polypeptide for a variety of cell types, and the prototype for a large family of structurally related cytokines. It plays an important role in almost every aspect of cellular processes, including cell proliferation, differentiation, hematopoiesis, apoptosis, tumorigenesis, migration, and extracellular matrix (ECM) production (Massague et al., 2000; Yue and Mulder, 2001). Of particular interest is the ability of TGF-b to potently inhibit the growth of many solid tumors of epithelial origin. However, many solid tumor cells become refractory to the growth inhi- bitory effects of TGF-b, and TGF-b can, in turn, promote the invasiveness of these tumors (Massague et al., 2000). Thus, it is important to elucidate TGF-b signal trans- duction pathways and to identify signaling components leading to the tumor-enhancing effects of TGF-b. Over the last few years, great progress has been made in elucidating signaling transduction pathways invol- ving TGF-b superfamily members. TGF-b binds and activates type I (RI) and type II (RII) TGF-b receptor heterocomplexes, which, in turn, activate downstream cellular components, including Smads and members of the Ras/Mapk pathways (Massague, 1996; Atfi et al., 1997; Heldin et al., 1997; Mulder, 2000; Yue and Mulder, 2001). Moreover, TGF-b may utilize multiple intersect- ing pathways to regulate downstream cellular events. The sum total effect of this intricate network of pathways functionally defines the TGF-b molecule. ß 2003 WILEY-LISS, INC. Jianbo Yue and Baodong Sun contributed equally to this work. Contract grant sponsor: National Institutes of Health Grants; Contract grant numbers: CA51452, CA90765; Contract grant sponsor: Department of Defense Grant (to K.M.M.); Contract grant number: DAMD 17-01-1-0592. Jianbo Yue’s present address is Department of Molecular Pharmacology, CCSR Building, Room 3160, 269 Campus Drive, Stanford University, Stanford, CA 94305. *Correspondence to: Kathleen M. Mulder, Department of Phar- macology MC H078, Pennsylvania State College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: kmm15@psu.edu Received 9 July 2003; Accepted 18 September 2003 DOI: 10.1002/jcp.10469