ORIGINAL PAPER Eun-Young Shin Æ Eun-Kyung Ma Æ Chi-Kyung Kim Sahng-June Kwak Æ Eung-Gook Kim Src/ ERK but not phospholipase D is involved in keratinocyte growth factor-stimulated secretion of matrix metalloprotease-9 and urokinase- type plasminogen activator in SNU-16 human stomach cancer cell Received: 5 July 2002 / Accepted: 6 September 2002 / Published online: 23 October 2002 Ó Springer-Verlag 2002 Abstract Purpose: We investigated the signaling path- way for keratinocyte growth factor (KGF)-induced invasion using human stomach cancer cell line, SNU-16. Methods: Alterations in the activities of Src, extracel- lular signal-regulated kinase (ERK), and phospholipase D (PLD) were measured using [c- 32 P] ATP for autop- hosphorylation of Src, phospho-specific ERK antibody, and [9,10- 3 H] myristic acid, respectively, while herbi- mycin A, PD98059 and butan-1-ol were used to inhibit their activities. Matrix metalloproteases (MMPs) and urokinase-type plasminogen activator (uPA) were quantified with zymography and Matrigel-coated Transwell was employed to estimate the invasiveness of SNU-16 cells. Results: Src, ERK, and PLD were activated in response to KGF treatment, and inhibi- tion of these enzymes – by their specific inhibitors – decreased KGF-induced invasion in a dose-dependent manner. However, only inhibition of Src and ERK could block KGF-stimulated secretion of uPA and MMP-9. Conclusion: Src, ERK, and PLD are suggested as mediators of KGF-induced invasion in SNU-16. uPA and MMP-9 are considered as downstream targets of Src and ERK whereas PLD is thought to utilize different pathways. Keywords Keratinocyte growth factor Æ Invasion Æ Src Æ ERK Æ Phospholipase D Introduction Fibroblast growth factors (FGFs), also called heparin- binding growth factors (HBGFs) have been character- ized as polypeptides with mitogenic properties for fibroblasts as well as for a variety of neuroectoderm- and mesoderm-derived cells. FGFs have also been implicated in processes related to differentiation of various cell types. Being a member (FGF-7) of the fibroblast growth factor family, keratinocyte growth factor (KGF) is ac- tively synthesized as a single-chain polypeptide of 28 kDa and secreted by the mesenchymal cells such as fibroblasts (Finch et al. 1989). Its high affinity receptor, FGFR2/IIIb, is expressed in the adjacent epithelial cells (Bottaro et al. 1990). Thus, the KGF/KGF receptor system represents an important signal communicator in epithelial-mesenchymal interaction, which plays a criti- cal role in early morphogenesis of many organs. Amplification of K-sam, a KGF receptor gene, was found in the gastric cancer cell line KATO-III (Hattori et al. 1990), and later was shown to occur preferentially in undifferentiated types of gastric cancer (Hattori et al. 1996). Increased expression of the KGF receptor has also been reported in various advanced cancers and cell lines of poor differentiation, and is thereby suggested as a potential prognostic marker in some cancers. In the case of gastric oncogenesis, since the KGF receptor gene is frequently amplified and its mRNA is overexpressed mainly in the late stage when tumors invade and me- tastasize, the KGF/KGF receptor system may contrib- ute to this process. In a previous study, we also presented evidence that the KGF receptor may be in- volved in gastric tumorigenesis (Shin et al. 2000). We J Cancer Res Clin Oncol (2002) 128: 596–602 DOI 10.1007/s00432-002-0388-4 E.-Y. Shin Æ E.-G. Kim (&) Department of Biochemistry, College of Medicine, Medical Research Institute and Biotechnology Research Institute, Chungbuk National University, San 48, Gaesin-dong, Heungduk-ku, Cheongju, 361–763, Korea E-mail: egkim@med.chungbuk.ac.kr Tel.: +82-43-261-2848 Fax: +82-43-274-9710 E.-K. Ma Æ C.-K. Kim Department of Microbiology and Biotechnology, and Biotechnology Research Institute, Chungbuk National University, Cheongju, 361-763, Korea S.-J. Kwak Department of Biochemistry, College of Medicine, Dankook University, Cheonan, 330–714, Korea