A B 1ls20 l e 1C00 d te80 a r ig60 f40 M o .o20 N 0 Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 Page 8 of 12 Control c-Met siRNA (500 nM) Axl shRNA (8 g) PDGFR- shRNA (8 g) Scramble shRNA (8 g) Control c-Met siRNA Axl shRNA PDGFR- shRNA Scramble shRNA C c-Met -actin Axl -actin PDGFR- -actin Ctrl Ctrl c-Met siRNA S Axl shRNA Ctrl PDGFR- shRNA S S Figure 5 The effect of c-Met, Axl and PDGFR-a expression on the migration of bladder cancer cells. (A) TSGH8301 bladder cancer cells (2 × 105 cells/200 μl DMEM) were plated in the upper chamber in the presence of 500 nM siRNA for c-Met, Axl and PDGFR-a, respectively, and analyzed for cell migration at 36 h. The filter was stained with 2% crystal violet, and the cells migrated to the opposite side of the filter were counted. (B) Quantification of (A). Data represents mean ± SD of three experiments. * *: p < 0.01, * * *: p < 0.001, by Student’s t test. (C) The inhibitory effect of siRNA on c-Met, Axl and PDGFR-a expression was analyzed by Western blot analysis. Expression of c-Met, Axl and PDGFR-a was suppressed only in those cells transfected with c-Met, Axl or PDGFR-a siRNA/shRNA. There was no reduction of c-Met, Axl or PDGFR-a proteins in cells transfected with scrambled siRNA/shRNA. Ctrl: control treatment; S: scramble siRNA. addition to c-Met, both Axl and PDGFR-a play a posi- tive role in the progression of human bladder cancer. Discussion and conclusions In this study, we showed that both Axl and PDGFR-a have a functional interaction with c-Met in vitro and in vivo. This is the first report showing their potential clin- ical importance in human bladder cancer. The results concur with co-expression of c-Met/PDGFR-a in all of 9 human bladder cancer cell lines reported by Black and his colleagues [40]. The interaction between c-Met and Axl or PDGFR-a was further corroborated by HGF sti- mulation and siRNA silencing experiments in vitro. The interaction among these three RTKs may be initiated by protein-protein interaction or signaling transduction. The former possibility was excluded by co-immunopre- cipitation assay (data not shown). In terms of signal reg- ulation, the successful inhibition of c-Met activation by PD98059, but not by FTI-277 (ras inhibitor) or PP2 (Src inhibitor), suggests a ras- and Src-independent MEK/ ERK 1/2 signaling in the transactivation of Axl and PDGFR-a. Our results seem to imply the existence of a novel mechanism by which c-Met transactivates the expression of Axl and PDGFR-a. Additional experi- ments are required to clarify whether protein kinase C is involved in this cross-talk in vivo. Further support for our hypothesis of regulation at transcriptional level comes from several prior reports. The Sp1/Sp3 cis-acting elements were demonstrated to activate the promoter of Axl in various cancer cell lines [41]. Moreover, Sp1 response elements are detected in PDGFR-a promoter region [42,43]. Given that c-Met induces the phosphorylation of Sp1 and enhances down- stream gene expression through MEK/ERK signaling pathway [44,45], c-Met might up-regulate the expression of Axl and PDGFR-a through Sp1. The dose-dependent