Overexpression of Sprouty 2 Inhibits HGF/SF-Mediated Cell Growth, Invasion, Migration, and Cytokinesis

Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis

Chong-Chou Lee1,2, Andrew J Putnam1,3, Cindy K Miranti1, Margaret Gustafson1, Ling-Mei Wang1,2, George F Vande Woude*,1 and Chong-Feng Gao1

1Van Andel Research Institute, 333 Bostwick Avenue, N.E., Grand Rapids, MI 49503, USA

A strict regulation of hepatocyte growth factor/scatter and signaling molecules can attenuate Met signaling in factor (HGF/SF)-Met signaling is essential for its lieu of sustained receptor activation (Hammond et al., appropriate function. Several negative regulators of Met 2001, 2003), suggesting the existence of inhibitory signaling have been identified. Here we report that human signals that target downstream Met signaling. Spry2 is induced by HGF/SF and negatively regulates Spry was first identified in Drosophila as a feedback HGF/SF-Met signaling. We show that overexpression of inhibitor of the FGF signaling pathway during tracheal Spry2 inhibits cell proliferation, anchorage-independent branching (Hacohen et al., 1998). Subsequently, it was cell growth, and migration in wound-healing and in vitro found that Spry also antagonized EGFR and other invasion assays. Measured in an electric cell-substrate receptor tyrosine kinase (RTK) signaling pathways impedance sensing biosensor, cell movement is restricted, during Drosophila organogenesis (Casci et al., 1999; because Spry2 dramatically facilitates cell attachment Kramer et al., 1999; Reich et al., 1999). Studies in and spreading by enhancing focal adhesions and increas- cultured Drosophila cells show that Spry negatively ing stress fibers. An analysis of cell cycle distribution regulates RTK signaling pathways by inhibiting the shows, unexpectedly, that Spry2-GFP cells are polyploid. Ras/MAP kinase pathway (Casci et al., 1999). To date, Thus, as with FGF and EGF receptors, Spry2-GFP four mammalian Spry genes have been identified tempers downstream Met signaling in addition to its (Hacohen et al., 1998; de Maximy et al., 1999; pronounced effect on cell adhesion, and it has properties Minowada et al., 1999). These proteins have highly suitable to be considered a tumor-suppressor protein. conserved, cysteine-rich C termini and highly variable N Oncogene (2004) 23, 5193–5202. doi:10.1038/sj.onc.1207646 termini. As in Drosophila, mammalian Spry proteins Published online 3 May 2004 also work as feedback inhibitors of FGF signaling during organogenesis (Minowada et al., 1999; Tefft Keywords: sprouty; hepatocyte growth factor/scatter et al., 1999; Mailleux et al., 2001). In cultured cells, the factor; met; signaling; invasion; migration overexpression of Spry1, Spry2, or Spry4 inhibits FGF- and VEGF-induced proliferation, migration, and differ- entiation by repressing pathways leading to MAP kinase activation (Gross et al., 2001; Impagnatiello et al., 2001; Lee et al., 2001). Spry1 and Spry2 can inhibit FGF- Introduction induced MAP kinase activity by preventing the recruit- ment of the Grb2–Sos complex to the FGF docking Inappropriate Met signaling has been implicated in adaptor protein FRS2 or Shp2 (Hanafusa et al., 2002), many types of human cancers (Birchmeier et al., 2003), or by acting downstream of the Grb2–Sos complex implying that strict regulation of Met signaling is (Gross et al., 2001). Spry can also exert its inhibitory essential for its normal function. Several mechanisms, effect at the level of Raf downstream of Ras (Yusoff including degradation and dephosphorylation of Met et al., 2002). It seems that different molecular mechan- protein and its substrates, have been reported to isms are selected to mediate this inhibitory function negatively regulate Met signaling (Jeffers et al., 1997; depending on the RTK, the cell type, or the experi- Petrelli et al., 2002). Mutant Met molecules escape Cbl- mental conditions. mediated downregulation, illustrating the important Spry2 also potentiates EGFR signaling by attenuating role of inhibitory signals in the control of Met signaling Cbl-mediated endocytosis of EGFR (Egan et al., 2002; (Peschard et al., 2001). However, downstream effector Wong et al., 2002; Guy et al., 2003). EGF stimulation induces the phosphorylation of Spry2 at an N-terminal *Correspondence: GF Vande; E-mail: [email protected] tyrosine, which is required for its interaction with the 2Current address: Vita Genomics, Inc. 7FL, No. 6, Sec. 1, Jungshing SH2 domain of c-Cbl (Fong et al., 2003; Rubin et al., Road, WuGu Shiang, Taipei 248, Taiwan 2003). The competitive binding of Spry2 to Cbl results in 3Current address: Department of Chemical Engineering & Materials Science, University of California, Irvine Irvine, CA 92697, USA prolonged cell surface exposure of EGFR and sustained Received 12 September 2003; revised 13 February 2004; accepted 13 signaling (Egan et al., 2002; Wong et al., 2002; Hall et al., February 2004; published online 3 May 2004 2003). Thus, Spry may negatively or positively regulate Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5194 EGFR signaling via differential interaction with various a Culture plates signal molecules. HGF/SF ECM Spry2 was shown to inhibit cell proliferation, migra- (min) 0 5 30 60 24h 0 24h tion, and angiogenesis in response to growth factors Spry2 (Gross et al., 2001; Impagnatiello et al., 2001; Lee et al., 2001). Although MAP kinase might be involved in those β -Actin cellular responses, EGF-induced proliferation of endothe- lial cells was inhibited by Spry1 and 2; even activation of p42/44 MAP kinase was not affected (Impagnatiello et al., b 2001). This observation suggests that Spry may inhibit signaling pathways other than that of MAP kinase. WT SK-LMS-1 Overexpression of hSpry2 resulted in an increase in the ARZ HT29 amount and activity of protein tyrosine phosphatase HGF/SF - + - + - + - + (PTP1B) in the soluble fraction of cells without influen- Spry2 cing the total amount of cellular PTP1B. An increase in soluble PTP1B activity contributes to the anti-migratory β -Actin action of Spry2 (Zhang et al., 2001; Yigzaw et al., 2003). Since RTK-induced biological effects are mediated by c several signaling pathways, the study of how Spry HGF/SF 0 15` 4h 24h influences other signaling pathways is of interest. Spry2 In this study, Spry2 was identified as an HGF/SF- inducible gene, and when ectopically overexpressed as a β -Actin green fluorescent protein (GFP) fusion protein (Spry2- GFP) in human cells, it markedly influences the cell Figure 1 HGF/SF induces Spry2 expression (a) HGF/SF-induced cycle, motility, and invasion in response to HGF/SF. Spry2 expression in SK-LMS-1 cells. Cells were either grown in We show that Spry2 promotes cell spreading and culture plates (Culture plates) or in Matrigel (ECM) and were serum-starved for 24 h and then treated with HGF/SF (50 ng/ml) adhesion and that the overexpressed protein is a for the indicated times. The expression level of Spry2 was analysed negative regulator of cell proliferation, migration, by Northern blotting. (b) HGF/SF-induced Spry2 expression in invasion, and anchorage-independent cell growth. renal cell carcinoma cell lines. ARZ and WT cells were serum- Moreover, Spry2-GFP expression delays G2/M in the starved for 24 h before adding HGF/SF to the medium for 1 h. Spry2 expression was examined by Northern analysis. b-Actin gene cell cycle, causing polyploidy, and the protein localizes expression served as the loading control. (c) Induction of Spry2 to the midbody. protein in SK-LMS-1 cells. Cells cultured in culture plates were serum-starved for 24 h before adding HGF/SF (50 ng/ml). Cells were isolated at the indicated times and the whole-cell extracts were Results subjected to Western blot analysis using anti-Spry2 antibody (Upstate Biotechnology) as described in Materials and Methods HGF/SF induces Spry2 gene expression The human leiomyosarcoma cell line SK-LMS-1 ex- pEGFP-N3 expression vector with the GFP tag at the C presses high levels of the HGF/SF receptor Met, and terminus. SK-LMS-1 cells stably overexpressing Spry2- exhibits various cellular responses in vitro upon HGF/ GFP (SK-Spry2) or GFP (SK-GFP) were established SF treatment (Jeffers et al., 1996). We identified in SK- and characterized by immunofluorescence (Figure 2). LMS-1 cells that Spry2 was one of the most prominent GFP protein was widely distributed throughout the cells genes induced after HGF/SF treatment (Figure 1a). and HGF/SF treatment had no effect on its distribution. Northern blot analysis with a PCR-amplified human Spry2-GFP was mainly localized in the cytoplasm and Spry2 cDNA probe revealed an approximately 2.5-kb was recruited to the plasma membrane upon HGF/SF Spry2 transcript that was upregulated within 24 h of stimulation. HGF/SF stimulation (Figure 1a). This induction of Spry2 proteins are known to modulate the Ras/MAP Spry2 mRNA peaked at 60 min after stimulation kinase pathway when induced by various activated (Figure 1a). HGF/SF-induced upregulation of Spry2 receptor tyrosine kinases (Casci et al., 1999; Reich et al., mRNA was not restricted to SK-LMS-1 cells. The renal 1999). Therefore, we tested the effects of Spry2-GFP on clear cell carcinoma cell lines WT and ARZ also showed HGF/SF-Met signal pathways. HGF/SF-induced acti- HGF/SF-induced expression (Figure 1b). By contrast, vation of ERK2 and AKT2 was evaluated by Western HT29 cells constitutively expressed a high level of Spry2 blotting with phosphorylation-specific antibodies in the absence of HGF/SF (Figure 1b). Spry2 protein (Figure 3). SK-LMS-1 cells treated with HGF/SF expression was also detected in SK-LMS-1 cells treated exhibited ERK phosphorylation within 10 min and with HGF/SF (Figure 1c). maintained high levels of phosphorylation for 30 min; some phosphorylation was detectable after 3 h. By Spry2 inhibits HGF/SF-induced ERK and AKT activation contrast, SK-Spry2 displayed a significant reduction in ERK phosphorylation after HGF/SF stimulation. To examine the biological activity of Spry2 in SK-LMS- HGF/SF-induced phosphorylation of AKT was drama- 1 cells, a full-length Spry2 cDNA was inserted into the tically blocked in SK-Spry2 cells, while SK-LMS-1 cells

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5195 C HGF/SF SK-LMS-1 SK-Spry2 HGF/SF (min) 0 10 30 180 0 10 30 180 Met

P-AKT

SK-GFP AKT

P-ERK

ERK β-Actin

Figure 3 Overexpression of Spry2-GFP inhibits HGF/SF-induced ERK2 and AKT signaling. The cells were serum-starved for 24 h and treated with HGF/SF (50 ng/ml) in a time course of 0, 10, 30,

SK-Spry2 and 180 min. The activation of ERK2 and AKT2 was analysed by Western blotting with phosphorylation-speciﬁc antibodies. b-Actin served as loading control

Figure 2 Ectopic expression of Spry2-GFP in SK-LMS-1 cells. Spry2-expressing cells (SK-Spry2) and control cells (SK-GFP) were serum-starved for 24 h and then treated with 50 ng/ml HGF/SF (HGF/SF) for 4 h or were not treated (C). The expression of GFP- Spry2 blocks HGF/SF-induced anchorage-independent Spry2 or GFP protein was observed with immunofluorescence cell growth microscopy ( Â 400). GFP was widely distributed in cells plus or minus HGF/SF, while Spry2-GFP was observed in the cytoplasm It is well established that anchorage-independent growth in granular particulates that change in distribution after 4 h of in soft agar is predictive of tumorigenicity in vivo.To exposure to HGF/SF determine whether Spry2 influenced anchorage-independent growth, we performed soft-agar colony-formation assays on SK-LMS-1, SK-GFP, and SK-Spry2 cells. As showed rapid AKT phosphorylation with kinetics shown (Figure 5), SK-LMS-1 and SK-GFP cells formed similar to those of ERK (Figure 3). Met protein level large colonies in soft agar after 14 days in the presence and stability was similar between parental cells and SK- of HGF/SF. By contrast, few, if any, colonies were Spry2 cells. These results indicated that Spry2-GFP has observed with SK-Spry2 cells (Figure 5). an inhibitory effect on HGF/SF-induced ERK and AKT activation when overexpressed in these cells. The expression of GFP-vector had no effect on the activa- Spry2 overexpression results in G2/M accumulation tion of ERK or AKT in SK-LMS-1 cells (data not shown). We observed, from FACS analysis (Table 1), a normal distribution of 64–85% of SK-LMS-1 cells in G1/G0 under serum-free, serum-containing, or serum þ HGF/ Spry2 inhibits HGF/SF-induced cell proliferation SF growth medium. However, only 36–49% of Spry2- and in vitro cell invasion GFP cells were present in this stage of the cell cycle and, The activation of HGF/SF-Met signaling induces notably, 29–34% of the Spry2 cells were in G2/M, with various cellular responses (Birchmeier et al., 2003). To 7–10% cells showing polyploidy. These results suggest explore the function of Spry2, we tested the effects of that Spry2 may work as a cell cycle regulator by Spry2 on HGF/SF-mediated cell proliferation and preventing cells from re-entering the G1 phase of the in vitro cell invasion. Equal numbers of SK-Spry2 cells next cell cycle. The polyploidy state of SK-Spry2 cells and control SK-LMS-1 cells were plated in 96-well was confirmed with nuclear staining with Hoechst33342 plates and tested for [3H]-thymidine incorporation. (Figure 6a). Similar basal levels of [3H]-thymidine incorporation In anaphase cells, Spry2-GFP predominantly loca- were observed in both cell types in the absence of serum lized in the midbody of the cleavage plane, where the (Figure 4a). However, HGF/SF (100 ng/ml) treatment contractile ring of actin/myosin filaments assembles of parental cells produced a threefold increase in under the cortex of the cells (Figure 6b). These results incorporation. By contrast, SK-Spry2 cells displayed show that Spry2 induces polyploidy and localizes to the only a slight increase in DNA synthesis in response to midbody. HGF/SF (Figure 4a). An even more dramatic effect of Spry2 was observed Spry2 facilitates cell attachment and spreading by on HGF/SF-induced cell invasion. SK-LMS-1 and SK- enhancing focal adhesions and increasing stress fibers GFP cells displayed significant HGF/SF-induced cell migration through Matrigel, while SK-Spry2 cells did Electric cell-substrate impedance sensing (ECIS) is a not (Figure 4b and c). sensitive device and method for measuring cell adhesion

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5196

Figure 4 Overexpression of Spry2 inhibits HGF/SF-induced proliferation and invasion. (a) DNA synthesis in SK-Spry2 cells. SK-Spry2 cells or SK-LMS-1 cells were plated in 96-well plates (2 Â 103 cells/well) and cultured for 24 h. After 30 h of serum starvation, the cells were treated with HGF/SF and [3H]-thymidine incorporation was determined as described in Materials and methods. The mean values (7s.e.) of [3H] activity from triplicate experiments are presented. (b, c) In vitro invasion assay. Cells (1 Â 104) were suspended in DMEM containing 1% BSA and seeded in the upper chamber. The lower chamber was ﬁlled with DMEM containing 1% BSA without HGF/SF (C) or with 100 ng/ml HGF/SF (HGF). At 18 h after incubation, in vitro invasion was examined following cell staining. The cells were photographed under phase-contrast microscopy ( Â 100). Representative areas are shown (b). The mean number (7s.e.) of invading cells from triplicate experiments is presented (c)

and spreading (Keese et al., 2004). Cells are plated on a By contrast, gap filling by SK-Spry2 cells was much gold-coated electrode to which a small AC signal is slower (Figure 8). applied. Cell attachment and spreading can be measured We examined the focal adhesion and spreading by increases in impedance, since the effective area architecture of Spry2-GFP cells to further study the available for current flow is influenced by the presence early attachment activity indicated by ECIS. Immuno- of the cells, which increases the impedance. After the fluorescent localization of vinculin (one of several initial increase, the impedance fluctuates with time, abundant structural focal adhesion proteins) revealed which results from small cellular movements called obvious qualitative differences in the relative size and micromotion (Giaever and Keese, 1991). As shown, SK- number of focal adhesions in Spry2-GFP cells compared Spry2 cells exhibited dramatic increases in attachment with the SK-LMS-1 parental cell lines (Figure 9a–c). and spreading that peaked by 2 h after cell plating The SK-LMS-1 parental cells lacked distinct adhesive (Figure 7). By 5 h, and afterwards, cell attachment structures and actin stress fibers, particularly at early activity reorganizes to the same level as observed in time points (3–8 h) after plating (Figure 9a). F-actin control cells. Notably, SK-LMS-1 cells display marked accumulation occurred primarily in the peripheral fluctuations in impedance, while the impedance cortex of the parental cells. By contrast, SK-Spry2 cells change in SK-Spry2 cells is smooth. These results had large, distinct, vinculin-containing focal adhesions suggest that Spry2 overexpression increases cell adhe- and robust stress fibers even at early time points sion and spreading but limits cell movement. Consistent (Figure 9b). Co-localization of Spry2-GFP with vinculin with these results, the ‘wound-healing’ assays show staining following adhesion to the cover slips suggested that parental cells in growth medium containing that Spry2-GFP might be targeted to the adhesion sites HGF/SF or 10% FBS fill the gap completely by 12 h. (Figure 9c). However, Spry2-GFP also accumulates in

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5197 a C HGF/SF other regions of the cell, particularly along the peripheral actin cortex (Figure 9c). The spreading of Spry2-GFP cells at 3 h is consistent with the early spreading indicated in the ECIS. SK-LMS-1 Discussion

We show here that HGF/SF induces expression of Spry2, and we have overexpressed Spry2-GFP in SK- SK-GFP LMS-1 cells to study its possible role in regulating the pleiotropic effects of Met signaling in vitro. Obviously, gross overexpression of Spry2-GFP must be viewed with some reservation, but under these conditions MAPK and AKT signaling are inhibited, as are a variety of cellular functions including cell proliferation and cell SK-Spry2 cycle progression, migration, invasion, and anchorage- independent cell growth. Interestingly, Spry2-GFP overexpression reduces progression through G2/M, b 140 induces polyploidy and enhances cell attachment, C spreading, and focal adhesion formation. 120 HGF Spry may bind to several proteins and regulate MAP kinase activation through different mechanisms. The 100 binding of Spry to c-Cbl potentiates MAP kinase 80 activation by stabilizing EGFR (Egan et al., 2002; Wong et al., 2002), while the binding of Spry to Grb2 or 60 Raf1 inhibits MAPK activation through Ras-dependent and -independent mechanisms, respectively (Hacohen et al., 1998; Yusoff et al., 2002; Sasaki et al., 2003). In

Number of colonies 40 EGF signaling, c-Cbl is the main partner of Spry, and 20 the overall effect of Spry expression is the sustained activation of MAP kinase. Met receptor is also down- 0 regulated in response to HGF/SF through the c-Cbl- SK-LMS-1 SK-GFP SK-Spry mediated pathway (Petrelli et al., 2002). However, Met Figure 5 Spry2 inhibits HGF/SF-induced anchorage-independent downregulation is not blocked in Spry2-expressing SK- growth on soft agar. Cells (1 Â 104) were grown in soft agar in six- Spry2 cells, suggesting that c-Cbl is not a major player in well plates in the presence of 100 ng/ml HGF (HGF) or in normal Spry function in this system. Consistent with this result, medium (C) for 14 days. (a) A representative area of colonies we have observed a signiﬁcant reduction of MAP kinase observed by phase-contrast microscopy ( Â 100). (b) Number of activation in Spry2-expressing cells. The overexpression visible colonies (40.2 mm in diameter) after crystal violet staining. The mean value of three independent experiments is presented. of Spry2 also suppressed HGF/SF-induced activation of Standard deviations are indicated by the error bars AKT2. Since FGF- or PDGF-induced AKT2 activity

Table 1 FACS analysis of SK-LMS-1/SK-Spry 2 cells Cell cycle distribution (% gated)

Conditions Cell type G1/G0 S G2/M Polyploidy

No serum Control 85.7 5.0 5.0 0.4 Spry2 49.1 9.5 29.7 7.3

Serum Control 72.8 11.6 9.7 0.8 Spry2 44.8 10.0 32.6 7.7

Serum+HGF Control 64.3 15.1 17.1 1.0 Spry2 36.2 11.8 34.4 9.9

HGF Control 78.0 9.5 8.4 0.6 Spry2 42.3 12.1 32.1 9.3

SK-LMS-1 cells (control) or SK-Spry2 cells (Spry2) were cultured for 48 h in DMEM (no serum), DMEM containing 10% fetal bovine serum (serum), DMEM containing 10% fetal bovine serum plus 100 ng/ml HGF/SF (serum+HGF), or DMEM containing 100 ng/ml HGF/SF (HGF). Cells were stained with propidium iodide and cell cycle distribution was analysed by ﬂuorescence activated cell sorting (FACS) as described in Materials and Methods

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5198

Figure 6 Overexpression of Spry2-GFP inhibits cytokinesis. (a) Overexpression of Spry2-GFP induces polyploidy. SK-Spry2 cells were seeded in a chamber and cultured for 24 h. Cells were ﬁxed in 4% formaldehyde and stained with Hoescht33342. A single cell is shown with UV(left panel) or visible light (right panel). ( b) Localization of Spry2-GFP at the midbody in mitotic cells. SK-Spry2 cells were observed under a microscope with UV(left panel) or visible light (right panel). Localization of Spry2-GFP was visualized by GFP (green)

attachment HGF/SF is similar to that by FGF and EGF. Sprouty and spreading movement appears to be the primary response mediated by a MAP kinase cascade (Hacohen et al., 1998; Ozaki et al., 2001). In SK-LMS-1 cells, HGF/SF-induced Spry2 expression SK-spry2 occurs within 30 min, suggesting that it is also a primary response to Met kinase signaling. Spry2 inﬂuences many phenotypes when overexpressed in SKL-MS-1 cells. Cell proliferation is retarded and cells become polyploid. Moreover, overexpression of Spry2 inhibits cell migration and totally blocks invasion while enhancing attachment and cell spreading. SK-LMS-1 Thus, Spry2 appears to be a general antagonist of downstream effectors in the HGF/SF-Met signaling pathway. Most importantly, overexpression of Spry2 inhibits anchorage-independent cell growth, a predictor of tumorigenicity in vivo. All these point to a role of Spry2 in modulating the tumorigenic activity of HGF/ Figure 7 Attachment and spreading of SK-Spry2 cells in growth SF-Met signaling. Taken together, we expect HGF/SF medium recorded as changes in ECIS impedance. SK-LMS-1 or SK-Spry2 cells (5 Â 104) were seeded with 10% fetal bovine serum Met-induced expression of Spry2 is a novel antagonist in each chamber; cell attachment and spreading was recorded as effector of Met signaling. Overexpression of Spry2 changes in ECIS impedance over 14 h. The two tracings are results in decreased activity of all cell functions tested representative of repeated analysis in two separate wells in vitro, especially migration and invasion, which may be due to enhanced cell attachment and spreading, corresponding with increased focal adhesion formation. was not affected by Spry1 or Spry2 (Gross et al., 2001), By these criteria, Spry2 could qualify as a tumor- the inhibition of AKT activation by Spry2 may be suppressor gene. Our ﬁndings support an evolutionarily unique to HGF/SF-Met signaling. Further experiments conservative function of Spry2 as a common antagonist are needed to clarify the exact mechanism of Spry action of growth factor signaling pathways in Drosophila, on the AKT pathway. The kinetics of Spry induction by mice, and humans.

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5199

Figure 8 Spry2-GFP inhibits cell migration in wound-healing assays. Cells were serum-starved 16 h before making a scratch (physical wound) in the cell layer. The cells were treated with 10% fetal bovine serum, EGF (50 ng/ml), or HGF/SF (50 ng/ml), and the migration of cells in the open space was observed under a phase-contrast microscope ( Â 200) at the indicated times

a b Vinculin (TRITC) F-actin (FITC) Triple

Vinculin (TRITC) F-actin (FITC) Triple

A.) 3 h

3 h

B.) 5 h

5 h

C.) 8 h 8 h

D.) 24 h 24 h

c t=33 hours h 5 t=5h hours 8 t=8h hours Vinculin (TRITC) Spry2-GFP Triple

Figure 9 Spry2-GFP enhances focal adhesion and stress fiber formation. Focal adhesions and stress fibers in SK-LMS-1 (a) and SK- Spry2 cells (b). Cells were seeded in cell chambers for the indicated times. The focal adhesions and stress fibers were revealed by staining with TRITC-conjugated anti-Vinculin antibody (red) and FITC-conjugated phalloidin (green), respectively. Nuclei were visualized by Hoechst staining (Triple). Representative images from multiple experiments are shown. (c) Localization of Spry2-GFP in focal adhesions. Focal adhesions were revealed by staining SK-Spry2 cells with TRITC-conjugated anti-vinculin antibody (red) and the localization of Spry2-GFP was visualized by GFP (green). The co-localization of vinculin and Spry2-GFP in focal adhesions is indicated as yellow. Representative images are shown

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5200 Materials and methods 1 h, followed by detection of the proteins with ECL reagents and then exposure to X-ray films. Cell lines SK-LMS-1 human leiomyosarcoma cells, HT29 colon cancer Thymidine incorporation assays cells, Von Hippel–Lindau (VHL) renal cell carcinoma cells The thymidine incorporation assay was carried out as follows. (ARZ), and the latter cells overexpressing wild-type VHL Cells were plated in 96-well plates (2 103 cells/well) and (WT) (Koochekpour et al., 1999) were obtained from the Â cultured in DMEM supplemented with 10% FBS for 24 h. The American Type Culture Collection (ATCC) and cultured in cells were starved in serum-free DMEM medium for 30 h and Dulbecco’s modified Eagle’s medium (DMEM) supplemented then stimulated with or without HGF/SF for 12 h. with 10% fetal bovine serum. [Methyl-3H]-thymidine (1 mCi, Perkin-Elmer Life Science) was added 4 h before analysis. Thymidine incorporation into Antibodies and reagents DNA was measured by cold 5% trichloroacetic acid precipita- tion, followed by extraction in lysis buffer (0.02 N NaOH, Anti-phospho-ERK and phospho-AKT were purchased from 3 New England BioLabs, and anti-Spry2 was obtained from 0.1% SDS), and then counted in the H channel of a liquid Upstate Biotechnology. Peroxidase-conjugated goat anti-rab- scintillation counter (TRI-CARB 3100TR, Packard). bit antibody and the ECL reagents were purchased from Pierce. Growth factor-reduced (GFR) Matrigel was obtained In vitro invasion assay from Becton Dickinson, and TRIzol was from Life Technol- ogies. Complete Protease Inhibitors Cocktail Tablets were The in vitro invasion assay was performed as previously obtained from Roche Diagnostics. described (Jeffers et al., 1996) using a 24-well size invasion chamber coated with GFR Matrigel (Becton Dickinson). Cells were plated in the invasion chamber (1 Â 104 cells/well) RNA isolation and Northern blot analysis with or without addition of HGF/SF (100 Units/ml) in the Total RNA was extracted using TRIzol according to the lower chamber. After 18 h, cells that invaded through manufacturer’s protocol, and 20 mg/lane was fractionated by Matrigel and membrane were stained with diff-Quik agarose gel electrophoresis and transferred to a nitrocellulose Stain Set (Dade Behring Inc.) and counted under a light membrane. Spry2 cDNA fragments (0.5 kb) were 32P-labeled microscope. with random primers (Stratagene). The hybridization process was performed under standard conditions (Stratagene). The Anchorage-independent growth assay blot was then exposed to X-ray film with an intensifying screen 4 at À801C. Cells (1 Â 10 ) were seeded in six-well plates with a bottom layer of 0.7% Bacto agar in DMEM and a top layer of 0.3% Bacto agar in DMEM. Fresh DMEM containing 10% FBS Constructs, transfection, and establishment of stable cell lines with or without HGF/SF (100 ng/ml) was added to the top of Human Spry2 full-length cDNA was amplified by RT–PCR the soft agar. The culture medium was changed twice a week. using the RNA extracted from human embryonic lung After 14 days, the colonies were stained with 0.005% crystal fibroblast cells (M426). A Spry2 cDNA fragment was violet and the visible colonies (40.2 mm in diameter) were generated using a forward primer, which has an internal KpnI counted. restriction site, of 50-GGGGTACCCCAAGACCTGATG- 0 0 GAGGCCAGA-3 , and a reverse primer, 5 -ACAG- Cell migration/wound-healing assay GATCCTGTTGGTTTTTCAAAGTTCCTAGG-30, which has an internal BamHI restriction site. The PCR product Transfected cells and control cells were plated separately was restriction-digested with KpnI and BamHI and subse- overnight to achieve a confluent cell layer in 35-mm culture quently inserted in a eukaryotic expression vector pEGFP-N3 plates. The growth medium was then switched to serum-free (Clontech). Sequences were verified by DNA sequencing. SK- medium for 16 h before making a scratch on the cell layer LMS-1 cells were transfected with Spry2 GFP expression with a needle tip. ‘Wound healing’ was visualized by vector in Lipofectamine (Life Science Technologies) as monitoring migrating cells in the gap during a 12-h described in the manufacturer’s standard procedure. Stably post-scratch period. transfected SK-LMS-1 cells were selected with G418 for further characterization. SK-LMS-1 cells transfected with a ECIS assay control vector were also established. Electrode arrays, a relay bank, and a lock-in amplifier for the ECIS measurement were obtained from Applied BioPhysics Western blot analysis (Troy, NY, USA). The ECIS device is based on AC impedance Cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.6, measurements using weak and noninvasive AC signals as 5mM EDTA, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM described (Keese et al., 2004). Briefly, a 4000-Hz AC signal b-glycerophosphate, 1 mM Na3VO4 Á 14H2O, and 5 mM with an amplitude of 1.0 Vwas supplied through a 1 M O Na4P2O7 Á 10H2O containing the protease inhibitor Completet resistor to create an approximately constant current of 1 mAto (Boehringer-Mannheim, Germany)). For Western blot analy- the sample. The voltage across the electrode system could be sis, proteins (5 mg/lane) were resolved in 10% SDS–PAGE and continuously monitored with the computer-controlled lock-in electrotransferred to a PVDF membrane using standard amplifier, while the data were stored and processed in a procedures. After blocking for 1 h with 3–5% nonfat dry milk personal computer. As cells attach and spread on the small in TBS-T buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, gold electrode surface, their plasma membranes act as 0.1% Tween-20), the blots were probed with primary insulators and block the current path. As a result, the antibodies for 1 h at room temperature. The blots were then impedance increases dramatically when cells block current reacted with a peroxidase-conjugated secondary antibody for flow. Fluctuations of the measured impedance reveal cell

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5201 motion and persist due to small dynamic changes in cell with 4% paraformaldehyde in PBS at 41C at the indicated morphology until cells reach confluence. times after plating (3, 5, 8, and 24 h). To record real-time electrical impedance measurements, Fixed cells were permeabilized in Tris-buffered saline 2 Â 104 cells were inoculated in 10% fetal bovine serum in a containing 0.1% Triton X-100 (TBS-T). Nonspecific binding growth chamber consisting of one gold electrode was blocked with 1% normal goat serum. Focal adhesions (4.9 Â 10À4 cm2). Each well has a surface area of 0.8 cm2 for were labeled by staining with either a monoclonal anti-vinculin cell attachment and growth and each well holds 500 mlof (Sigma) or a monoclonal anti-paxillin (BD Transduction Labs) growth medium. The resistance across the monolayer is related antibody diluted in TBS-T containing 2% BSA, followed by to cell spreading and the distance between cells. The cells in Alexa546-conjugated goat anti-mouse IgG (Molecular culture were monitored with an applied electric field for 15 h as Probes). F-actin was visualized using OregonGreen-conju- described (Tiruppathi et al., 1992). gated phalloidin (Molecular Probes). Cells were visualized on a Nikon TE300 microscope equipped for epi-fluorescence using Immunofluorescent localization of focal adhesions a CCD camera (Hammamatsu) attached to an Apple Macintosh G4. Images were compiled using OpenLab software Immunofluorescent localization of focal adhesions in SK- (Improvision) and cropped for publication using Adode LMS-1 parental and SK-Spry2 cells was performed following a Photoshop 5.0. standard cell adhesion assay protocol (Miranti, 2002). Briefly, cells were starved overnight (approximately 18 h) in serum-free Fluorescence-activated cell-sorting analysis DMEM medium. Cover slips were incubated at 371C with DMEM þ 10% FBS overnight, rinsed twice with PBS, and SK-LMS-1 or SK-Spry2 cells (1 Â 106) cultured under subsequently blocked with PBS containing 1% bovine serum different conditions were collected by trypsinization and albumin for 2 h. During this blocking step, serum-starved cells stained with PI solution (0.1% sodium citrate, 0.1% Triton were harvested by washing once with calcium- and magne- X-100, and 0.005% propidium iodide in PBS) for 1 min. The sium-free PBS and then were trypsinized with 0.0125% cells were washed with PBS and suspended in PI solution trypsin-EDTA in PBS þ 5mM EDTA. Trypsin was neutralized without Triton X-100. The samples were analysed with FACS by adding soybean trypsin inhibitor (1 mg/ml) in PBS þ 5mM Caliber (Becton Dickinson).We thank Han-Mo Koo for EDTA. Cells were collected by centrifugation and then assistance with the FACS analysis and David Nadziejka and resuspended in DMEM þ 10% FBS before being subsequently Michelle Reed for assistance with the preparation of the plated onto the cover slips. Adherent cells were fixed for 20 min manuscript.

References

Birchmeier C, Birchmeier W, Gherardi E and Vande Woude Koochekpour S, Jeffers M, Wang PH, Gong C, Taylor GA, GF. (2003). Nat. Rev. Mol. Cell Biol., 4, 915–925. Roessler LM, Stearman R, Vasselli JR, Stetler-Stevenson Casci T, Vinos J and Freeman M. (1999). Cell, 96, 655–665. WG, Kaelin Jr WG, Linehan WM, Klausner RD, de Maximy AA, Nakatake Y, Moncada S, Itoh N, Thiery JP Gnarra JR and Vande Woude GF. (1999). Mol. Cell. Biol., and Bellusci S. (1999). Mech. Dev., 81, 213–216. 19, 5902–5912. Egan JE, Hall AB, Yatsula BA and Bar-Sagi D. (2002). Proc. Kramer S, Okabe M, Hacohen N, Krasnow MA and Hiromi Natl. Acad. Sci. USA, 99, 6041–6046. Y. (1999). Development, 126, 2515–2525. Fong CW, Leong HF, Wong ES, Lim J, Yusoff P and Lee SH, Schloss DJ, Jarvis L, Krasnow MA and Swain JL. Guy GR. (2003). J. Biol. Chem., 278, 33456–33464. (2001). J. Biol. Chem., 276, 4128–4133. Giaever I and Keese CR. (1991). Proc. Natl. Acad. Sci. USA, Mailleux AA, Tefft D, Ndiaye D, Itoh N, Thiery JP, 88, 7896–7900. Warburton D and Bellusci S. (2001). Mech. Dev., 102, 81–94. Gross I, Bassit B, Benezra M and Licht JD. (2001). J. Biol. Minowada G, Jarvis LA, Chi CL, Neubuser A, Sun X, Chem., 276, 46460–46468. Hacohen N, Krasnow MA and Martin GR. (1999). Guy GR, Wong ES, Yusoff P, Chandramouli S, Lo TL, Lim J Development, 126, 4465–4475. and Fong CW. (2003). J. Cell Sci., 116, 3061–3068. Miranti CK. (2002). Methods Cell Biol., 69, 359–383. Hacohen N, Kramer S, Sutherland D, Hiromi Y and Krasnow Ozaki K, Kadomoto R, Asato K, Tanimura S, Itoh N and MA. (1998). Cell, 92, 253–263. Kohno M. (2001). Biochem. Biophys. Res. Commun., 285, Hall AB, Jura N, DaSilva J, Jang YJ, Gong D and Bar-Sagi D. 1084–1088. (2003). Curr. Biol., 13, 308–314. Peschard P, Fournier TM, Lamorte L, Naujokas MA, Hammond DE, Carter S, McCullough J, Urbe S, Vande Band H, Langdon WY and Park M. (2001). Mol. Cell, 8, Woude G and Clague MJ. (2003). Mol. Biol. Cell, 14, 995–1004. 1346–1354. Petrelli A, Gilestro GF, Lanzardo S, Comoglio PM, Migone N Hammond DE, Urbe S, Vande Woude GF and Clague MJ. and Giordano S. (2002). Nature, 416, 187–190. (2001). Oncogene, 20, 2761–2770. Reich A, Sapir A and Shilo B. (1999). Development, 126, Hanafusa H, Torii S, Yasunaga T and Nishida E. (2002). 4139–4147. Nat. Cell Biol., 4, 850–858. Rubin C, Litvak V, Medvedovsky H, Zwang Y, Lev S and Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Yarden Y. (2003). Curr. Biol., 13, 297–307. Cotten M and Christofori G. (2001). J. Cell Biol., 152, Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki 1087–1098. M, Kuriyama M, Saito N, Shibuya M and Yoshimura A. Jeffers M, Rong S and Vande Woude GF. (1996). Mol. Cell. (2003). Nat. Cell Biol., 5, 427–432. Biol., 16, 1115–1125. Tefft JD, Lee M, Smith S, Leinwand M, Zhao J, Bringas Jr P, Jeffers M, Taylor GA, Weidner KM, Omura S and Vande Crowe DL and Warburton D. (1999). Curr. Biol., 9, Woude GF. (1997). Mol. Cell. Biol., 17, 799–808. 219–222. Keese CR, Wegener J, Walker SR and Giaever I. (2004). Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR and Proc. Natl. Acad. Sci. USA, 101, 1554–1559. Giaever I. (1992). Proc. Natl. Acad. Sci. USA, 89, 7919–7923.

Oncogene Sprouty 2-GFP and HGF/SF-mediated cell activity C-C Lee et al 5202 Wong ES, Fong CW, Lim J, Yusoff P, Low BC, Yusoff P, Lao DH, Ong SH, Wong ES, Lim J, Lo TL, Leong Langdon WY and Guy GR. (2002). EMBO J., 21, HF, Fong CW and Guy GR. (2002). J. Biol. Chem., 277, 4796–4808. 3195–3201. Yigzaw Y, Poppleton HM, Sreejayan N, Hassid A and Patel Zhang S, Lin Y, Itaranta P, Yagi A and Vainio S. (2001). TB. (2003). J. Biol. Chem., 278, 284–288. Mech. Dev., 109, 367–370.

Oncogene