Mitogenic Synergy Through Multilevel Convergence of Hepatocyte Growth Factor and Interleukin-4 Signaling Pathways

Mitogenic synergy through multilevel convergence of hepatocyte growth factor and interleukin-4 signaling pathways

Regina M Day1,2,3, Lilian Soon1,2,3, Diane Breckenridge1,2,3, Benjamin Bridges1,2,3, Bharvin KR Patel1,2,3, Ling Mei Wang1,2,3, Seth J Corey1,2 and Donald P Bottaro*,1,2,3

1Laboratory of Cellular and Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; 2Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 3Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Hepatocyte growth factor (HGF) regulates various Introduction physiological and developmental processes in concert with other growth factors, cytokines and hormones. We Hepatocyte growth factor (HGF) stimulates mitogen- examined interactions between cell signaling events esis, motogenesis, and morphogenesis in a wide range elicited by HGF and the cytokine interleukin (IL)-4, in of cellular targets including epithelial and endothelial the IL-3-dependent murine myeloid cell line 32D cells, hematopoietic cells, neurons, melanocytes, as well transfected with the human HGF receptor, c-Met. as hepatocytes (reviewed in Michalopoulos and De- HGF was a potent mitogen in these cells, and prevented Frances, 1997; Rubin et al., 1993; Zarnegar and apoptosis in response to IL-3 withdrawal. IL-4 showed Michalopoulos, 1995). These pleiotropic eects play modest anti-apoptotic activity, but no signi®cant mito- important roles during development, organogenesis genic activity. IL-4 synergistically enhanced HGF- and tissue regeneration. HGF is essential for the stimulated DNA synthesis, whereas only additive pre- normal development of both liver and placenta vention of apoptosis was observed. IL-4 did not enhance (reviewed in Birchmeier and Gherardi, 1998), con- HGF-dependent tyrosine phosphorylation of c-Met or tributes to neural development (reviewed in Streit and Shc. In contrast, HGF-stimulated activation of MAP Stern, 1997), branching morphogenesis in various kinases was enhanced by IL-4, suggesting that the IL-4 organs (reviewed in Birchmeier et al., 1997), and and HGF signaling pathways converge upstream of these promotes kidney and lung regeneration (Yanagita et events. Although phosphatidylinositol 3-kinase (PI3K) al., 1993; Balkovetz and Lipschutz, 1999). inhibitors diminished HGF-induced mitogenesis, anti- Among hematopoietic tissues, HGF is produced by apoptosis, and MAP kinase activation, IL-4 enhanced bone marrow stromal cells (Weimar et al., 1998), and c- HGF signaling persisted even in the presence of these Met is expressed in CD34+ hematopoietic progenitor inhibitors. IL-4 enhancement of HGF signaling was cells (HPC) derived from bone marrow, peripheral partially blocked in 32D/c-Met cells treated with blood, and cord blood (Weimar et al., 1998; Kmiecik et inhibitors of MEK1 or c-Src kinases, completely blocked al., 1992; Ikehara, 1996). HGF promotes the prolifera- by expression of a catalytically inactive mutant of Janus tion, adhesion, and survival of CD34+ HPC (Weimar et kinase 3 (Jak3), and increased in 32D/c-Met cells al., 1998; Kmiecik et al., 1992), and acts synergistically overexpressing STAT6. Our results suggest that the with interleukin (IL)-3 or granulocyte-macrophage IL-4 and HGF pathways converge at multiple levels, and colony stimulating factor (GM-CSF) to increase colony that IL-4-dependent Jak3 and STAT6 activities mod- forming units in culture by CD34+ HPC in vitro ulate signaling events independent of PI3K to enhance (Kmiecik et al., 1992; Ikehara, 1996). Erythropoietin HGF-dependent mitogenesis in myeloid cells, and (EPO) and HGF also act synergistically to induce the possibly other common cellular targets. formation of erythroid burst-forming unit colonies from Oncogene (2002) 21, 2201 ± 2211. DOI: 10.1038/sj/ CD34+ HPC (Ikehara, 1996; Iguchi et al., 1999). HGF onc/1205289 compensates for de®ciencies in stem cell proliferation observed in mice harboring dominant mutations in the Keywords: growth factor; cytokine; signal transduc- genes encoding stem cell factor (SCF) or its receptor, c- tion; cell proliferation kit (Yu et al., 1998). HGF enhances antibody production in mature B cells (Delaney et al., 1993), and stimulates rapid cytoskeletal changes, adhesion, and migration in peripheral blood T cells (Adams et al., 1994). In addition to roles in normal development and adult *Correspondence: DP Bottaro, Department of Cell and Molecular homeostasis, HGF signaling is strongly linked to cancer, Biology, EntreMed, Inc., Rockville, Maryland, MD 20850 USA; including colon, breast, lung, thyroid and renal E-mail: [email protected] Received 2 May 2000; revised 3 January 2002; accepted 7 January carcinomas, melanoma and several sarcomas, as well as 2002 glioblastoma (reviewed in Vande Woude et al., 1997). HGF and IL-4 mitogenic synergy RM Day et al 2202 Among hematopoietic tumors, many human leukemia Results cell lines secrete HGF in vivo and in vitro, including those derived from adult T-cell leukemia, acute lymphoblastic Mitogenic synergy between HGF and IL-4 leukemia, multiple myeloma (MM), acute undieren- tiated leukemia, chronic myelocytic leukemia, acute The mitogenic response of 32D/c-Met cells to HGF is myeloblastic leukemia, and acute promyelocytic leuke- shown in Figure 1a. Increased DNA synthesis was mia (Gohda et al., 1995). HGF promotes the adhesion of detectable at 50 ng/ml (0.55 nM) HGF, and reached B-lymphoma cells to the extracellular matrix (Weimar et maximal levels at 1 mg/ml (11 nM;ED50=3.3 nM). al., 1997), and the overproduction of HGF associated 32D/c-Met cells also express low levels of IL-4 with MM has been linked to the extensive bone marrow receptor, but IL-4 is not mitogenic in these cells (Wang angiogenesis and bone destruction characteristic of that et al., 1993). As shown in Figure 1b, IL-4 at disease (Borset et al., 1999). concentrations of up to 100 ng/ml did not stimulate An emerging theme in our understanding of HGF signi®cant [3H]-thymidine incorporation. However, IL- action is the modulation of its biological activities by 4 signi®cantly enhanced HGF-stimulated [3H]-thymi- other cytokines. Among the many cytokines whose dine incorporation in a dose-dependent manner (Figure actions coincide spatially and temporally with those of 1b). These results show that the eects of HGF with HGF, few are as ubiquitous as Interleukin (IL)-4. IL-4 IL-4 far exceeded the sum of either mitogen adminis- is produced predominantly by T cells, mast cells and tered alone, indicating mitogenic synergy between IL-4 basophils (Paul, 1991). The IL-4 receptor, IL4Ra,a and HGF in 32D/c-Met cells. We estimate that IL-4 member of the hematopoietin/cytokine receptor super- (1 ng/ml) increased the ED50 of HGF approximately family, is expressed on both hematopoietic and non- 3 ± 5-fold. Similar eects have been observed in hematopoietic cells (Cosman, 1993). IL-4 stimulates the epithelial cells, such as Balb/MK keratinocytes (JS proliferation and dierentiation of B cells, the Rubin and WG Taylor, personal communication). proliferation of T cells and mast cells, and regulates Heparan sulfate proteoglycan (HSPG) has been the lymphokine producing phenotype of CD4+ T shown to modulate HGF signaling in several target cell lymphocytes (Keegan et al., 1994). In non-hematopoie- types, including Baf3 and 32D, which lack abundant cell tic cells such as ®broblasts and epithelial cells, IL-4 surface HSPG (Day et al., 1999; Schwall et al., 1996; modulates extracellular matrix production, causes Sakata et al., 1997). Soluble heparin (10 mg/ml), which changes in morphology and cytoskeletal organization, may substitute for cell-surface HSPG to some extent, and stimulates chemotaxis (Postlethwaite and Seyer, enhanced HGF mitogenic activity in 32D/c-Met cells at 1991; Postlethwaite et al., 1992). IL-4 can also least threefold, but did not impart mitogenic activity to stimulate the dierentiation of human carcinoma cells, the truncated HGF isoform, HGF/NK2 (Figure 2). The and thereby block their proliferation (Hoon et al., eects of heparin were most striking at low levels of 1991; Morisaki et al., 1992, 1994). The biological HGF (2 and 20 ng/ml), and decreased at very high eects of IL-4 are also in¯uenced by other cytokines (200 ng/ml) HGF concentrations, as maximal mitogenic and growth factors. IL-4 complements IL-2 in T-cell stimulation was reached (Figure 2a). The simultaneous tumor lines for ex vivo autocrine growth and for in vivo addition of both soluble heparin and IL-4 to 32D/c-Met tumorigenicity (Hassuneh et al., 1997), acts synergisti- cells increased the mitogenic activity of HGF to a greater cally with platelet-derived growth factor (PDGF) in extent than either factor alone. As shown in Figure 2a, mitogenesis (Patel et al., 1996), and inhibits HGF- IL-4 (1 ng/ml) or heparin (10 mg/ml) alone enhanced stimulated migration of colon carcinoma cells (Uchiya- mitogenic activity of HGF (2 ng/ml) about fourfold. In ma et al., 1996). the presence of both IL-4 and heparin, HGF-dependent We investigated the eects of IL-4 on HGF mitogenic activity was more than 20-fold higher than biological activities in the myeloid cell line 32D that of HGF alone (Figure 2a). In addition to dierences transfected with human HGF receptor (32D/c-Met in their presumed sites of action, the ability of soluble cells; Day et al., 1999). HGF stimulated mitogenesis heparin to further enhance the HGF/IL-4 mitogenic and prevented apoptosis in response to IL-3 with- synergy supports the hypothesis that IL-4 and heparin drawal. IL-4, in contrast, showed modest anti- facilitate HGF mitogenicity by distinct mechanisms: apoptotic activity and no mitogenic activity. However, heparin at the level of HGF-c-Met interaction, and IL-4 IL-4 synergistically enhanced HGF-stimulated DNA at the level of intracellular signaling. synthesis, which was re¯ected biochemically at the level Interestingly, the combination of IL-4 and high of MAP kinase, but not phosphatidylinositol 3-kinase concentrations (1 mg/ml) of HGF/NK2 was mitogenic, (PI3K) activation. HGF/IL-4 mitogenic synergy was while neither factor alone stimulated signi®cant DNA partially dependent on Src kinases, required the synthesis (Figure 2b). In most cell lines, HGF/NK2 activation of Jak3, and was enhanced by overexpres- stimulates increased cell motility, but not mitogenesis sing STAT6. Our results suggest that the HGF and IL- (Stahl et al., 1997; Chan et al., 1991). In 32D/c-Met 4 signaling pathways converge at multiple levels cells, HGF and HGF/NK2 potently stimulate the downstream of HGF-stimulated PI3K activation, and activation of c-Met kinase, phosphatidylinositol 3- that IL-4-activated Jak3 and STAT6 are critical kinase (PI3K), and MAP kinase, yet the biological contributors to HGF/IL-4 mitogenic synergy in response to HGF/NK2 is limited to increased cell myeloid cells. motility (Day et al., 1999). Thus, HGF/NK2 appears

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2203

Figure 1 Dose-dependent modulation of HGF mitogenic activity by IL-4. 32D/c-Met cells were starved for 36 h in RPMI+10% FBS with the indicated concentrations of growth factor. [3H]-Thymidine incorporation into DNA was measured as described in Materials and methods. (a) HGF alone; (b) increasing concentrations of IL-4 in the absence (open circles) or presence (solid squares) of HGF (100 ng/ml). Values represent mean+standard deviation of triplicate samples; results are representative of at least three experiments. Where no standard deviations are visible, the error is smaller than the symbol size

synthesis. The eects of IL-4 on signaling by HGF and HGF/NK2 suggest that the critical elements not activated in response to HGF/NK2 could be common to both HGF and IL-4 signaling pathways. In contrast to some other cultured cell models (Schwall et al., 1996), the combination of HGF/NK2 and soluble heparin did not stimulate signi®cant mitogenesis in 32D/c-Met cells, even at very high concentrations of HGF/NK2 (Figure 2b). Moreover, the mitogenicity observed in response to the combination of HGF/NK2 and IL-4 was not further enhanced in the presence of heparin (Figure 2b).

HGF and IL-4 protect 32D/c-Met cells from apoptosis 32D cells have been used extensively to study the anti- apoptotic eects of IL-3 and IL-4 (Wang et al., 1992, 1993; Zamorano et al., 1996). We observed that within 36 h of IL-3 deprivation, approximately 31% of 32D/c- Met cells enter apoptosis, and that the addition of 1 ng/ml IL-3 reduced this to 0.2% (Figure 3). The addition of HGF at concentrations of 50 and 200 ng/ ml prevented 75 and 92% of apoptosis stimulated by IL-3 deprivation, respectively (Figure 3). Consistent Figure 2 IL-4 and soluble heparin independently enhance HGF with previous reports showing that IL-4 is non- mitogenic activity in heparan sulfate proteoglycan-de®cient 32D/ mitogenic but anti-apoptotic (Wang et al., 1992, c-Met cells. Intact cells were treated with either HGF (a) or HGF/ 1993), this cytokine (1 ng/ml) reduced apoptosis NK2 (b) at the indicated concentrations in the presence or associated with IL-3 withdrawal in 32D/c-Met cells absence of and/or soluble heparin. [3H]-Thymidine incorporation into DNA was measured as described in Materials and methods. by approximately 25% (Figure 3). IL-4 treatment Open bars: HGF or HGF/NK2 alone; black bars:+IL-4 (1 ng/ enhanced the anti-apoptotic eects of HGF, reducing ml); gray bars:+soluble heparin (10 mg/ml); stippled bars:+IL-4 apoptosis 38% (from 8.2 to 5.1%) for 50 ng/ml HGF, and soluble heparin. Values represent mean+standard deviation and 66% (from 2.6 to 0.9%) for 200 ng/ml HGF of triplicate samples; results are representative of at least three (Figure 3). The anti-apoptotic eects of HGF in experiments. Where no standard deviations are visible, the error was too small to indicate graphically combination with IL-4 were approximately additive, suggesting some degree of independence in their signaling pathways. IL-4 caused no further reduction of apoptosis in the presence of IL-3 (data not shown). to activate a subset of the intracellular pathways that HGF/NK2 had no anti-apoptotic activity on 32D/c- are activated by full-length HGF, but fails to activate Met cells, even in combination with IL-4, above the some as yet unidenti®ed eector(s) critical to DNA eects of IL-4 alone (data not shown).

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2204

Figure 3 IL-4 modulates HGF anti-apoptotic activity. 32D/c- Met cells were treated with the indicated concentrations of HGF with or without IL-4 (1 ng/ml), or with IL-3 (1 ng/ml). FACS analysis was used to determine the percentage of apoptotic cells as described in Materials and methods. Open bars: without IL-4; gray bars:+IL-4. Values represent mean+standard deviation of triplicate samples; results are representative of at least three Figure 4 IL-4 does not enhance HGF stimulated c-Met, Shc or experiments. Where no standard deviations are visible, the error PI3K activation. 32D/c-Met cells were treated with HGF, IL-4, was too small to indicate graphically HGF+IL-4 or IL-3. Cell lysates were immunoprecipitated with anti-pY and immunoblotted for either c-Met (a) or shc (b), or assayed for PI3K activity (c). HGF concentrations are indicated in mg/ml; `+' indicates HGF at 200 ng/ml. IL-3 and IL-4 were IL-4 enhances HGF mitogenicity downstream of c-Met, added at 100 ng/ml, ®nal concentration. The results are representative of three or more experiments Shc, and PI3K To explore the mechanism(s) by which IL-4 synergized with HGF, we investigated the activation of c-Met, Shc Figure 4c, HGF stimulated PI3K activation while IL-4 and PI3K. After treatment of 32D/c-Met cells with did not, and IL-4 did not appear to enhance PI3K HGF, IL-4 or IL-3, cell lysates were immunoprecipi- activation by HGF. Higher levels of PI3K activity were tated with anti-phosphotyrosine and immunoblot observed in response to higher concentrations of HGF, analysis was performed using anti-c-Met. As shown indicating the level of PI3K activity observed in in Figure 4a, signi®cantly more c-Met was immuno- response to both HGF and IL-4 was submaximal precipitated with anti-phosphotyrosine after treatment (data not shown). These data are consistent with a with HGF, indicating HGF-stimulated c-Met kinase point of convergence between the HGF and IL-4 autophosphorylation and activation in these cells. mitogenic signaling pathways downstream of PI3K Neither IL-4 nor IL-3 alone stimulated signi®cant c- activation. Met tyrosine phosphorylation, and neither cytokine Several HGF-stimulated biological activities are enhanced HGF-stimulated c-Met activation (Figure dependent on PI3K in epithelial HGF target cell types 4a), suggesting that IL-4 did not enhance the activation (Day et al., 1999; Graziani et al., 1991; Rahimi et al., of c-Met by HGF. 1996). To assess the PI3K dependence of HGF- Shc is an adapter protein implicated in the mitogenic stimulated mitogenic and anti-apoptotic activities in pathways of several growth factors, and is thought to 32D/c-Met cells, and to further explore the contribu- participate in the activation of the PI3K and SOS-Ras- tion of IL-4 to these activities, each was examined in MAP kinase pathways (Bon®ni et al., 1996). In 32D/c- the presence and absence of speci®c PI3K inhibitors Met cells, Shc was tyrosine phosphorylated in response (Figure 5). Neither Wortmannin (100 nM) nor to IL-3, and, to a lesser extent, in response to HGF LY294002 (10 mM) exhibited strong inhibitory eects (Figure 4b). IL-4 alone did not increase Shc phosphor- on IL-3-stimulated 32D/c-Met cell growth (Figure 5a). ylation on tyrosine, and IL-4 did not appear to In contrast, DNA synthesis elicited by HGF was enhance HGF-stimulated Shc tyrosine phosphoryla- strongly suppressed: Wortmannin inhibited 95% of tion, suggesting that IL-4 acts independently or HGF-induced mitogenesis, while LY294002 inhibited downstream of Shc in HGF signaling. 88% (Figure 5a). Thus HGF-stimulated mitogenesis in Upon its activation by HGF, c-Met has been shown 32D/c-Met cells exhibited a degree of PI3K dependence to associate with, and activate, PI3K in epithelial cells similar to those reported for other HGF target cell (Graziani et al., 1991), as well as in 32D/c-Met cells types (Rahimi et al., 1996). Immunoblotting with anti- (Day et al., 1999). PI3K activation is required for active Akt antibody con®rmed that the activation of HGF-induced mitogenesis in SP1 mammary carcinoma targets directly downstream of PI3K was completely cells and Mv1Lu lung epithelial cells (Rahimi et al., blocked under these conditions (data not shown). In 1996). We investigated the eects of IL-4 on HGF- the presence of both HGF and IL-4, Wortmannin stimulated PI3K activity in 32D/c-Met cells by inhibited 84% of HGF-stimulated mitogenesis, while measuring PI3K activity in anti-phosphotyrosine im- LY294002 blocked 60% of this activity (Figure 5a). munoprecipitates prepared from intact cells treated Close scrutiny of these data revealed that despite with each factor alone or in combination. As shown in potent blockade of HGF mitogenic activity by PI3K

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2205 inhibitors, signi®cant enhancement of HGF mitogenic examined the eects of HGF and IL-4 on MAP kinase signaling by IL-4 was still observed, again suggesting activation by immunoblot analysis using antibody raised that convergence between the HGF and IL-4 mitogenic against active (phosphorylated at Y204) p44/42 MAP signaling pathways occurred downstream of PI3K. kinases. HGF and IL-3 stimulated the activation of Wortmannin did not signi®cantly inhibit IL-3- these MAP kinases above the level of phosphorylation dependent anti-apoptotic activity, but HGF-dependent observed in untreated control 32D/c-Met cells (Figure anti-apoptotic activity was strongly suppressed by this 6a). Increased activation of MAP kinase was seen drug (Figure 5b). The percentage of cells entering primarily in the level of phosphorylation of ERK-2. apoptosis in the presence of HGF with Wortmannin IL-4 enhancement of HGF-stimulated ERK-2 phos- increased ®vefold over the percentage treated with phorylation was observed, while IL-4 alone had no HGF alone (Figure 5b). IL-4 enhanced HGF-mediated detectable eect on MAP kinase activation (Figure 6a). protection from apoptosis even in the presence of To con®rm the apparent increase in MAP kinase Wortmannin, reducing the level of apoptosis observed activity indicated by immunoblotting experiments, the in cells treated with both HGF and Wortmannin by time course of MAP kinase activity toward a synthetic approximately 30% (Figure 5b). These data are myelin basic protein (MBP) peptide in vitro was consistent with previous ®ndings that IL-4 inhibited analysed in anti-MAP kinase immunoprecipitates apoptosis via a PI3K-independent pathway (Yashiro et prepared from control and growth factor treated al., 1996). 32D/c-Met cells. MAP kinase-catalysed MBP peptide phosphorylation in vitro increased threefold above IL-4 enhances HGF-stimulated MAP kinase activation The p44 and p42 MAP kinases (ERK-1 and -2) are activated by a wide variety of growth factors and cytokines, and are critical for many mitogenic pathways (Cowley et al., 1994; Mansour et al., 1994). We

Figure 6 IL-4 enhances MAP kinase activation by HGF. (a) 32D/c-Met cells were treated for 10 min at 378C with HGF at the Figure 5 HGF-stimulated mitogenic and anti-apoptotic activ- indicated concentrations (mg/ml), or IL-4 (100 ng/ml), or IL-3 ities are PI3K-dependent in 32D/c-Met cells. (a) DNA synthesis (100 ng/ml), or left untreated (lane 6), and cell lysates were was measured in 32D/c-Met cells treated with IL-3 (1 ng/ml), immunobloted with anti-phospho-MAP kinase. (b) 32D/c-Met HGF (200 ng/ml), or HGF+IL-4 (1 ng/ml), as indicated below cells were treated for the indicated times at 378C with HGF the x-axis. Open bars represent no drug addition, black bars (200 ng/ml; open squares) or HGF+IL-4 (200 ng/ml and 100 ng/ represent treatment with Wortmannin (0.1 mM), and gray bars ml, respectively; open circles). Anti-MAP kinase immunoprecipi- represent treatment with LY-294002 (10 mM). Values shown are tates were then assayed for MAPK activity in vitro using myelin mean+standard deviation of triplicate samples. The results are basic protein (MBP) peptide as substrate, as described in representative of three or more experiments. (b) Apoptosis of Materials and methods. Values shown are mean+standard 32D/c-Met cells that had been left untreated (control), or treated deviation of triplicate samples; where error bars are not visible with HGF, HGF+IL-4, or IL-3 at the same ®nal concentrations the standard deviation is smaller than the symbol size. (c) 32D/c- listed in panel (a) was measured after 36 h as described in Met cells were treated for 10 min at 378C with HGF (200 ng/ml), Materials and methods. Open bars represent no drug addition, IL-4 (100 ng/ml), IL-3 (100 ng/ml), or HGF+IL-4, in the black bars represent treatment with Wortmannin (0.1 mM). Values presence and absence of Wortmannin (0.1 mM) as indicated. shown are mean+standard deviation of triplicate samples. The Lysates were immunobloted with monoclonal anti-phospho-MAP results are representative of three or more experiments kinase. The results are representative of three experiments

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2206 basal levels in response to HGF treatment, and the and data not shown). In contrast, partial inhibition combination of HGF with IL-4 increased MAP kinase (approximately 35%) of the increase in DNA synthesis activity more than sevenfold (Figure 6b). These stimulated by the combination of HGF and IL-4 was experiments also revealed that IL-4 enhanced primarily consistently observed (Figure 7a), suggesting that part the magnitude of MAPK activation, without aecting of the synergistic eect of IL-4 might occur through the kinetics or duration of activation (Figure 6b). The MEK1 activation. results of both immunoblot analysis and in vitro kinase Src kinases mediate the biological actions of a assays suggest that the modulation of HGF signaling variety of cytokines and growth factors, including by IL-4 can occur, at least in part, upstream of MAP HGF (Sunitha et al., 1999). Although we have kinase. observed increased tyrosine phosphorylation of pp60 HGF-stimulated MAP kinase activation is thought c-Src in 32D/c-Met cells in response to HGF treatment to be PI3K-dependent in epithelial cells (Rahimi et al., (data not shown), the individual roles of the several Src 1996), and immunoblot analysis of growth factor- kinase family members expressed in HGF target cells treated 32D/c-Met cells using the anti-active MAP have not yet been thoroughly characterized. The recent kinase antibody revealed that MAP kinase activation development of selective Src kinase inhibitors has induced by HGF, but not IL-3, was strongly inhibited provided a means to rapidly evaluate the impact of by Wortmannin (Figure 6c). Consistent with the eects Src kinases on speci®c biological activities. The Src of Wortmannin on DNA synthesis driven by HGF and inhibitors PP1 and PP2 are eective on members of IL-4, evidence of IL-4 enhancement of HGF-mediated MAP kinase activation persisted in the presence of Wortmannin, although the total MAP kinase activity detected was signi®cantly decreased (Figure 6c). Buers used in these assays contained dimethyl sulfoxide (DMSO) to increase the solubility of Wortmannin, and the presence of DMSO was associated with an equal activation of both p42 and p44 MAP kinases by HGF and IL-3 (Figure 6c), in contrast to the predominant activation of p42 by these factors in the absence of DMSO (Figure 6a). The mechanism by which this occurs is unknown. Nevertheless, our results show unambiguously that HGF-stimulated ERK-2 activation is PI3K-dependent in 32D/c-Met cells, and that this activation was enhanced by IL-4. The persistence of IL-4 enhancement of HGF-stimulated MAP kinase activation in the presence of Wortmannin also indicates convergence of the HGF and IL-4 signaling pathways upstream of MAP kinase activation.

Partial blockade of IL-4 enhanced HGF signaling by MEK1 and Src inhibitors To identify eectors at the convergence of the HGF and IL-4 mitogenic signaling pathways that might act downstream of PI3K and upstream of MAP kinases, we characterized the eects of two selective kinase Figure 7 Mek1 and Src kinase inhibitors diminish the enhance- inhibitors on DNA synthesis stimulated by HGF and ment of HGF mitogenic signaling by IL-4. (a) DNA synthesis by IL-4: the MEK1 inhibitor PD98059 (Alessi et al., 32D/c-Met cells left untreated (open bars) or treated with HGF 1995), and the Src inhibitor PP2 (Hanke et al., 1996). (50 ng/ml; gray bars) or HGF+IL-4 (50 ng/ml and 10 ng/ml, MEK1 has been shown to be an important activator of respectively; black bars). The Mek1 inhibitor PD98059 was added at a concentration of 10 or 20 mM, as indicated. Controls for the MAP kinases, the latter being critical for HGF- presence of low levels of DMSO in PD98059-containing media stimulated mitogenesis and motogenesis in certain consisted of samples treated with identical amounts of DMSO epithelial HGF target cell types (Vande Woude et al., alone, as indicated. (b) DNA synthesis by 32D/c-Met cells treated 1997). In 32D/c-Met cells, however, PD98059 was not with the Src kinase inhibitor PP2 at 1, 3, or 10 mM concentrations 15 min prior to treatment with HGF (30 ng/ml; open bars) or found to be a potent inhibitor of HGF-stimulated cell HGF+IL-4 (30 ng/ml and 5 ng/ml, respectively; gray bars), as migration (Day et al., 1999). Consistent with those indicated. Controls for the presence of low levels of DMSO in observations, PD98059 had no signi®cant eect on PP2-containing media consisted of samples treated with identical HGF-stimulated DNA synthesis in 32D/c-Met cells at amounts of DMSO alone, as indicated. For both panels, values represent mean+standard deviation of triplicate samples; where concentrations up to 20 mM, despite complete blockade no standard deviation is visible, the value was too small to of MAP kinase activation con®rmed by immunoblot- indicate graphically. The results are representative of at least three ting with anti-active MAP kinase antibody (Figure 7a experiments

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2207 this kinase family at concentrations of 0.1 ± 10 mM, activation by IL-4R, and overexpression of STAT6 can whereas eects on other tyrosine kinase families such enable mitogenic signaling by IL-4 in this system (Patel as EGF receptor require at least 10-fold higher et al., 1996). To further explore the impact of STAT6 concentrations (Hanke et al., 1996). We found that on the mitogenic synergy between HGF and IL-4, HGF-stimulated DNA synthesis in 32D/c-Met cells DNA synthesis was compared among clonal 32D cell was only modestly inhibited by PP2 over a range of 1 ± lines expressing both human c-Met and STAT6 at 10 mM (Figure 7b). However, PP2 caused more various expression levels (Table 1). Even very high substantial inhibition of HGF+IL-4 stimulated DNA expression of STAT6, relative to endogenous levels, synthesis (Figure 7b). While the inhibition of combined was not associated with transformation, and all cell HGF+IL-4 mitogenic activity was partial, maximal lines remained IL-3-dependent for proliferation (data eects of 40 ± 50% at 10 mM PP2 were consistently not shown). Relative to 32D/c-Met cells, c-Met/STAT6 seen. These results, together with those obtained with cells expressed approximately twofold more STAT6 the MEK1 inhibitor PD98059, suggest that the HGF protein, c-Met/STAT6.1 cells expressed about 20-fold and IL-4 signaling pathways converge at multiple more STAT6 protein, and the STAT6 protein content levels. of c-Met/STAT6.2 was about 30-fold over endogenous STAT6 levels (data not shown). Tyrosine phosphorylation of STAT6 in c-Met/STAT6 cells was more HGF/IL-4 mitogenic synergy mediated by Jak3 and potently stimulated by IL-4, but phosphorylation in STAT6 response to HGF was not detected (Figure 8). More- In parallel with our analysis of HGF-activated path- over, HGF did not signi®cantly enhance IL-4-stimu- ways for biochemical evidence of HGF/IL-4 mitotic lated STAT6 phosphorylation in any of these cell lines synergy, we examined IL-4 activated pathways to (Figure 8 and data not shown). However, DNA characterize their potential roles in this phenomenon. synthesis stimulated by the combination of HGF and IL-4Ra activation can lead to the tyrosine phosphor- IL-4 was substantially increased in these cells relative ylation of Jak1, Jak3, insulin receptor substrate (IRS) - to the parental 32D/c-Met cell line (Table 1). Twofold 1, IRS-2, STAT6, and various other eector molecules overexpression of STAT6 protein was associated with a (Keegan et al., 1994; Witthuhn et al., 1994; Wang et modest but consistent increase in the HGF/IL-4 al., 1992, 1993). In 32D cells, which do not express IRS-1 or IRS-2, IL-4 is unable to stimulate the activation of PI3K and its downstream eectors, and lacks mitogenic activity (Figure 1b). However, IL-4 does stimulate the activation of Jak1, Jak3, and STAT6 in 32D cells (Patel et al., 1996). We examined the role of Jak1 activation in HGF/IL-4 mitogenic synergy by comparing ligand-stimulated DNA synthesis in 32D/c-Met cells with that observed in the same cells transfected with a dominant-negative, kinase dead (kd) Jak1 mutant. Although expression of the mutant Jak1 protein was well in excess of endogenous Jak1, no signi®cant eect on HGF/IL-4 mitogenic synergy was observed (data not shown). We examined the role of Jak3 in HGF/IL-4 mitogenic synergy using the same strategy outlined for Jak1. 32D/c-Met cells were transfected with a kinase dead mutant of human Jak3, and clonal cell lines expressing mutant Jak3 in excess of endogenous Jak3 were selected for further analysis. A comparison of ligand-stimulated STAT6 tyrosine phosphorylation in parental 32D/c-Met cells and a representative c-Met/ Jak3kd cell line revealed that it was severely reduced in Figure 8 Jak3 and STAT6 contribute to IL-4 enhanced HGF the presence of the dominant negative Jak3 mutant signaling. 32D/cMet cells (left four lanes), and two 32D/cMet- (Figure 8). Associated with the strong suppression of derived clones transfected with a Jak3 kinase-dead mutant (c- STAT6 tyrosine phosphorylation was the complete loss Met/Jak3kd; center four lanes), or human STAT6 (c-Met/STAT6; of mitogenic synergy between HGF and IL-4 in c-Met/ right four lanes) were treated with HGF (200 ng/ml), IL-4 (100 ng/ml), HGF+IL-4, or left untreated, as indicated above Jak3kd cells (Table 1). These results suggest that Jak3 each lane. Cells were lysed and equal amounts of protein were is more critical than Jak1 for HGF+IL-4 signaling in immunoprecipitated with anti-STAT6. STAT6 immunoprecipi- 32D/c-Met cells, and that IL-4 stimulated Jak3 tates were resolved by SDS ± PAGE and immunoblotted with activation is required for IL-4 enhancement of HGF anti-pY (top panel), or anti-STAT6 (bottom panel). Note that mitogenic signaling. because human STAT6 is not as well-recognized as murine STAT6 by the anti-STAT6 antibody used for immunoblotting, Tyrosine phosphorylation and activation of STAT the rightmost four lanes in the bottom panel underestimate the proteins is an important consequence of Jak kinase total STAT6 protein content of these samples

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2208 Table 1 HGF+IL-4-stimulated DNA synthesis by 32D/cMet cell carcinoma cells in a cell-matrix invasion assay lines (Uchiyama et al., 1996). In that study, IL-4 also Cell line DNA synthesisa inhibited HGF-stimulated expression of matrix metal- 32D/c-Met 162+8 loproteinases (MMP)-1, -2, and -9. Results from our c-Met/Jak3kd 80+9 own experiments (not shown), where HGF-stimulated c-Met/STAT6 200+17 cell migration was measured using a modi®ed Boyden c-Met/STAT6.1 340+11 chamber system (Day et al., 1999), indicated that IL-4 c-Met/STAT6.2 510+16 had no eect on HGF- or HGF/NK2-stimulated a[3H]-Thymidine incorporation into DNA was measured as described migration of 32D/c-Met cells. It is important to note in Materials and methods. 32D cells expressing human c-Met, kinase- that comparative analysis of HGF signaling in dead JAK3, or STAT6 at various expression levels were treated with epithelial and myeloid cells suggests that while some HGF (100 ng/ml) or HGF+IL-4 (100 ng/ml and 2 ng/ml, respec- intracellular eectors are common to both cell types, tively). Values shoen are mean+standard error of the mean of triplicate samples, and are expressed as a percentage of DNA certain events leading to HGF-stimulated mitogenesis synthesis stimulated by HGF treatment alone. Results are represen- and migration in these two cell types are distinct (Day tative of at least three experiments et al., 1999). Despite emerging evidence that modulation of HGF signaling may occur frequently in vivo, the intracellular mitogenic synergy: 162 vs 200%, relative to HGF mechanisms responsible remain to be elucidated. The treatment alone (Table 1). Higher levels of STAT6 results presented here suggest that IL-4 does not aect expression were associated with greater increases in HGF-stimulated c-Met autophosphorylation, Shc HGF/IL-4 synergy: 340 and 510% for c-Met STAT6.1 phosphorylation, or the overall level of PI3K activa- and c-Met/STAT6.2 cells, respectively, relative to HGF tion. IL-4 did enhance HGF-stimulated MAP kinase treatment alone (Table 1). These results con®rm that activation in 32D/c-Met cells, and consistent with this, STAT6 is an important contributor to the IL-4 the MEK1 inhibitor PD98059 partially blocked HGF/ signaling pathway, and suggest that STAT6 also IL-4 mitogenic synergy. In contrast, the mitogenic mediates, at least in part, the HGF/IL-4 mitogenic synergy between HGF and bFGF, dbcAMP, or MGF synergy observed in this system. reported earlier was not accompanied by synergistic or additive increases in MAP kinase activity (Rubin et al., 1993). Interestingly, we found that the MEK1 inhibitor Discussion PD98059 had little signi®cant eect on DNA synthesis stimulated by HGF alone, suggesting that in myeloid Various growth factors, cytokines and hormones acting cells such as 32D, MAP kinase activation might be in concert regulate complex physiological processes, mediated by other factors such as PKC, PLC-g and/or and one of the current challenges of cell biology and Src family kinases (Machide et al., 1998; van Dijk et physiology is understanding the integration of dierent al., 1997; Torigoe et al., 1992). In any event, the incoming signals by target cells. HGF is involved in substantial reduction in HGF-stimulated MAP kinase embryogenesis, organogenesis, homeostasis and onco- activation associated with Wortmannin treatment genesis, yet relatively little is known regarding the suggests that the mediators of HGF-stimulated MAP modulation of HGF-stimulated biological activities by kinase activation lie downstream of PI3K. other signaling molecules. HGF stimulates motility in To identify molecules downstream of PI3K that melanocytes, but HGF-stimulated mitogenesis in that might mediate the observed mitogenic synergy between system appears to require the presence of other factors, HGF and Il-4, we investigated the role of Src kinases such as basic ®broblast growth factor (bFGF), mast in the IL-4 and HGF mitogenic pathways, since both cell growth factor (MGF), or dibutyryl cAMP (Rubin factors are known to activate the Src kinase family et al., 1993). HGF in combination with other growth members. We found that the Src inhibitor PP2 blocked factors may support the growth and migration of approximately 50% of the HGF/IL-4-stimulated DNA primary melanoma cells (Rubin et al., 1993). HGF synthesis above that observed in response to HGF appears to cooperate with nerve growth factor to alone, suggesting that a portion of the synergy was the enhance axonal outgrowth from cultured dorsal root result of Src-related kinase activity. Hematopoietic ganglion neurons (Streit and Stern, 1997). Mitogenic stem cell lines are known to express four of the eight synergy has also been observed between HGF and known Src-family members; Lyn, Fyn, Yes and Hck insulin-like growth factor 1 (IGF-1) in cultured (Torigoe et al., 1992). Of these kinases, Fyn (but not keratinocytes and mammary epithelial cells (WG Lyn) has been shown to be activated by IL-4 in a Taylor and JS Rubin, personal communication). human Burkitt lymphoma B-cell line, DND39 (Ikizawa In addition to positive or cooperative interactions et al., 1994). Indeed, c-Met is known to associate with between HGF and other signaling pathways, evidence c-Src and Fyn (Bardelli et al., 1992). Studies are of negative modulation also exists. Activation of PKC underway to further de®ne the role of Src-kinase can lead to the phosphorylation of c-Met on serine activation by IL-4 and HGF in mitogenic signaling 985, and in turn, inhibition of the receptor's intrinsic using the 32D/c-Met model system. tyrosine kinase activity (Gandino et al., 1994). IL-4 Through the activation of its receptor, and in turn, blocked the HGF-stimulated migration of colon Jak kinases and STAT proteins, IL-4 can protect 32D

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2209 cells from apoptosis in a PI3K-independent manner HGF-enhancement of IL-4-stimulated STAT6 activa- (Wang et al., 1992, 1993; Zamorano et al., 1996). To tion, nor did we observe the activation of STAT6 or investigate the role of these PI3K-independent events STAT3 in 32D/c-Met cells within 20 min of HGF in the mitogenic synergy between HGF and IL-4, we treatment (Figure 8 and data not shown). Further compared ligand-stimulated DNA synthesis by par- studies are needed to determine whether HGF can ental 32D/c-Met cells with that of cells expressing stimulate de novo synthesis of STAT3 or STAT6 proteins dominant negative Jak mutants, and separately with that may, in turn, contribute to mitogenic signaling. cells overexpressing STAT6. We found that expression Our results demonstrate mitogenic synergy between of a kinase-dead Jak1 mutant well in excess of HGF and IL-4, two factors that are widely expressed endogenous Jak1 levels had no eect on the mitogenic throughout development and adulthood, each acting synergy between HGF and Il-4 (data not shown). In on a broad spectrum of target cell types, including contrast, similar relative expression levels of a mutant hematopoietic cells. Investigation of the mechanism by Jak3 protein completely blocked the ability of IL-4 to which this synergy occurs revealed pathway conver- synergize with HGF. The Jak3 mutant also strongly gence downstream of PI3K, which plays a central role suppressed IL-4-stimulated tyrosine phosphorylation of in HGF-stimulated mitogenesis in both epithelial and STAT6, providing a clear biochemical link to this hematopoietic cells, and upstream of MAP kinase intracellular eector. activation. We present evidence that Src-related kinases STAT6 activation is critical for IL-4-stimulated contribute to the observed synergy between HGF and mitogenicity, which occurs in myeloid cells and IL-4, but may not appear to be critical for DNA ®broblasts that express PI3K binding proteins such as synthesis stimulated by HGF alone in myeloid cells. IRS-1 and IRS-2 (Patel et al., 1996; Wang et al., 1993). Independent of PI3K, we demonstrate that IL-4- The absence of IRS family members in 32D cells, and stimulated activation of Jak3 and in turn, STAT-6, thus a physical link between PI3K and the IL-4 also contribute to the observed synergy between this receptor, is responsible for the failure of IL-4 to elicit cytokine and HGF. These results suggest that the HGF mitogenicity in this cell line (Wang et al., 1992, 1993; and IL-4 signaling pathways converge at multiple Zamorano et al., 1996). In 32D/c-Met cells HGF levels: at least once upstream of MAP kinase stimulates PI3K activation, and IL-4-stimulated activation, and further downstream at the level of cell STAT6 activation may provide a complementary cycle regulators under the transcriptional control of mitogenic signal that contributes to the observed STAT6. synergy between these two factors. In support of this hypothesis, overexpression of STAT6 in 32D/c-Met cell lines resulted in enhanced STAT6 tyrosine phosphorylation and HGF/IL-4 mitogenic synergy, relative to Materials and methods the parental cell line. The mechanism by which IL-4- stimulated STAT6 activation complements HGF Reagents, cell lines and cDNA transfections signaling is not yet known. In 32D cells, mitogenic Full-length HGF was produced as described (Cioce et al., synergy between IGF-1 and IL-4 mediated by the Jak/ 1996). The truncated HGF isoform HGF/NK2, which STAT6 pathway is associated with upregulation of c- contains the HGF N-terminal domain and ®rst two kringle myc expression (Soon et al., 1999). Others have shown domains, was produced as described (Stahl et al., 1997). that STAT proteins control lymphocyte proliferation HGF/NK2 is a potent motogen, but lacks the mitogenic and by downregulating the expression of p27Kip1, an morphogenic activities of full-length HGF (Stahl et al., 1997; inhibitor of cell cycle progression (Kaplan et al., Chan et al., 1991; Montesano et al., 1998). Wortmannin 1998). Clearly much more experimentation will be (Sigma Chemical Company; 11.6 mM stock), LY294002 required to identify the targets of STAT6 signaling that (Alexis; 20 mM stock), PD98059 (Calbiochem; 50 mM stock), contribute critically to the mitogenic synergy between and PP2 (Calbiochem; 17 mM stock) were dissolved in DMSO. The murine IL-3 dependent cell line 32D was HGF and IL-4. cultured in RPMI 1640, 15% fetal bovine serum (FBS), Recently, STAT3 was shown to be activated by HGF and 5% WEHI-3B conditioned medium as a source of IL-3. in human hepatocytes, hepatoma cells and MDCK 32D/c-Met cells co-expressing STAT6 or a Jak3 kinase-dead epithelial cells (Boccaccio et al., 1998; Schaper et al., (Jak3kd) mutant were generated by transfection of 32D/c- 1997). Blockade of HGF-stimulated STAT3 activation Met cells with STAT6 (Patel et al., 1996) or Jak3kd in epithelial cells resulted in the inhibition of tubulogen- (Witthuhn et al., 1994) cDNA plasmids as described (Pierce esis, but not HGF-stimulated cell motility or prolifera- et al., 1988). Stable cell lines were selected for expression of c- tion (60 Boccaccio et al., 1998). The time course of Met and Stat6 by immunoblotting lysates of neomycin- STAT3 DNA binding (5 ± 7 h after HGF stimulation) resistant clonal cell lines. suggests that STAT3 may be activated as a result of de novo synthesis of signaling molecules (Schaper et al., Mitogenicity assays 1997). In contrast, STAT6 activation by IL-4 and DNA synthesis was measured in 32D transfectants as PDGF, which is critical to the mitogenic synergy described (Pierce et al., 1988). Brie¯y, 32D transfectants between those factors observed in ®broblasts, occurs were incubated in RPMI, 15% FBS with or without growth within 20 min and is not inhibited by the addition of factors at 26105 cells/ml in 24-well plates for 36 h. [3H]- cycloheximide (Patel et al., 1996). We did not detect Thymidine (1 mCi/ml) was added for 3.5 h and cells were

Oncogene HGF and IL-4 mitogenic synergy RM Day et al 2210 collected on a ®lter using an automatic cell harvester MAP kinase assays (Skatron). [3H]-Thymidine incorporation was determined by scintillation counting. 32D transfectants were starved for 3 h in DMEM, treated with growth factors, and extracted in buered non-ionic detergent at 48C as described (Day et al., 1999). Lysates were Detection of apoptosis and cell cycle analysis immunoprecipitated with anti-ERK-1/2 and GammaBind 32D cell transfectants were incubated at 26105 cells/ml in Plus Sepharose for 2 h at 48C. The sepharose beads were DMEM, 15% FBS with growth factor(s) for 36 h. Cells were processed for kinase reactions as described (Day et al., 1999). washed twice in ice cold phosphate buered saline (PBS), and 32P-phosphorylated MBP peptide was determined by liquid ®xed by the addition of cold ethanol (80% in water). One scintillation counting. hour before FACS analysis, cells were pelleted and resuspended in 0.5 ml propidium iodide (PI) staining solution Phosphatidylinositol 3-kinase assays (0.05 mg/ml RNase A, 4.5% PI (w/v) in PBS). Cells were analysed using a FacScan and FacStation software (Becton Phosphatidylinositol 3-kinase (PI3K) assays were performed Dickinson). Calculations of cell populations were determined as described (Day et al., 1999; Wang et al., 1992). Cells were using ModFit LT Cell Cycle Analysis software (Verity starved 4 h in DMEM prior to treatment with growth Software House). factors. Cells were then washed, lysed, and lysates were immunoprecipitated 4 h at 48C with GammaBind Plus Sepharose and anti-PY. Immunoprecipitates were washed Immunoblot analysis three times and processed as described (Day et al., 1999). Cells were extracted in lysis buer and cleared by Kinase reaction products were subjected to thin layer centrifugation as described (Day et al., 1999). Equal amounts chromatography, and visualized by autoradiography. of protein were immunoprecipitated for 2 h at 48C with antiserum (1 : 100) and GammaBind Plus Sepharose (1 : 10) (Pharmacia Biotech). Proteins were separated by SDS ± PAGE, transferred to Immobilon-P (Millipore), blocked in PBS+5% BSA at 378C for 2 h, and probed with antibodies against human c-Met, ERK-1/2 (c-16; Santa Cruz Biotech- nology), phosphotyrosine (anti-PY; 4G10; Upstate Biotech- Acknowledgments nology), phospho-p44/42 MAP kinase (New England We thank Drs Jacalyn Pierce, Jerey Rubin, William Biolabs), Shc (Transduction Laboratories) or STAT6 (Santa Taylor, William LaRochelle, and Yuichiro Suzuki for Cruz Biotechnology). Immunoreactive bands were visualized helpful discussions. We also thank Nelson Ellmore for using enhanced chemiluminescence (Amersham Life Science). technical assistance.

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