Research Article
99
Dissection of HEF1-dependent functions in motility and transcriptional regulation Sarah J. Fashena, Margret B. Einarson, Geraldine M. O’Neill, Christos Patriotis and Erica A. Golemis* Fox Chase Cancer Center, 7701 Burholme Ave, Philadelphia, PA 19111, USA *Author for correspondence (e-mail:
[email protected])
Accepted 24 September 2001 Journal of Cell Science 115, 99-111 (2002) © The Company of Biologists Ltd
Summary Cas-family proteins have been implicated as signaling intermediaries in diverse processes including cellular attachment, motility, growth factor response, apoptosis and oncogenic transformation. The three defined Cas-family members (p130Cas, HEF1/Cas-L and Efs/Sin) are subject to multiple forms of regulation (including cell-cycle- and cell-attachment-mediated post-translational modification and cleavage) that complicate elucidation of the function of specific Cas proteins in defined biological processes. To explore the biological role of HEF1 further, we have developed a series of cell lines in which HEF1 production is regulated by an inducible promoter. In this system, HEF1 production rapidly induces changes in cellular morphology and motility, enhancing cell speed and haptotaxis towards
fibronectin in a process partially dependent on intact ERK and p38 MAPK signaling pathways. Finally, cDNA expression array analysis and subsequent studies indicate that HEF1 production increases levels of mRNA transcripts encoding proteins that are associated with motility, cell transformation and invasiveness, including several metalloproteinases, MLCK, p160ROCK and ErbB2. Upregulation of such proteins suggests mechanisms through which misregulation of HEF1 may be involved in cancer progression.
Introduction Integrin-dependent signaling contributes to cellular decisions to initiate diverse programs such as proliferation, apoptosis, differentiation and migration (Giancotti and Ruoslahti, 1999; Miyamoto et al., 1995; Ruoslahti and Reed, 1994; Sheetz et al., 1998). Hence, integrin signaling affects a variety of physiological processes including development, tissue remodeling, wound healing and tumor cell growth and metastasis (Petersen et al., 1998). Efforts to understand the mechanisms through which integrin signaling regulates these cellular processes have focused on analysis of focal adhesions. Focal adhesion sites consist of integrin receptors clustered following their engagement by extracellular ligand and an associated complex of intracellular proteins including actin filaments, actin-binding and -cross-linking proteins, and a number of tyrosine kinases, phosphatases and docking/adaptor proteins (Dedhar and Hannigan, 1996). In addition to providing a physical scaffold connecting cells to basement membranes, focal adhesions also act as signaling centers, which generate and convey information from the cell periphery to downstream effector molecules. Thus, focal adhesions integrate the mechanical signals derived from morphological changes with the chemical signals triggered by receptor engagement (Chen et al., 1997; Chicurel et al., 1998; Huang et al., 1998). The contributions of a number of kinases and docking/ adaptor proteins to the signaling capacity of focal adhesions have been elucidated. For example, focal adhesion kinase (FAK) and family members are tyrosine kinases that localize to focal adhesion sites, undergo autophosphorylation
following integrin receptor engagement and contribute to focal adhesion regulation (Schlaepfer and Hunter, 1998). Members of the Src family of tyrosine kinases localize with FAK and phosphorylate components of focal adhesions (Schlaepfer and Hunter, 1998). Significantly, modulation of FAK signaling affects both cell motility and the induction of apoptosis, suggesting that these cellular processes have components in common (Frisch et al., 1996; Hungerford et al., 1996; Ilic et al., 1995). Substrates of FAK and Src (Hanks and Polte, 1997), which include actin binding proteins such as paxillin and adaptor proteins such as Crk and the Cas (Crkassociated substrate) family of signaling proteins, have become the targets of scrutiny as FAK/Src effectors in these dual processes. The Cas family of adaptor proteins (O’Neill et al., 2000) includes p130Cas (Sakai et al., 1994), human enhancer of filamentation 1 (HEF1; also known as CasL) (Law et al., 1996; Minegishi et al., 1996) and Efs (also known as Sin) (Ishino et al., 1995; Alexandropoulos and Baltimore, 1996). Members of this family were initially identified as components of viral transformation signaling pathways (Ishino et al., 1995; Kanner et al., 1990; Sakai et al., 1994) and/or as modulators of cell growth and morphology (Law et al., 1996). Intriguingly, recent clinical studies have indicated that enhanced Cas family expression correlates with significant differences in cancer progression in humans, whereas induction of p130Cas overexpression enhances resistance to the action of antiestrogens (Brinkman et al., 2000; van der Flier et al., 2000). The Cas proteins have a conserved domain structure composed of an N-terminal SH3 domain, a substrate domain containing
Key words: HEF1, Cell Spreading, Migration, Apoptosis, Cas Family, cDNA Array
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multiple tyrosine motifs that are recognized by SH2 domain proteins following phosphorylation, a serine-rich region and a C-terminal dimerization motif (O’Neill et al., 2000). HEF1, p130Cas and Efs localize to focal adhesion sites via interaction of their SH3 domains with FAK (Law et al., 1996; Ohba et al., 1998; Polte and Hanks, 1995; Tachibana et al., 1997) and contribute to the assembly of signaling complexes downstream of the integrin receptor following ligand binding (O’Neill et al., 2000). An important question is whether the function of discrete Cas family members at focal complexes is equivalent or whether individual Cas proteins are associated with promotion of different biological effects. A number of studies characterizing HEF1 and p130Cas have underscored the potential for functional divergence between these Cas family proteins. HEF1 and p130Cas are differentially regulated; HEF1 is produced at maximal levels in cells of epithelial and lymphoid origin (Law et al., 1996; Law et al., 1998; Minegishi et al., 1996), whereas p130Cas is produced ubiquitously (Sakai et al., 1994). Moreover, HEF1 is also regulated in a cell cycle dependent manner and is processed at the G2/M boundary by caspases to truncated isoforms that localize to distinct subcellular compartments (Law et al., 1998). Most notably, we have recently found that HEF1 overproduction mediates apoptosis in epithelially derived cell lines, including MCF7 and HeLa cells (Law et al., 2000), which is contrary to the pro-survival activity described for p130Cas (Almeida et al., 2000; Cho and Klemke, 2000). Finally, recent studies in lymphoid cells showed that HEF1 (CasL) expression contributes to T cell migration induced by ligation of CD3 and β1 integrin (Ohashi et al., 1999; van Seventer et al., 2001). Taken in sum, these results demonstrate that HEF1 differs from p130Cas in both the manner in which it is regulated and its spectrum of effector function. We have begun to elucidate the mechanisms underlying HEF1-induced cellular responses by performing cDNA array analyses to identify downstream transcriptional targets that are upregulated as a consequence of HEF1 overproduction. Using a tetracycline-regulated HEF1-producing MCF7 cell line, we find a dramatic effect of HEF1 overproduction on cell morphology and motility, characterized by the development of a crescent shape, enhanced ruffling and increased cell spreading. HEF1-induced populations contain more highly motile cells and demonstrate increased haptotaxis towards fibronectin. Using DNA array analysis, we find that this enhanced motility is accompanied by upregulation of a set of genes associated with enhanced migration and invasion, including those encoding myosin light chain kinase (MLCK), p160ROCK, eight matrix metalloproteinases (MMPs) and ErbB2. Overall, these data suggest that the spectrum of biological effects attributable to HEF1 is complex and potentially includes promigratory and prometastatic activity.
Materials and Methods Expression plasmids The complete coding sequence of HEF1 was cloned in the pBPSTR1 retroviral vector (Paulus et al., 1996) downstream of a tetracycline responsive element to create pBPSTR1-HEF1. A second plasmid, pTet-tTAK, encodes the tTA protein, a fusion of the Tet repressor DNA binding domain and the transcriptional activation domain of VP16 (Gibco/BRL).
Cell culture To prepare stable, regulated clonal cell lines, MCF7 breast adenocarcinoma cells were transfected with pBPSTR1-HEF1, pTettTAK and MSCVhygroR (which provides a hygomycin resistance gene; kindly provided by J. Testa) using Lipofectamine (Gibco/BRL). Transfected cells were selected in media containing 2 µg ml–1 puromycin (to retain the tetracycline-regulated pBPSTR1HEF1), 400 µg ml–1 hygromycin and 1 µg ml–1 tetracycline (to repress HEF1 production during selection). Cell lines were derived from isolated single colonies, expanded and examined for inducible HEF1 production. Unless otherwise stated, experiments were carried out in DMEM plus 10% FBS. Induction of HEF1, cell lysis, immunoprecipitation and western analysis Cells were plated at low cell density (~600,000 cells per 100-mm culture dish) in the presence (uninduced) or absence (induced) of tetracycline for up to 24 hours prior to lysis, as noted in the figure legends. Adherent monolayers were washed twice with phosphate buffered saline and then lysed in Triton X-100 lysis buffer (50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 10 mM Na4P2O7) supplemented with 1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg ml–1 aprotinin and 1 µg ml–1 leupeptin. The protein concentration of total cell lysates was quantitated using a BCA protein determination kit (Pierce). Total cell lysate was separated by SDS-PAGE and transferred to polyvinyl difluoride membranes (Immobilon). Membranes were blocked using 5% fat-free milk, probed with rabbit polyclonal antisera specific for HEF1 [αHEF1-SB-R1 (Law et al., 1998)] or p130Cas [Transduction Labs; cross-reactive with HEF1, as noted by Law et al. (Law et al., 1998)] and developed using a chemiluminescent system (NEN). As indicated, membranes were also probed with the following antibodies, following the manufacturer’s protocols: mouse monoclonal antibody specific for ERK (MAPK; Transduction Laboratories, Lexington, KY) and rabbit polyclonal antisera specific for p38 kinase (Santa Cruz Biotechnology, Santa Cruz, CA) and activated forms of MAPK and p38 kinase (Promega Corporation, Madison, WI). For p130Cas immunoprecipitation, cells were harvested in PTY buffer (O’Neill and Golemis, 2001) at the indicated time points and immunoprecipitated with antibody to p130Cas using ProteinG/Sepharose (Gibco/BRL). Precipitates were subjected to SDS-PAGE and transfer (see above), and tyrosine phosphorylation was assessed using primary antibody 4G10 (Upstate Biotechnology) and secondary antibody and development as described above, except using bovine serum albumin (BSA) as blocking agent. Subsequently, blots were stripped and reprobed with antibody to p130Cas or HEF1, as described above. Cell spreading analysis Cells were initially plated at ~60% confluence in the presence or absence of tetracycline for 18 hours. Cells were then detached by incubation in PBS + 5mM EDTA for 15 minutes at 37°C, re-plated onto either uncoated glass coverslips in Dulbecco’s modified Eagle’s medium (DMEM) plus fetal bovine serum (FBS) or human fibronectin (FN) (Gibco/BRL) coated coverslips (6 µg ml–1) in serum-free DMEM and maintained in either inducing or non-inducing conditions for the indicated times prior to fixation in 3.5% paraformaldehyde. In each field, cell area measurements were determined using Inovision ISEE software to outline the perimeters of individual cells and to calculate the number of pixels encompassed. Immunofluorescence detection Cells cultured on coverslips were fixed in 3.5% paraformaldehyde,
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Fig. 1. Generation of MCF7 stable cell lines in which HEF1 expression is regulated by tetracycline. (A) MCF7 cells were transfected with a plasmid encoding the tTA transactivator and a tetracycline-regulatable expression plasmid either without (CM1,2) or with a HEF1 cDNA insert (HEF1.M1-M5). Total cell lysates (35 µg) derived from uninduced (+ lanes) or induced/mock induced (− lanes) clones were processed by immunoblotting with αHEF1-SBR1 antisera to assess HEF1 production levels. The p105 and p115 proteins are differently phosphorylated forms of HEF1 (Law et al., 1998). (B) Lysates (30 µg) were isolated from HEF1.M1 cells induced for the indicated time intervals (in hours, labeled above each lane). Negative controls include lysates isolated after 24 hours from uninduced HEF1.M1 (lane 1) or mock induced CM1 cells (lane 9), probed with antibody αHEF1-SB-R1, which is specific for HEF1. (C) Lysates from CM.1, HEF1.M1 and HEF1.M2 cells induced for the indicated time intervals (in hours) or maintained in tetracycline (+T) were immunoblotted with antibody to p130Cas (bottom). Corresponding levels of HEF1 at the same time points are also shown (top). (D) Cell lysates from CM1, HEF1.M1 or HEF1.M2 cells prepared at 0, 9 or 24 hours after induction, or in cells maintained in tetracycline (+T) were immunoprecipitated with antibody to p130Cas and then probed with antibody to phosphotyrosine (top), stripped and reprobed with antibody to p130Cas (bottom).
permeabilized with 0.2% Tween-20 and blocked with 0.1% BSA in Tris buffer (10 mM Tris (pH 7.5), 150 mM NaCl). Cells were incubated with anti-HEF1 rabbit antisera (αHEF1-SB-R2) (Law et al., 1998) or anti-paxillin mouse monoclonal antibodies (Transduction Labs) as primary and either rhodamine-conjugated anti-rabbit antibodies (Molecular Probes), biotin-conjugated anti-rabbit antibodies plus Texas-Red-conjugated streptavidin (Vector Laboratories) or dichlorotriazinylaminofluorescin (DTAF)-conjugated anti-mouse antibodies (Jackson Immunological Labs) as secondary antibody. FITC- or TRITC-conjugated phalloidin (Molecular Probes) was included in a final incubation to visualize actin. A Bio-Rad MRC 600 laser scanning confocal microscope (Cell Imaging Facility, Fox Chase Cancer Center) was used to analyze images. Motility assays For measurements of haptotaxis, 10,000 cells per 35 mm-well were plated onto the porous membrane (top well) of a modified Boyden chamber (tissue-culture treated, 8-µm pores, Transwell; Costar, Cambridge, MA). Both top and bottom of the Boyden chamber contained DMEM with or without tetracycline. Soluble human plasma FN (Gibco/BRL) was added (4 µg ml–1, as indicated) to the bottom wells just before cell plating to coat the underside of the porous membrane. Cells on the upper side of the membrane were removed by scraping. Cells attached to the bottom membrane were fixed and stained with modified Giemsa stain. For measurements of haptotaxis in the presence of pharmacological inhibitors, the above procedure was scaled down. Briefly, 2000 cells per well (24-well Transwell plates, 8-µm pores) were added to the top chamber of a modified Boyden chamber in the absence or presence of the following compounds: 25 µM PD98059 (Sigma), 25 µM SB202190 (Sigma) and
DMSO (in which PD98059 and SB202190 were dissolved) as control. Migratory cells in five to ten randomly selected fields (10× objective) per condition were counted. For the speed analysis, cell lines were plated at low cell density in DMEM plus 10% FBS with or without tetracycline for 4-6 hours prior to the start of time-lapse video microscopy imaging. Phase contrast images were recorded at 5-minute intervals for calculation of cell speed for 18-24 hours. Cells were tracked for 70 intervals using Isee and Nanotrack imaging software, and the results were analyzed using Excel. Atlas array analysis and RT-PCR confirmation Total RNA was purified from HEF1.M1 and CM1 cells that were uninduced or induced for 9 hours, treated with RNase-free DNase I (Gibco/BRL) and used to synthesize 33P-labeled cDNA probes using the protocols and cDNA-probe synthesis kit provided with the Clontech Atlas 1.2 Human Cancer gene arrays. Each of the obtained cDNA probes were hybridized in parallel to an Atlas 1.2 Human Cancer array filter for ~14 hours, washed and exposed to BioMax film with an LE transcreen (Kodak) for 24-72 hours, according to the manufacturer’s instructions. The obtained autoradiographic array images were scanned at 16 bits per pixel and 1200 dpi (25 µm) resolution, exported as 8-bit bitmap files and images processed using the Arrayexplorer software (Patriotis et al., 2001) and further analysed in Excel. Each gene array data set was normalized on the basis of the expression values of nine housekeeping genes included in the array (details available in Clontech Atlas array manual). Array data sets were subjected to pair-wise correlation analysis to establish reproducibility between experiments. The ratios between the gene intensities were calculated for each pair of data sets and the genes
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series of stable MCF7 clonal cell lines that expressed a tetracycline-regulatable HEF1 transgene or carried the parental vector were generated and characterized. Immunoblots of total cell lysates derived from representative HEF1-inducible clones (HEF1.M1-M.5) probed with HEF1-specific antibodies (αHEF1-SB-R1) showed that transgene expression was tightly regulated, as demonstrated by the appearance of full length HEF1 protein following removal of tetracycline (Fig. 1A). By contrast, the parental vector clones CM1 and CM2 demonstrated no change in HEF1 production levels in the presence or absence of tetracycline (Fig. 1A). To enhance visualization of tetracycline-induced HEF1 expression, the western blot shown was exposed relatively briefly. For this reason, endogenous HEF1 is not well visualized in this experiment. To determine the kinetics of HEF1 production, total cell lysates were isolated from the HEF1.M1 clone at the indicated time intervals following induction upon tetracycline removal and analyzed on immunoblots probed with αHEF1-SB-R1 antibodies to evaluate HEF1 protein levels (Fig. 1B). By six hours post-induction, HEF1 levels were significantly enhanced in induced HEF1.M1 lysates relative to those of uninduced HEF1.M1 lysates. Maximal HEF1 production was achieved by 9 hours of induction and maintained over the course of an induction spanning 24 hours. By contrast, Fig. 2. HEF1 induces a morphological conversion to crescent-shaped lysates derived from the uninduced HEF1.M1 clone and cells. HEF1.M1 (A,B), HEF1.M2 (C,D) and CM1 (E,F) cells were either the mock induced CM1 clone produced low levels of uninduced (A,C,E) or induced/mock induced (B,D,F) for HEF1 HEF1 protein, reflecting the expression of the production. Phase contrast CCD images were acquired with a 40× endogenous protein, at all time intervals examined (Fig. objective. Bar, ~25 µm. 1B, lanes 1 and 9, respectively). Because HEF1 is closely related in sequence to p130Cas and enhanced levels of HEF1 may compete with undergoing significant change in expression (greater than twofold) were identified. p130Cas for shared interactive partners, characterizing the For reverse-transcription PCR (RT-PCR), total RNA was isolated status of p130Cas in the context of HEF1 induction constituted from HEF1.M1 cell populations that were either uninduced or induced an important control. First, in the control CM1 cells and in to express HEF1 for 9 hours and DNase treated as described above. HEF1.M1 and HEF1.M2 cells, we have analyzed p130Cas Following the protocol outlined in the Advantage RT-for PCR Kit levels in cells induced for 0-24 hours or left uninduced. Levels (Clontech) with minor modifications, cDNA was generated from these of p130Cas remain constant throughout the experiment, samples and normalized using quantitative competitive template whereas levels of HEF1 increase in the HEF1.M1 and (QCT) RT-PCR with primers specific for actin using the Gene Express HEF1.M2 cell lines (Fig. 1C). Second, although the similar System 1A. Parallel PCRs were performed on a panel of dilutions of migration of p130Cas species in samples prepared at different the two cDNA samples that were spiked with a constant amount of time points suggested that phosphorylation of p130Cas was not actin competitive template (CT). Following normalization for actin template levels, specific PCR analyses were performed using primer affected by HEF1 induction, we tested this point directly. The pairs specific for MLCK, p160ROCK, MDA7 and disintegrin/ p130Cas and HEF1 proteins were immunoprecipitated from metalloprotease. For direct analyses of proteins nominated by mRNA CM1, HEF1.M1 or HEF1.M2 cells that were induced for 0, 9 analysis, lysates were generated from parallel populations of or 24 hours or left uninduced. Tyrosine phosphorylation was HEF1.M1 cells (uninduced or induced for ~20 hours) and analyzed assessed using antibody to phosphotyrosine; blots were by immunoblotting using mouse monoclonal antibodies for human cstripped and reprobed to compare phosphotyrosine levels to ErbB2 (NeoMarkers, Union City, CA), MMP1 and MMP14 levels of immunoprecipitated p130Cas or HEF1. As shown, (Chemicon International, Temecula, CA). levels of phosphotyrosine were constant for p130Cas (Fig. 1D), whereas robust tyrosine phosphorylation of induced HEF1 was Results observed (results not shown). Establishment of cell lines that inducibly produce HEF1 To examine the consequences of HEF1 overproduction, we HEF1 production induces crescent morphology and cell created stable cell lines derived from MCF7 breast spreading adenocarcinoma cells. As endogenous HEF1 is produced at We previously demonstrated that HEF1 overproduction significant levels in MCF7 cells and many cell lines of mediates apoptosis in epithelial cells (Law et al., 2000), epithelial origin (Law et al., 1998), it appeared likely that whereas separate reports have shown that modulating HEF1 HEF1 protein partners would be present in this context. A
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Fig. 3. HEF1 localizes to prominent focal adhesion sites. Immunofluorescent staining of either uninduced (A-C) or induced (D-I) HEF1.M1 cells was performed using antisera specific for HEF1 (A,D,G) and antibodies specific for paxillin (B,E) or phalloidin-stained Factin (H). Merged images (C,F,I) demonstrate the pronounced co-localization of HEF1 and paxillin to the prominent focal adhesion sites in the leading edge lamellipodia (F), with HEF1 present at the distal ends of F-actin-rich stress fibers (I, arrowheads). Images depict 1.4 µm sections, acquired using a Bio-Rad MRC 600 confocal microscope (60× objective). Bar, ~25 µm.
levels can contribute to T-cell migration (Ohashi et al., 1999; van Seventer et al., 2001). To address the question of whether HEF1 also regulates epithelial cell shape and motility or whether cell-type-specific differences influence the spectrum of HEF1 activities, we characterized the MCF7-based cell lines for HEF1-dependent changes in cell morphology, substratedependent adhesion and movement. As prolonged overproduction of HEF1 induces apoptosis in MCF7 cells (Law et al., 2000), we focused on the first 24 hours after tetracycline removal for this analysis. Morphological changes consequent on HEF1 production were evident within 4-6 hours of induction, concomitant with the increase in HEF1 protein levels (Fig. 1B; Fig. 2). Phase contrast microscopy of HEF1.M1 and HEF1.M2 following 18 hours of HEF1 induction revealed that the cells had undergone dramatic morphological changes. These were typified by the appearance of crescent-shaped cells with large leading edge lamellipodia, enhanced ruffling and a pronounced trailing edge (Fig. 2, compare A to B and C to D), similar to the morphology typifying highly motile cells (Cooper and Schliwa, 1986). Analysis of time-lapse video microscopy images taken from six experiments indicated that between 47% and 75% of the examined population of cells were crescents by 18-20 hours post-induction (data not shown). By contrast, crescent-shaped cells were not detected in uninduced HEF1 cells (Fig. 2A,C) and uninduced or mock induced CM1 cells (Fig. 2E,F). Qualitatively similar morphological changes were observed with other inducible HEF1 lines (results not shown). In addition, we performed a titration of tetracycline removal for the HEF1.M1 and HEF1.M2 lines, comparing samples
incubated with 1 µg ml–1, 0.5 µg ml–1, 0.25 µg ml–1 and 0 µg ml–1 for HEF1 induction and morphological phenotype. Graded changes in HEF1-dependent phenotypes were observed, with reduced phenotypes at 0.25 µg ml–1 versus 0 µg ml–1 and marginally detectable phenotypic differences at 0.5 µg ml–1. HEF1 and other Cas family proteins localize to focal adhesions in adherent cells (Law et al., 1996; O’Neill et al., 2000). Immunofluorescence analysis with antibody to HEF1 (Fig. 3A,D,G) and the focal adhesion proteins paxillin (Fig. 3B,E) and FAK (not shown) indicated that HEF1 resided predominantly at focal adhesions in the induced HEF1.M1 cell line and was not mislocalized owing to overproduction. Most focal adhesions observed were found in the leading edge lamellipodia (Fig. 3D-F). Visualization of HEF1 (Fig. 3G) and the actin cytoskeleton (Fig. 3H) revealed that HEF1 was concentrated at the distal ends of actin stress fibers, coinciding with focal adhesion sites (Fig. 3I, arrowheads). HEF1producing cells also exhibited reorganization of the actin cytoskeleton, with actin bundles arranged radially at the lamellipodial front and in stress fibers radiating out from the perinuclear region to the leading edge (Fig. 3H,I). Based on observed paxillin and FAK staining, focal adhesions were more prominent in HEF1-producing cells than in uninduced HEF1 lines (Fig. 3, compare B and E) or in either uninduced or mock induced vector control cell lines (data not shown). Together, these results suggest that enhanced levels of HEF1 might contribute to the recruitment of other focal adhesion component proteins. Finally, to evaluate the contribution of increased HEF1 production to cell spreading, HEF1.M1 cells
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FBS FN
CELL AREA (# of pixels)
35000
N=95
30000 25000
N=123
20000
N=161
15000
N=177
N=166
-
+
N=179
10000
N=196
N=189
-
+
5000 0
CM1
-
+
HEF1.M1
CM1
-
+
HEF1.M1
Fig. 4. HEF1 production increases cell spreading. HEF1.M1 cells were maintained for 18 hours in either non-inducing (A,C,E,G,I) or inducing (B,D,F,H,J) conditions and then replated on glass coverslips coated with 6 µg ml–1 human FN (1.25 µg cm–2). Cells were fixed at 30 minutes (A,B), 1 hour (C,D), 2 hours (E,F), 3 hours (G,H) and 6 hours (I,J) after plating. Phase contrast CCD images were acquired with a 40× objective. Cells were maintained in inducing or non-inducing conditions for the duration of the experiment. The graphs show the quantitation of increased cell spreading. HEF1.M1 or CM1 cells maintained for 18 hours in either non-inducing (–) or inducing (+) condition were replated for 6 hours on glass coverslips either uncoated in the presence of 10% FBS (gray) or coated with human FN in serum-free medium (black). CCD images were acquired with a 40× objective (8-10 fields per condition) and the area determined using Inovision ISEE. Results shown are the means of three independent experiments±standard error.
and control cells were induced for 18 hours to facilitate the production of high levels of HEF1 and then replated and allowed to spread on FN-coated coverslips in serum-free media under non-inducing or inducing conditions (Fig. 4). HEF1producing cells were more highly spread at earlier time points (Fig. 4, compare E and F, 2 hours after plating) and maintained an enhanced degree of spread relative to uninduced or control cells at 6 hours post-plating (Fig. 4, compare I and J). Cell spreading and focal complex assembly depend on contributions from both serum factors and extracellular matrix (ECM) components that engage integrin receptors (Clark et al.,
1998; Hotchin and Hall, 1995). To evaluate the contribution of serum factors and integrin engagement to HEF1-enhanced cell spreading, HEF1.M1 cells and controls were induced for 18 hours and then replated for an additional 6 hours with induction on uncoated coverslips in media containing serum or on FNcoated coverslips in serum-free media. Quantification of mean cell area for induced and uninduced populations revealed that HEF1 production resulted in a 2.2-times (uncoated coverslips) or a 1.75-times (FN-coated coverslips) increase in cell area (Fig. 4, right panel). Analysis of controls demonstrated that the mean cell area of control uninduced HEF1.M1 cells was
HEF1-dependent motility and transcription
A
50
Migrating Cells (%)
40
Uninduced
30
Induced
20
Fold Induction Migration (4 µg/ml FN)
A
105
45 40 35 30 25 20 15 10 5 0
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+
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B
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Fig. 5. HEF1 production enhances cell motility by increasing cell speed. The average speed of uninduced or induced HEF1.M1 (A) or CM1 (B) cells was determined by analyzing the movement of individual cells at 5-minute intervals over the course of ~6 hours using Inovision ISEE nanotracking software. (A) The average speed of uninduced (gray) or induced (black) HEF1.M1 cells, grouped into speed ranges. (B) The average speed of uninduced (gray) or mock induced (black) CM1 cells, grouped into speed ranges; the speed of CM1 cells was not altered by mock induction. These data represent the average speed of HEF1.M1 cells (uninduced, N=59; induced, N=55) and CM1 cells (uninduced, N=76; induced, N=60) derived from two independent experiments.
similar to that of uninduced or mock induced CM1 cells. These results indicate that FN is sufficient to enable HEF1-dependent spreading in the absence of any other serum factors. HEF1 production enhances cell motility The HEF1-dependent shape changes described above result in a morphology typical of highly motile cells. To examine the effect of HEF1 production on cellular motility directly, we followed two approaches. First, we measured the speed of movement of HEF1-induced versus control cells (Fig. 5). To this end, phase contrast video images of uninduced and
FN
/D
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/S B2
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Experimental conditions
Fig. 6. HEF1 production augments FN mediated haptotaxis. (A) Increase in the number of cells that traversed the membrane in a Boyden chamber assay in response to soluble FN, as opposed to the absence of stimulus. HEF1.M1 (black) or CM1 (gray) cells were seeded into the top well of Boyden chambers and assessed for their ability to migrate towards FN in either non-inducing or inducing (for 20 hours) conditions. The haptotactic response of populations maintained in either non-inducing (–) or inducing (+) conditions were grouped separately and normalized against the number of cells that traversed the membrane for each condition in the absence of stimulus. Results shown are the mean of multiple independent experiments for each cell line±standard error. (B) Induced lysates were probed with anti-phosphorylated-MAPK and antiphosphorylated-p38 antibodies (top), and in parallel with anti-MAPK (lanes 1,2) and anti-p38 (lanes 3,4) (bottom) (C) Inhibition of HEF1.M1 cell haptotaxis toward FN in a Boyden chamber assay (as described above) following treatment with drug inhibitors for MAPK kinase (PD98059, 25 µM), p38 (SB202190, 25 µM) and control (DMSO).
induced cell lines were recorded at 5-minute intervals using a CCD camera, compiled and analyzed to determine cell speed. The average speed of HEF1-producing HEF1.M1 cells (3.33 nm second–1±0.16 standard error) reflected a 26% increase over that of uninduced cells (2.65 nm second–1±0.11 standard error). By contrast, parallel analyses of the CM1 clone
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Table 1. Transcriptional regulation of 29 genes in response to HEF1 induction
A
80000
Mock induced
70000
Induction with vector‡
+ (NTH) 2.3× 2.7× + + (NTH) + (NTH)
NS 1× 1× 0.5× 0.02× 0.1×
+ 4× 1.8× 4× + 2.5× + +
NS 0.8× 1× 1.4× NS 1.1× + (NTH) NS
Extracellular matrix components FN precursor (X02761) Collagen 11 Collagen 4 precursor Cadherin 11 (L34056) Cadherin 4 (L34059) Desmocollin Heparin sulfate proteoglycan (M85289)
2.7× 2.4× + (NTH) + 2.2× 2.1× + (NTH)
NS
