Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells
- Salman Rahman†1Email author,
- Yatin Patel†1,
- Jacqueline Murray2,
- Kirti V Patel1,
- Rushika Sumathipala1,
- Michael Sobel2 and
- Errol S Wijelath†2
© Rahman et al; licensee BioMed Central Ltd. 2005
Received: 09 August 2004
Accepted: 17 February 2005
Published: 17 February 2005
The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes.
Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1.
These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease.
The generation and repair of blood vessels in adult life requires the regulation of endothelial cell survival, migration, proliferation and their differentiation from lineage-committed progenitors by the coordinated action of several classes of vaso-active agents including growth factors, cytokines, and the extracellular matrix (ECM) [1–4]. Elucidating the molecular mediators of these signals and their mechanism of action is vital to understanding the fine regulation of neo-vessel development and maintenance.
There is growing evidence pointing to a close collaboration between growth factors and the ECM in several biological processes including vasculogenesis and post-natal revascularization. Studies have shown that the response of cells to growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are potentiated by integrin ligation to specific ECM glycoproteins [5–8]. In a previous report, we showed that VEGF-induced endothelial cell migration was augmented by fibronectin (FN) . We also presented evidence that the VEGF/VEGFR-2 pathway is coupled to the integrin α5β1 through a mechanism involving the promotion of an integrin α5β1-VEGFR-2 signalling moiety generated as a consequence of receptor ligation by a VEGF-FN complex. These events promoted the sustained activity of Erk kinase, which was coupled to the migratory response. More recently, we presented data demonstrating that FN significantly enhanced VEGF-mediated migration of CD34+ cells and their differentiation into endothelial cells . In addition to the VEGF pathway, in vitro studies have highlighted the importance of hepatocyte growth factor (HGF) as a pro-angiogenic mediator. HGF, also termed scatter factor, has a well-established role in tumourogenesis but may be an important mediator of neovascularization since studies show that HGF induces the expression of VEGF in endothelial cells in vitro and that HGF synergises with VEGF to promote capillary-tube assembly in collagen matrices [11, 12]. In addition, neovascularization in the rat cornea was also elevated by co-administration of HGF and VEGF compared to either growth factor in isolation . The emerging significance of HGF as a pro-angiogenic mediator was further highlighted by a recent study of a large cohort of patients (1090 patients, CAPTURE trial) with acute coronary syndromes and identified serum levels of HGF as a positive indicator of patients' prognosis associated with a significantly lower event rate and increased collateralization of the target vessel . Although the pro-angiogenic effects of HGF are known, the detailed mechanism of HGF action on the vascular cells, including the identity of intracellular mediators remains poorly understood.
In the present work we show that HGF forms a specific physical complex with FN and VN and that these complexes are present in degranulated platelet suspensions implicating a putative role in vivo. Significantly, we show that HGF-FN and HGF-VN molecular complexes induce a unique and enhanced intracellular signal employing Ras, thereby highlighting an important mechanism of growth factor receptor tyrosine kinase and integrin cooperation in promoting pro-angiogenic responses.
Identification of novel HGF binding domains on FN and VN
Platelets release HGF complexed to FN and VN
To establish whether HGF-FN and HGF-VN molecular complexes occur in vivo we examined platelets, a rich source of growth factors, for the presence of these complexes. Washed human platelet suspensions were stimulated with thrombin (1 U/ml) to promote degranulation and the derived supernatants were immunoprecipitated with antibodies directed to FN or VN. The resulting immune complexes were analysed for co-precipitation of HGF (Fig. 1E &1F). Immunoprecipitation of FN from thrombin-stimulated platelet supernatants resulted in significant co-precipitation of HGF (Fig. 1E). In contrast, minimal levels of HGF was observed in samples derived from unstimulated platelet supernatants or from samples derived from thrombin-stimulated platelet supernatants when an isotype-matched control antibody was employed in the experiment. Probing of the same blot with antibodies to FN confirmed that the primary precipitation of FN was responsible for the co-precipitation of HGF (Fig. 1E, lower panel). In a parallel experiment, immunoprecipitation of VN also co-precipitated HGF to a similar if not greater extent than FN (Fig. 1F). These experiments demonstrate that HGF is released from platelets and is found in the form of soluble molecular complexes with both FN and VN, confirming the results of the ligand binding studies in vitro.
HGF-Induced endothelial cell migration is dependent upon co-stimulation with ECM
To further characterize the degree and identity of integrin involvement in the observed migratory response, we investigated the consequences of blocking integrin receptors on HMVEC with specific integrin antibodies prior to HGF-ECM stimulation. Antibodies directed to the integrin α5β1 completely inhibited HGF-FN-induced endothelial migration (Fig. 2C). In contrast, an antibody with specificity for the αv-subunit (LM142) had no inhibitory effect on endothelial cell migration. However, antibodies to the αvβ3 integrin (LM609) did inhibit endothelial cell migration to HGF-FN by 20% suggesting an ancillary role for this integrin in mediating HGF-FN responses. When endothelial cell migration was induced by HGF-VN complexes, the integrin dependence shifted as expected (Fig. 2D). Under these conditions endothelial cell migration was predominantly dependent on αv-integrins for mediating the migratory signal with some apparent involvement of the integrin α5β1 (approximately 30%). This latter effect may be a consequence of integrin signal cross-talk (transdominant integrin regulation), as reported previously [14, 15]. These experiments demonstrate that for HMVEC, HGF induced cell migration is dependent upon the ligation of integrins by ECM molecules.
Met associates with αvβ3 and α5β1 integrins
To elucidate the role of Met activation in the formation of the Met-integrin signalling complex, endothelial cells were treated with HGF in the absence of ECM glycoprotein and with HGF-FN and HGF-VN complexes and the kinetics of Met tyrosine phosphorylation investigated (Fig. 3B). These experiments demonstrated that HGF in the absence of ECM glycoprotein could activate Met transiently with a strong signal present at 15 min but absent at 1 hour. In contrast, cells stimulated with HGF-FN and HGF-VN showed strong activation of Met at 15 min, which was sustained at 1 hour and was evident, although reduced, at 2 hours post-stimulation. Cell lysates derived from samples stimulated for 15 mins were also assessed for the presence of a Met-integrin complexes. As shown in Fig. 3C, HGF in the absence of FN or VN did not promote a significant association of Met with the integrins α5β1 or αvβ3. However, cells treated with HGF-VN and HGF-FN for 15 min contained significant levels of Met in a physical association with these integrins. These studies show that Met activation by HGF is insufficient to promote a physical association with integrins.
HGF binding domains on FN and VN promote enhanced intracellular signals
HGF/ECM-Induced endothelial migration is coupled to the PI-3 kinase pathway
To determine the intracellular pathway(s) that were coupled to the migratory response, HMVECs were treated with specific inhibitors of MEK and PI-kinase. In cell migration assays, LY294002 but not U1026 inhibited endothelial cell migration induced by HGF-FN (Fig. 4 C) and HGF-VN (Fig. 4 D), clearly demonstrating that the PI-3 kinase pathway was predominantly coupled to the migratory response and not the Map kinase pathway. Other inhibitors of potential down stream effectors were also tested. HGF-FN stimulated cells pre-treated with PP1, U73122, and piceatannol showed maximal migratory responses indicating that Src, PLCβ and Syk were not components of the migratory signal (data not shown).
HGF-Induced endothelial proliferation is coupled to the Erk-pathway
Ras is a specific, upstream regulator of Erk and PI-3 kinase pathways in cells stimulated with HGF-FN and HGF-VN complexes
The model in Fig. 9C is supported by the following observations. The enhanced responses of HMVEC to HGF-FN and HGF-VN complexes is consistent with the observation that in these cells the activity of Ras, PI-3 kinase (AKT phosphorylation) and Erk 1/2 phosphorylation were sustained, and in the case of Ras and Akt, were 2–3 fold higher than observed in cells stimulated with HGF and collagen-1 (Fig. 6). The distinct signalling mechanisms induced by the co-activation of endothelial cells with HGF in the presence of a binding and non-binding ECM glycoprotein partner was also supported by the observation that treatment of cells with the inhibitor of Ras farnesylation, FPT III, reduced the phosphorylation of both Erk 1/2 and Akt kinases in cells stimulated with HGF-FN but not HGF plus collagen-1 (Fig. 8A &8B). Furthermore, Ras co-precipitated with the integrin α5β1 derived from endothelial cell lysates stimulated with HGF-FN complexes but not with the integrin α2β1 derived from cells stimulated with HGF and collagen-1 (Fig. 8C). These results are consistent with the pioneering work by Rodriquez-Viciana and colleagues who demonstrated the regulation of p85 PI-3 kinase by Ras via direct molecular interaction. It is now known that the regulatory subunit of all type 1, PI-3 kinases contain a Ras binding domain that associates with activated Ras (GTP-Ras) [17–20]. Therefore, our data and model demonstrating the sustained activation of Ras and PI-3 kinase by stimulation of endothelial cells with HGF-FN and HGF-VN complexes is consistent with previous work showing Ras to be a key regulator of PI-3 kinase. The identity of the Ras subtypes mediating the regulation of PI-3 kinase in our cell system is currently under investigation.
The results of the present study both support and extend our previous observations of the enhanced endothelial cell migration induced by VEGF-FN molecular complexes . In that study, VEGF binding domains identified on FN drove the formation of VEGF-FN complexes that upon receptor ligation promoted the association of the integrin α5β1 with VEGFR-2. This co-receptor activation gave rise to a sustained activation of the Erk kinase activity, which promoted an enhanced migratory response. Similarly, the present work has shown that HGF-FN and HGF-VN molecular complexes induce the formation of Met-integrin signalling complexes promoting the transduction of a unique Ras-dependent signal. Several studies have illustrated the significance of the cooperation between integrins and growth factor receptor tyrosine kinases in mediating cellular responses. For example, the proliferation and migration of fibroblasts in response to PDGF-BB was enhanced in the presence of VN and was accompanied by the physical association of the αvβ3 integrin with the PDGF-β receptor [5, 7]. Furthermore, it was recently demonstrated that HGF in combination with FN prolongs the survival of GM-colony-forming cells  and enhanced the adhesion and motility of MTLn3 breast carcinoma cells . In addition, integrins αvβ3 and αvβ5 were shown to be necessary for mediating FGF-2 and VEGF mediated angiogenesis respectively by the differential regulation of components of the Erk kinase pathway . However, the present study extends these observations and is, to our knowledge, the first description of a distinct signalling pathway employed by the activity of growth factor-ECM molecular complexes as opposed to growth factors and ECM proteins functioning independently through ligation of their respective receptors. The identification of a Ras-dependent pathway in endothelial cells specifically activated with HGF-FN and HGF-VN complexes as opposed to HGF in the presence of collagen-1 is significant and correlates with Met-integrin association. Although the precise nature of the interaction between the Met tyrosine kinase and integrins was not elucidated, the role of Ras in this system appears important for the sustained and enhanced activation of the PI-3 kinase and Erk kinase pathways.
In contrast to the migratory signals promoted by VEGF-FN molecular complexes , HGF-FN and HGF-VN complexes induce a response in endothelial cells characterized by a tight coupling of the PI-3 kinase pathway to cell migration. Several additional pro-angiogenic mediators such as sphingosine 1-phosphate and NO, or the activation of CD40 and Eph B4 receptors by their counter ligands, promote endothelial cell migration through activation of the PI-3 kinase pathway [24–28]. In addition, HGF on its own was shown to stimulate smooth muscle cell migration in a PI-3 kinase dependent manner . However, the lack of a significant migratory response, coupled with the absence of Akt phosphorylation observed in the present study, suggests that in primary endothelial cells the Met receptor is unable to activate PI-3 kinase without cooperative signals from the ECM/integrins. This observation is intriguing bearing in mind that Met has been shown to activate PI-3 kinase in epithelial cells via recruitment and activation of Gab-1, which directly interacts with the p85 subunit . Consistent with our observation of an integrin dependency for signal transduction, Trusolino et al showed that in carcinoma cell lines Met induced signals were considerably amplified as a consequence of its constitutive association with the integrin α6β4. Intriguingly, in this system the authors showed that the role of the integrin α4 subunit was independent of extracellular integrin ligation since a truncated α4 construct lacking its extracellular portion could mediate HGF/Met responses and signals to downstream effectors provided that its ability to recruit the adaptor Shc was not affected . In contrast, our studies using primary endothelial cells showed that integrin ligation was essential for generating a significant migratory signal via PI-3 kinase and in the case of HGF-FN and HGF-VN complexes, for promoting the association of Met with the integrins α5β1 and αvβ3 respectively. Indeed, Met association with the integrins α5β1 and αvβ3 was dependent upon the activation of both Met and integrins through ligation of their cognate ligands since tyrosine phosphorylation of Met by HGF alone could not induce integrin association (Fig. 3C). These observations support the contention of a signalling mechanism requiring the formation HGF-ECM molecular complexes as a prerequisite for Met-integrin association and consequent signal amplifiation as proposed in Fig. 9C. However, the importance of integrin cytoplasmic domains in recruiting Ras and Ras-binding partners appears to reflect a common mechanism of HGF signal transduction between these cellular systems.
The results of the present work demonstrate an important mechanism by which integrins collaborate with growth factor receptor tyrosine kinases on endothelial cells and predict that HGF binding domains on both FN and VN may play a significant role in promoting wound healing and post-natal neovascularization. In support of this contention, HGF-FN and HGF-VN complexes were identified in the supernatants derived from degranulated platelet suspensions indicating that these complexes do exist in vivo and may be deposited at sites of vessel perturbation or injury. This observation is similar to the identification of VEGF-FN molecular complexes in platelet supernatants in our previous report  and suggests that HGF and VEGF may act synergistically in vivo. Indeed, recent studies have shown that HGF synergises with VEGF to promote capillary-tube assembly in collagen matrices and neovascularization in the rat cornea . Furthermore, HGF positively regulates VEGF expression and down regulates TSP-1, an inhibitor of angiogenesis, thereby promoting angiogenesis . It is noteworthy that the HGF binding domains for FN were located in the same proteolytic fragments as those of VEGF, namely the N-terminal 70 kDa and C-terminal 40 kDa fragments. Further studies involving the fine mapping and characterization of the binding domains for VEGF and HGF on FN and VN should help decipher the mechanism of interplay between these important pro-angiogenic mediators.
Solid phase assay and Surface Plasmon Resonance Analysis (SPR)
ECM proteins and FN peptides were purchased from Sigma and Gibco and were further purified by gel filtration and ion exchange chromatography. The assay was performed as described previously . 125I-HGF (NEN) in binding buffer (PBS containing 2% BSA) were added to the microtitre plates and incubated for 30 min at room temperature (RT) before washing and counting to determine bound radioactivity. SPR analysis was performed on the BIAcore X (Biacore Herts UK) as described previously . HGF (30–500 nM) was injected across the FN 70 kDa fragment immobilised on a CM5 chip in HEPES saline (pH 7.4) supplemented with 1 mM MgCl2, 2 mM CaCl2 and the sensograms recorded. The data was analysed by the ASSAY programme (Biosoft, UK) in order to determine the EC50 value and Kd.
Migration and proliferation assays
Human dermal microvessel endothelial cells (HMVEC) were maintained in EBM-2 growth medium (Clonetics Corp). Migration studies were carried out essentially as described previously  using serum starved Calcein AM-loaded HMVEC in a modified Boyden chamber assay using Fluorblok transwell chambers (BD Bioscience) as described by the manufacturer. Cell migration was detected by fluorescence measurement (within the lower chamber compartment). Membranes of transwell chambers were coated with either FN or VN or collagen-1 (10 μg/ml) overnight at 4°C and preliminary experiments were performed to assess the optimal dosage of both HGF and ECM protein. With antibody inhibition studies, the transwell chamber was coated with poly-L-lysine (Sigma) to facilitate cell attachment to the filters as opposed to adhesion using ECM glycoproteins. HMVEC were pre-treated with αvβ3 and α5β1 integrin blocking antibodies for 30 min at room temperature prior to application to the upper transwell chamber. The levels of cell adhesion to ECM-coated transwell filters were determined by allowing HGF-stimulated HMVEC to adhere to transwells (coated overnight with ECM glycoprotein (10 μg/ml) and then blocked by incubation in 3.5 mg/ml BSA in basal culture medium) for 1 hour at room temperature followed by extensive washes with basal culture medium. The remaining cells were measured using a fluorescence plate reader (measuring fluorescence in the upper transwell compartment). For proliferation experiments, cell division was measure by fluorescence labeling of DNA (CyQuant, Molecular Probes). HMVEC was plated on poly-D-lysine coated 48-well plates and cultured overnight in MCDB-131 medium containing 5% FBS. After washing plates with PBS, endothelial cells were then cultured in MCDB-131 medium + 0.1% FBS containing HGF (10 ng/ml) in the presence or absence of VN, FN or Collagen-1 (10 μg/ml). Cells incubated for 48 h and HGF/ECM was added every 24 hours. Cell proliferation was quantified using a fluorescence plate reader.
Phosphorylation analysis and ras activation
HMVEC were assessed for the activation profiles of Erk1/2 and Akt using phosphospecific antibodies (Cell Signalling Technology) to Erk (Thr202/Tyr204) and Akt (Ser473) respectively by Western blotting. These studies were performed with both cells in suspension and with adherent populations. Cells were grown to 80% confluence and serum starved for 2 hours prior to harvesting. Cells were resuspended in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA (resuspension buffer) at a concentration of 1–5 × 106 cells /ml. The cell suspensions were challenged 10 ng/ml HGF supplemented with 2 μg/ml collagen-1, or FN or VN for various durations ranging from 2 to 120 min at room temperature. Cells were harvested by centrifugation at 4°C and lysed in 10 mM Tris pH 7.4, 145 mM NaCl supplemented with 0.1% Triton X-100 and protease inhibitors. For inhibitor studies, serum-starved HMVEC suspensions were pre-treated with the inhibitor for 45 min prior to stimulation with HGF and ECM molecules for a further 60 min at room temperature. The cells were pelleted, washed in ice-cold resuspension buffer without BSA and lysed in a lysis buffer containing 1% (v/v) Triton X-100. Cell lysates were analysed by Western blotting using protocols specific for the phosphospecific antibodies according to the manufacturer's recommendations. Blots were cut along appropriate marker divides and probed with antibodies to phopho Erk 1/2 and Akt (Ser473) (Cell Signaling Technologies) simultaneously. For GTP-Ras pull down assays, serum-starved HMVEC were stimulated with HGF and ECM molecules for a desired time point and the cells were spun down and washed in ice-cold resuspension buffer without BSA. Cell pellets were lysed in MLB buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 % Igepal CA-630, 10% Glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate and protease inhibitor cocktail) and 500 μg of cell lysate was mixed with a 10 μl suspension of RBD-Sepharose (Upstate Biotechnology) for each reaction at 4°C for 60 min. Sepharose beads were spun down and washed in MLB prior to solubilization and analysis by Western blotting probing for Ras using a monoclonal antibody (Upstate Biotechnology). For Ras inhibition studies cells were pre-incubated with FPT-III (100 μM) and GGTI (2 μM) (approx 40 × IC50 values) for 45 min at room temperature prior to cell stimulation for 60 min with HGF and ECM.
Human microvessel endothelial cells (HMVEC) in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA were plated on collagen, FN and VN coated petri dishes in the absence or presence of HGF (50 ng/ml) for 15 min to 1 hour at room temperature. Cells were then harvested as described previously  and integrin immunoprecipitation was performed with monoclonal antibodies (Chemicon) to α2β1 (clone JBS2), α5β1 (clone JBS5) and αvβ3 clone (LM609). After analysis by SDS-PAGE and protein transfer, the blot was then probed with a monoclonal to Met (clone DL-21, Upstate Biotechnology) and developed by chemiluminescence. For Met tyrosine phosphorylation analysis, cells were stimulated with HGF alone or HGF-FN and HGF-VN complexes for various time points ranging from 15 mins to 2 hours at room temperature. Lysed samples were immunoprecipitated with a polyclonal anti-phosphoMet antibody (Cell Signalling) and the immune complexes analysed by SDS-PAGE and Western blotting using a monoclonal ant-Met antibody (Upstate). Met was visualised using chemiluminescence technology (Pierce).
Immunoprecipitation of FN-HGF and VN-HGF complex from platelet supernatants
Supernatants from non-stimulated and thrombin-stimulated washed platelet suspensions were prepared as previously described . Supernatants were immunoprecipated with an antibody to FN or VN (Chemicon) or an isotype matched control reagent (IgG). Following SDS-PAGE and immunoblotting, HGF was detected with a polyclonal antibody (Santa Cruz) by chemiluminescent development.
hepatocyte growth factor
human microvessel endothelial cells.
SR and YP thank Grifols UK and Centeon for sponsorship respectively. This study was funded in part by a grant from the American Heart Association to ESW
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