The effect of TNP-470 on cell proliferation and urokinase-type plasminogen activator and its inhibitor in human lung cancer cell lines

The plasminogen activator system has been found to modulate neoplastic spread and angiogenesis in tumors. An angiogenesis inhibitor, TNP-470, has been shown to possess potent antitumor activities in various types of cancer cells. We therefore investigated the inhibitory effect of TNP-470 on cancer cell proliferation, and the suppressive effect of TNP-470 on urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1), in human lung cancer cell lines in vitro. TNP-470 inhibited cell proliferation in a dose-dependent manner in both cell lines. u-PA and PAI-1 in culture supernatants were suppressed with the concentrations of TNP-470, in association with a decrease in viable cancer cells. Unchanged serum levels of u-PA and PAI-1 would be of great advantage to the diseased patients, since the plasminogen activator system has a crucial function in the process of distant metastasis.     

Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-alpha. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17beta-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors alpha and beta, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone, at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor-mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.     

The fibrinolytic system in neoplasia

During activation of the fibrinolytic system plasminogen is converted to plasmin by tissue plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). t-PA is predominantly released from endothelial cells, u-PA primarily by renal parenchymal cells. The activation of plasminogen is regulated by plasminogen activator inhibitor-1 (PAI-1), plasmin is controlled by alpha 2-plasmin inhibitor. The fibrinolytic system is not only involved in the intravascular dissolution of fibrin (thrombi), it also plays a vital role in normal physiologic reproduction, wound repair, angiogenesis, and tissue remodeling. Fibrinolysis is also a vital component in the pathogenesis of neoplastic disease. It is essential in releasing cells from their primary site of origin, providing nutrition for neoplastic cell growth and promoting cell mobility and motility. In neoplastic cells the degradation of the extracellular matrix proteins is facilitated by excessive expression of u-PA, t-PA, and u-PAR. In many forms of carcinoma increased expression of u-PAR and u-PA is associated with significantly shorter survival. Greater expression of u-PA in breast cancer cells, for example, is associated with shorter survival and increased relapse rate. Progressively aggressive neoplastic cells evidence high expression of u-PA and u-PAR activities, variable expression of t-PA, and enhanced PAI-1 and PAI-2 activities. In acute nonlymphocytic leukemias, poor outcome correlates with high t-PA levels. In acute progranulocytic leukemia there is a high incidence of DIC. Neoplastic prostatic tissue also expresses high u-PA activity and the more aggressive the cell line, the greater the number of u-PAR and the higher the u-PA activity. In gynecologic malignancies, a greater expression of u-PA in combination with cathepsin D is associated with widespread disease and poor prognosis. High u-PA values were also seen in patients with brain, gastric, and hepatic malignancies. It is evident that the plasminogen-plasmin system is a vital component in the biology of neoplastic disease and that it is, in theses conditions, in no way beneficial to the host.     

Antisense targeting of the urokinase receptor blocks urokinase-dependent proliferation, chemoinvasion, and chemotaxis of human synovial cells and chondrocytes in vitro

Proliferation and invasion of synovial pannus in rheumatoid arthritis and cartilage remodeling in osteoarthritis are key events in development of disability of arthritic joints. The mechanisms that trigger these events are still poorly understood. The production of urokinase-type plasminogen activator (u-PA) by synovial cells and chondrocytes and the subsequent interaction of u-PA with its membrane receptor (u-PAR) is under the control of a variety of growth factors and cytokines released within the inflamed joints. Here we show that u-PA, on interaction with the specific receptor, regulates movement and invasion as well as proliferation of human synovial cells and chondrocytes. Targeting the urokinase receptor with an antisense oligonucleotide blocks the u-PA-dependent synoviocyte and chondrocyte proliferation and chemoinvasion, suggesting a possible use for this new class of drugs in the progression of the disease in rheumatoid arthritis and osteoarthritis.     

Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively.     

Characterization of tTA and its functional domain in tetracycline repressor-mediated gene repression system

The tissue concentrations of urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were investigated by an ELISA technique in normal and malignant samples of the prostate from 24 patients undergoing radical prostatectomy for organ-confined prostate cancer. The median concentration of u-PA was significantly higher in cancerous than in normal prostate tissue (p = 0.006). No significant increase of u-PAR, PAI-1 and t-PA was found in cancer tissue in comparison with the benign samples (p > 0.05). Assessment of the relationship between fibrinolytic proteins and DNA ploidy revealed an increased u-PA, u-PAR and PAI-1 in diploid prostate cancer as compared with the normal controls. However, in aneuploid cancer u-PA remained high but u-PAR and PAI-1 were decreased. This led to a higher local concentration of u-PA in aneuploid samples than in normal prostate and in diploid prostate cancer. No alteration of median t-PA was found in benign prostate or in diploid or aneuploid prostate cancer. The altered expression of u-PA, u-PAR and PAI-1 in diploid and aneuploid prostate cancer suggests a possible role of fibrinolytic proteins in the different biologic behavior of tumors, and may be one explanation for the higher metastatic potential of aneuploid tumors.     

Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor

The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.     

Effects of excitatory amino acids on cerebral oxygen consumption and blood flow in rat

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.     

Decreased fibronectin expression in Met/HGF-mediated tumorigenesis

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor (HGF)/scatter factor are involved in the etiology and progression of a number of human cancers. Coexpression of Met and HGF in mesenchymal cells increases the tumorigenic and metastatic potential of the cells. In the studies described here, we used differential display screening to identify changes in gene expression that are initiated by Met/HGF, and that may lead to these phenotypes. We learned that Met/HGF signaling resulted in greatly decreased fibronectin mRNA production in three different human and mouse tumor cell lines; these decreases in fibronectin mRNA were paralleled by decreases in fibronectin protein. We also found a progressive decrease in fibronectin in tumor explants and metastases derived from the Met/HGF transformed cells. The absence of fibronectin expression is a frequent cancer phenotype; our results indicate that decreases in fibronectin correlate with, but are not essential for, MetHGF/SF-mediated tumorigenesis.     

In vitro urokinase type plasminogen activator levels and total plasminogen activator activity in squamous cell carcinomas of the head and neck

OBJECTIVE: Determine total plasminogen activator (PA) activity and urokinase-type plasminogen activator (u-PA) levels in cell-free supernatants derived from primary and metastatic squamous cell carcinoma of the head and neck. DESIGN: Plasminogen activator activity was measured by spectrophotometric assay with chromogenic substrate Val-Leu-Lys-para-nitroanilide. Urokinase-type plasminogen activator levels were measured with enzyme-linked immunosorbent assay technique. RESULTS: Fourteen established squamous cell carcinoma lines from patients with head and neck cancer were assayed for both total PA activity and u-PA levels at 24 to 48 hours of incubation. Compared with control and fibroblast-conditioned media, cell lines established from squamous cell carcinoma of the head and neck had significantly (P < .005) higher levels of both total PA activity and u-PA levels. Linear regression analysis showed a positive correlation (r = .65, P = .007) between total PA activity and u-PA levels. CONCLUSIONS: Squamous cell carcinomas of the head and neck are able to activate plasminogen and produce u-PA in vitro. The production of PA by squamous cell carcinomas of the head and neck may play an important role in the biology of invasion and metastasis.     

The cleavage of pro-urokinase type plasminogen activator by stromelysin-1

Membrane binding of urokinase type plasminogen activator (u-PA) is thought to play a pivotal role in connective tissue remodeling and invasive processes. We compare the ability of different matrix-metalloproteinases involved in connective tissue turnover to cleave pro-urokinase type plasminogen activator between the catalytic domain and the receptor binding part to investigate a potential role for matrix-metalloproteinases in the regulation of membrane-associated proteolytic activity. We employed several forms of human stromelysin-1 (full length, C-truncated, and recombinant catalytic domain), rabbit C-truncated stromelysin-1, the human gelatinases A and B and the human catalytic domain of neutrophil collagenase. The gelatinases and the collagenase did not separate the receptor binding domain of pro-urokinase type plasminogen activator from the catalytic domain, whereas all stromelysin-1 forms cleaved the glutamic acid 143-leucine 144 bond of pro-urokinase type plasminogen activator. This reaction could be inhibited by specific inhibitors of matrix metalloproteinases and was not affected by inhibitors of serine proteinases. The M(r) 31000 cleavage product with leucine 144 as N-terminus displayed no proteolytic activity towards the pro-urokinase type plasminogen activator substrate pyroGlu-Gly-Arg-pNA-HCI (S2444), but it could be activated by an additional treatment with plasmin. Comparison between full length stromelysin-1 and its C-truncated forms, showed that both exhibited the same cleavage properties towards pro-urokinase type plasminogen activator. Thus, the cleavage of pro-urokinase type plasminogen activator by stromelysin-1 is not influenced by the presence or absence of the C-terminal domain. The recombinant catalytic domain of MMP-3 generated pro-urokinase type plasminogen activator, whereas incubation of pro-urokinase type plasminogen activator with the native forms of human or rabbit stromelysin-1 led to a moderate activation of pro-uPA due to an additional cleavage that is catalyzed by a serine proteinase.     

Requirement of receptor-bound urokinase-type plasminogen activator for integrin alphavbeta5-directed cell migration

The urokinase plasminogen activator (uPA) interacts with its cell surface receptor (uPAR), providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. Evidence is provided for a novel function of cell surface-associated uPA.uPAR. Specifically, induction of cell surface expression of uPA. uPAR by growth factors or phorbol ester was necessary for vitronectin-dependent carcinoma cell migration, an event mediated by integrin alphavbeta5. Cell migration on vitronectin was blocked with either a soluble form of uPAR, an antibody that disrupts uPA binding to uPAR, or a monoclonal antibody to alphavbeta5. Moreover, plasminogen activator inhibitor type 2 blocked this migration event but did not affect adhesion, suggesting a direct role for uPA enzyme activity in this process and that migration but not adhesion of these cells is regulated by uPA.uPAR. Growth factor-mediated induction of uPA.uPAR on the carcinoma cell surface promotes a specific motility event mediated by integrin alphavbeta5, since cells transfected with the beta3 integrin subunit expressed alphavbeta3 and migrated on vitronectin independently of growth factors or uPA.uPAR expression. This relationship between alphavbeta5 and the uPA.uPAR system has significant implications for regulation of motility events associated with development, angiogenesis, and tumor metastasis.     

Methylesters of L-arginine and N-nitro-L-arginine induce nitric oxide synthase in Staphylococcus aureus

Urokinase type plasminogen activator (u-PA) secreted by cancer cells is considered to play a key role in promoting invasion and metastasis of cancer cells. This study was designed to evaluate the expression and prognostic value of u-PA in Dukes B and C colorectal cancer. u-PA expression was investigated in 57 Dukes B or C colorectal cancers using a monoclonal antibody against u-PA. u-PA expression was mainly observed on the cytoplasm of cancer cells, and was associated with relapse, especially hematogenous metastasis (p=0.025, the chi2 test). Patients with high u-PA expression had a lower rate of DFS (9/22 events) compared to those with low u-PA expression (6/35 events) (p=0.061, log-rank test). This study demonstrated that u-PA expression might be a novel prognostic factor in Dukes B and C colorectal cancer.     

c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells

Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types of human cancers, suggesting a critical role for Met in growth and development of malignant cells. However, the molecular mechanism by which Met contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has been implicated as a modulator of cell proliferation, spreading, and migration; these functions are also regulated by Met. To explore whether c-Src kinase is involved in HGF-induced cell growth, a mouse mammary carcinoma cell line (SP1) that co-expresses HGF and Met and a nonmalignant epithelial cell line (Mv1Lu) that expresses Met but not HGF were used. In this study, we have shown that c-Src kinase activity is constitutively elevated in SP1 cells and is induced in response to HGF in Mv1Lu cells. In addition, c-Src kinase associates with Met following stimulation with HGF. The enhanced activity of c-Src kinase also correlates with its ability to associate with Met. Expression of a dominant negative double mutant of c-Src (SRC-RF), lacking both kinase activity (K295R) and a regulatory tyrosine residue (Y527F), in SP1 cells significantly reduced c-Src kinase activity and strongly blocked HGF-induced motility and colony growth in soft agar. In contrast, expression of the dominant negative c-Src mutant had no effect on HGF-induced cell proliferation on plastic. Taken together, our data strongly suggest that HGF-induced association of c-Src with Met and c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas and further support the notion that the presence of paracrine and autocrine HGF loops contributes significantly to the transformed phenotype of carcinoma cells.     

Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis

BACKGROUND: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro. METHODS: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgen-independent), and LNCaP (androgen-dependent). We studied the expression of the HGF receptor c-met proto-oncogene (c-met) by Western blot analysis, and also determined the effects of HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay. RESULTS: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells. CONCLUSION: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Urokinase plasminogen activator colocalizes with CD25+ cells in atherosclerotic vessels

Proteolytic activity in vascular tissue is necessary for cellular migration, remodelling of extracellular matrix and the development of atherosclerotic lesions. Inflammatory cells, mainly macrophages, are numerous in atherosclerotic plaques and may synthesize and secrete proteolytic enzymes. The principal activator of plasminogen in tissues is urokinase plasminogen activator (u-PA). To determine if an activated phenotype of inflammatory cells colocalizes with local expression of u-PA in atherosclerotic vessels, vascular biopsies from 15 patients with peripheral atherosclerotic disease were analyzed by immunohistochemistry on consecutive sections. Anti-CD68 antibodies were used as markers for macrophages and were positive in 14/15 specimens. Anti-CD25 (interleukin-2 receptor-alpha) antibodies were used to identify inflammatory cells with an activated phenotype and were positive in 9/14 CD68+ specimens. The same 9 specimens were positive for u-PA. A positive reaction for u-PA was found only in specimens with CD25+ cells. Specimens with positive reactions for all three antibodies were further analyzed with computer-assisted image analysis. The colocalization with u-PA was higher for CD25 compared to CD68 in all specimens. Mean percentage of the u-PA-positive area in regions positive for cellular markers was 52% (SEM 6%) for CD25 and 19% (SEM 5%) for CD68 (p < 0.01). The results indicated that the activation of macrophages in atherosclerotic vessels may modulate local proteolysis and be of importance in plaque development and stability.     

Immunopathogenesis and immunotherapy of psoriasis

The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.     

Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons

The development of the nervous system is a dynamic process during which factors act in an instructive fashion to direct the differentiation and survival of neurons, and to induce axonal outgrowth, guidance to, and terminal branching within the target tissue. Here we report that mice expressing signaling mutants of the hepatocyte growth factor (HGF) receptor, the Met tyrosine kinase, show a striking reduction of sensory nerves innervating the skin of the limbs and thorax, implicating the HGF/Met system in sensory neuron development. Using in vitro assays, we find that HGF cooperates with nerve growth factor (NGF) to enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons. HGF also enhances the neurotrophic activities of NGF in vitro, and Met receptor signaling is required for the survival of a proportion of DRG neurons in vivo. This synergism is specific for NGF but not for the related neurotrophins BDNF and NT3. By using a mild signaling mutant of Met, we have demonstrated previously that Met requires signaling via the adapter molecule Grb2 to induce proliferation of myoblasts. In contrast, the actions of HGF on sensory neurons are mediated by Met effectors distinct from Grb2. Our findings demonstrate a requirement for Met signaling in neurons during development.