miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.     

Hepatocyte growth factor and Met in tumor biology and therapeutic approach with NK4

Hepatocyte growth factor (HGF) and Met/HGF receptor tyrosine kinase play a role in the progression to invasive and metastatic cancers. A variety of cancer cells secrete molecules that enhance HGF expression in stromal fibroblasts, while fibroblast-derived HGF, in turn, is a potent stimulator of the invasion of cancer cells. In addition to the ligand-dependent activation, Met receptor activation is negatively regulated by cell-cell contact and Ser985 phosphorylation in the juxtamembrane of Met. The loss of intercellular junctions may facilitate an escape from the cell-cell contact-dependent suppression of Met-signaling. Significance of juxtamembrane mutations found in human cancers is assumed to be a loss-of-function in the negative regulation of Met. In attempts to block the malignant behavior of cancers, NK4 was isolated as a competitive antagonist against HGF-Met signaling. Independently on its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF. In experimental models of distinct types of cancers, NK4 inhibited Met activation and this was associated with inhibition of tumor invasion and metastasis. NK4 inhibited tumor angiogenesis, thereby suppressing angiogenesis-dependent tumor growth. Cancer treatment with NK4 suppresses malignant tumors to be "static" in both tumor growth and spreading.     

Low Dose Geldanamycin Inhibits Hepatocyte Growth Factor- and Hypoxia-Stimulated Invasion of Cancer Cells

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.     

Hepatocyte growth factor enhances proteolysis and invasiveness of human nasopharyngeal cancer cells through activation of PI3K and JNK

The hepatocyte growth factor (HGF) receptor, Met, is frequently overexpressed in nasopharyngeal cancer (NPC). Here, we showed for the first time that human NPC cells with high Met expression were more sensitive to the cell motility and invasion effect of HGF. The downregulation of Met by small interfering RNA decreased tumor cell invasion/migration. HGF significantly increased matrix metalloproteinase-9 production. This was inhibited by blocking phosphatidylinositide 3-kinase (PI3K) and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways. We also demonstrated that PI3K induced activation of JNK, with Akt as a potential point of this cross-talk. These results provide new insights into the molecular mechanism responsible for NPC progression and metastasis.     

c‐Met function requires n‐linked glycosylation modification of pro‐Met

c‐Met, the receptor for hepatocyte growth factor (HGF), is cell surface tyrosine kinase that controls cancer cell growth, survival, invasion, and metastasis. Post‐translational modification, such as glycosylation, plays an essential role in regulating the function of cell surface molecules. Whether glycosylation modification regulates the enzymatic properties of c‐Met is unknown. In this study, we investigated the effect of glycosylation on the function of c‐Met. We found that c‐Met is an N‐linked glycosylated protein. Both pro‐Met and p145Met (the β subunit of mature c‐Met) have N‐linked glycosylation. Glycosylation inhibitor studies revealed that the N‐glycosylation modification of p145Met is from pro‐Met, but not due to the further modification of pro‐Met. Importantly, blocking the N‐glycosylation targets pro‐Met to cytoplasm and initiates its phosphorylation independent of HGF engagement. Nonglycosylated pro‐Met activates c‐Met downstream pathways to a certain extent to compensate for the degradation of p145Met induced by glycosylation blocking‐mediated endoplasmic reticulum (ER) stress. J. Cell. Biochem. 114: 816–822, 2013. © 2012 Wiley Periodicals, Inc.     

Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC₅₀ of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC₅₀ of ∼ 20 μM. The 2D ¹H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.     

Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis

Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis.     

肝细胞生长因子抑制剂——NK4的抗肿瘤作用

肝细胞生长因子(hepatocyte growth factor, HGF)是一种多功能的细胞因子,其生物学活性由c-Met蛋白所介导.HGF/c-Met信号通路在肿瘤生成、侵袭、转移以及肿瘤新生血管生成方面起重要促进作用. 因此, HGF/c-Met信号转导通路可以作为抗肿瘤药物设计的靶点.其中,HGF-α链N端447个氨基酸组成的NK4蛋白是HGF的特异性拮抗剂,它不仅通过抑制HGF/c-Met系统的信号转导发挥抗肿瘤效应;而且可以通过拮抗HGF和其它血管生成因子如成纤维细胞生长因子（fibroblast growth factors, FGF)、血管内皮生长因子（vascular endothelial growth factor, VEGF）的活性,进而抑制肿瘤新生血管生成,最终导致肿瘤细胞的凋亡.NK4的这种双重抗肿瘤功能使其成为一类很有前景的新型抗肿瘤药物.本文就NK4对肿瘤的抑制作用及其机制的研究进展进行综述.     

Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal-epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis-driven HF regression (catagen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild-type controls, transgenic mice overexpressing HGF/SF under the control of the MT-1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met-mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.-Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling.     

Met degradation: more than one stone to shoot a receptor down

The receptor tyrosine kinase Met and its high-affinity ligand, the hepatocyte growth factor/scatter factor (HGF/SF), are essential to embryonic development. Deregulation of their signaling is associated with tumorigenesis and metastasis, notably through receptor overexpression. It is thus important to understand the mechanisms controlling Met expression. The ligand-dependent internalization of Met and its subsequent degradation in the lysosomal compartment are well described. This process is known to attenuate downstream Met signaling pathways. Yet internalized Met takes part directly in intracellular signaling by chaperoning signaling factors in the course of its trafficking. Furthermore, recent studies describe various new degradation mechanisms of membrane-anchored Met, involving proteolytic cleavages or association with novel partners. Although all these degradations are ligand-independent, they share, to different extents, some common features with canonical HGF/SF-dependent degradation. Interestingly, activated Met variants display resistance to degradation, suggesting defective degradation is involved in tumorigenesis. Conversely, forced degradation of Met through reinduction of one or more degradation pathways is a promising therapeutic strategy.     

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright © 2012 John Wiley & Sons, Ltd.     

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion

Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF‐receptor c‐Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression‐free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c‐Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c‐Met, inducing the release of soluble c‐Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c‐Met shedding, but has limited impact on surface c‐Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro‐invasive role and may contribute to UM progression.     

Brain-Derived Neurotrophic Factor (BDNF)-Induced Tropomyosin-Related Kinase B (Trk B) Signaling Is a Potential Therapeutic Target for Peritoneal Carcinomatosis Arising from Colorectal Cancer

Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential in vitro and in vivo. Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a (a selective pharmacological pan-Trk inhibitor) were used for in vitro cell viability, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Tissue BDNF mRNA was associated with liver and peritoneal metastasis. Tissue TrkB mRNA was also associated with lymph node metastasis. The co-expression of BDNF and TrkB was associated with liver and peritoneal metastasis. Patients with higher BDNF, TrkB, and co-expression of BDNF and TrkB had a significantly poor prognosis. BDNF increased tumor cell viability, migration, invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal metastatic nodules was found to be reduced, as compared with control mice. BDNF/TrkB signaling may thus be a potential target for treating peritoneal carcinomatosis arising from colorectal cancer.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in Met binding and signaling

Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its alpha/beta heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the alpha-chain binds Met with high affinity. Recently, we reported that the protease-like HGF beta-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325-2335). Here we demonstrate that the zymogen-like form of HGF beta also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions. Moreover, the crystal structure of HGF beta determined at 2.53 A resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the "active site region" including "triad" residues Gln(534) [c57], Asp(578) [c102], and Tyr(673) [c195] and neighboring "activation domain" residues Val(692), Pro(693), Gly(694), Arg(695), and Gly(696) [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Activation mechanisms of the urokinase-type plasminogen activator promoter by hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. One of the effectors of HGF/SF is the urokinase-type plasminogen activator, a serine protease that facilitates tumor progression and metastasis by controlling the synthesis of the extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells that were stably transfected with the human Met receptor (NIH 3T3-Methum) with HGF/SF induced a trans-activation of the urokinase promoter and urokinase secretion. Induction of the urokinase promoter by HGF/SF via the Met receptor was blocked by co-expression of a dominant-negative Grb2 and Sos1 expression construct. Further, the expression of the catalytically inactive mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the Mek1-specific inhibitor PD 098059 abrogated the stimulation of the urokinase promoter by HGF/SF. A sequence residing between -2109 and -1870 base pairs (bp) was critical for stimulation of the urokinase gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an AP-1 site at -1880 bp or a combined PEA3/AP-1 site at -1967 bp showed binding of nuclear factors from NIH 3T3-Methum cells. Expression of an expression plasmid that inhibits DNA binding of AP-1 proteins (A-Fos) abrogated inducible and basal activation of the urokinase promoter. Nuclear extract from unstimulated NIH 3T3-Methum cells contained more JunD and showed a stronger JunD supershift with the AP-1 oligonucleotides, compared with HGF/SF-stimulated cells. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF. These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-++ +Jun-dependent mitogen-activated protein kinase pathway.