Identification of a competitive HGF antagonist encoded by an alternative transcript.

We identified a naturally occurring hepatocyte growth factor (HGF) variant, whose predicted sequence extends only through the second kringle domain of this plasminogen-related molecule. This smaller molecule, derived from an alternative HGF transcript, lacked mitogenic activity but specifically inhibited HGF-induced mitogenesis. Cross-linking studies demonstrated that the truncated molecule competes with HGF for binding to the HGF receptor, which has been identified as the c-met protooncogene product. Thus, the same gene encodes both a growth factor and its direct antagonist.

Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.

An expression cloning strategy was devised to isolate the keratinocyte growth factor (KGF) receptor complementary DNA. NIH/3T3 fibroblasts, which secrete this epithelial cell-specific mitogen, were transfected with a keratinocyte expression complementary DNA library. Among several transformed foci identified, one demonstrated the acquisition of specific high-affinity KGF binding sites. The pattern of binding competition by related fibroblast growth factors (FGFs) indicated that this receptor had high affinity for acidic FGF as well as KGF. The rescued 4.2-kilobase complementary DNA was shown to encode a predicted membrane-spanning tyrosine kinase related to but distinct from the basic FGF receptor. This expression cloning approach may be generally applicable to the isolation of genes that constitute limiting steps in mitogenic signaling pathways.

Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B.

Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma are dominantly inherited cancer syndromes. All three syndromes are associated with mutations in RET, which encodes a receptor-like tyrosine kinase. The altered RET alleles were shown to be transforming genes in NIH 3T3 cells as a consequence of constitutive activation of the RET kinase. The MEN2A mutation resulted in RET dimerization at steady state, whereas the MEN2B mutation altered RET catalytic properties both quantitatively and qualitatively. Oncogenic conversion of RET in these neoplastic syndromes establishes germline transmission of dominant transforming genes in human cancer.

Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.

Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the beta subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase. Covalent cross-linking of 125I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF.

The acidic domain and first immunoglobulin-like loop of fibroblast growth factor receptor 2 modulate downstream signaling through glycosaminoglycan modification.

Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

Disassociation of met-mediated biological responses in vivo: the natural hepatocyte growth factor/scatter factor splice variant NK2 antagonizes growth but facilitates metastasis.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates numerous cellular activities capable of contributing to the metastatic phenotype, including growth, motility, invasiveness, and morphogenetic transformation. When inappropriately expressed in vivo, an HGF/SF transgene induces numerous hyperplastic and neoplastic lesions. NK1 and NK2 are natural splice variants of HGF/SF; all interact with a common receptor, Met. Although both agonistic and antagonistic properties have been ascribed to each isoform in vitro, NK1 retains the full spectrum of HGF/SF-like activities when expressed as a transgene in vivo. Here we report that transgenic mice broadly expressing NK2 exhibit none of the phenotypes characteristic of HGF/SF or NK1 transgenic mice. Instead, when coexpressed in NK2-HGF/SF bitransgenic mice, NK2 antagonizes the pathological consequences of HGF/SF and discourages the subcutaneous growth of transplanted Met-containing melanoma cells. Remarkably, the metastatic efficiency of these same melanoma cells is dramatically enhanced in NK2 transgenic host mice relative to wild-type recipients, rivaling levels achieved in HGF/SF and NK1 transgenic hosts. Considered in conjunction with reports that in vitro NK2 induces scatter, but not other activities, these data strongly suggest that cellular motility is a critical determinant of metastasis. Moreover, our results demonstrate how alternatively structured ligands can be exploited in vivo to functionally dissociate Met-mediated activities and their downstream pathways.

The juxtamembrane regions of the epidermal growth factor receptor and gp185erbB-2 determine the specificity of signal transduction.

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

c-Met autocrine activation induces development of malignant melanoma and acquisition of the metastatic phenotype.

The molecular and genetic events that contribute to the genesis and progression of cutaneous malignant melanoma, a complex and aggressive disease with a high propensity for metastasis, are poorly understood due in large part to the dearth of relevant experimental animal models. Here we used transgenic mice ectopically expressing hepatocyte growth factor/scatter factor (HGF/SF) to show that the Met signaling pathway is an important in vivo regulator of melanocyte function, whose subversion induces malignant melanoma. Tumorigenesis occurred in stages, beginning with the abnormal accumulation of melanocytes in the epidermis and dermis and culminating in the development of metastatic melanoma. Oncogenesis in this model was driven by creation of HGF/SF-Met autocrine loops through forced expression of the transgenic ligand and apparent selection of melanocytes overexpressing endogenous receptor, rather than paracrine stimulation or mutational activation of c-met. Preference for liver as a metastatic target correlated with high HGF/SF-Met autocrine activity, consistent with the notion that such activity may influence colonization. Although basic fibroblast growth factor and its receptor were both weakly expressed in the majority of melanomas examined, high levels were found only in those rare neoplasms with low or undetectable HGF/SF and Met expression, suggesting that these two tyrosine kinase receptor autocrine loops serve a critical overlapping function in melanocytic tumorigenesis. Our data support a causal role for HGF/SF-Met signaling in the development of melanoma and acquisition of the metastatic phenotype. Moreover, this transgenic mouse should serve as a highly useful model, facilitating our understanding of mechanisms by which human melanoma progresses to malignancy and expediting the development of efficacious therapeutic modalities designed to constrain metastasis.

Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/Thr protein phosphatases.

The c-myb proto-oncogene encodes a nuclear phosphoprotein that plays a crucial role in normal hematopoiesis. It is a short-lived transcription factor rapidly degraded by the 26S proteasome. Although it has been shown that instability determinants reside in its carboxyl terminus, the molecular mechanism of c-Myb degradation is unknown. Here, we report the first evidence that phosphorylation plays a role in targeting the protein to the proteasome. Inhibition of cellular serine/threonine protein phosphatase activity by okadaic acid resulted in hyperphosphorylation of c-Myb and extremely rapid turnover. The hyperphosphorylation resulted in a protein with altered properties that was indicative of conformational changes. Its mobility on gel electrophoresis was altered as well as its recognition by specific monoclonal antibody. The altered hyperphosphorylated protein still bound to DNA with an affinity similar to that of the hypophosphorylated form. Phosphorylation of three previously identified sites, serines 11, 12, and 528, does not appear to be involved in the proposed changes in conformation or stability. However, phosphoamino acid analyses of the hyperphosphorylated form of c-Myb revealed increased c-Myb phosphorylation mainly on threonine residues that correlated with other okadaic acid-induced alterations of c-Myb. These findings indicate that Ser/Thr phosphatases prevent conformational changes that may play an important role in controlled degradation of c-Myb. Oncogene (2000) 19, 2846 - 2854

Differential signaling by alternative HGF isoforms through c-Met: activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis.

HGF/NK2, a naturally occurring truncated HGF isoform, antagonizes the mitogenic and morphogenic activities of full length HGF, but stimulates cell scatter, or the motogenic response to HGF. We studied postreceptor signaling by these HGF isoforms in the human breast epithelial cell line 184B5, and in murine myeloid progenitor 32D cells transfected with c-Met, the human HGF receptor (32D/c-Met). HGF stimulated DNA synthesis in 184B5 and 32D/c-Met cells, while HGF/NK2 was mitogenically inactive, despite the ability of HGF/NK2 to stimulate c-Met autophosphorylation, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) in both cell systems. In 184B5 cells, HGF stimulated sustained MAPK activation, while activation by HGF/NK2 declined rapidly. In contrast, both isoforms activated MAPK with rapidly attenuated kinetics in 32D/c-Met cells. In both cell systems the increased motility observed in response to either HGF or HGF/NK2 treatment was more potently blocked by the PI3 kinase inhibitor wortmannin, than by PD98059, an inhibitor of MAPK kinase (MEK1). These data suggest that (1) alternative HGF isoforms signaling through c-Met generate both common and distinct biological responses, (2) the extent and duration of ligand-stimulated c-Met and MAPK activities are dependent on the cellular context and are not predictive of mitogenic signaling, and (3) in at least some HGF target cells, the activation of both MAPK and PI3K signaling pathways is insufficient for mitogenesis elicited through c-Met.

Induction of tissue inhibitor of metalloproteinases-3 is a delayed early cellular response to hepatocyte growth factor.

Hepatocyte growth factor (HGF) stimulates mitogenic, motogenic, and morphogenic responses in various cell types. We analysed HGF-responsive cells by differential display PCR to identify HGF-induced genes that mediate these biological events. One of the genes identified encoded a member of the tissue inhibitor of metalloproteinases (TIMP) family, TIMP-3. HGF transiently induced TIMP-3 mRNA in keratinocytes as well as kidney and mammary epithelial cells maximally between 4 and 6 h post-stimulation. Increased TIMP-3 protein secretion returned to basal levels within 18 h, while the expression of gelatinases A and B remained unchanged, suggesting that temporary suppression of matrix degradation is a delayed early response to HGF. Ectopic overexpression of TIMP-3 in cultured leiomyosarcoma cells conferred an epithelial morphology, reduced cell growth rate, anchorage-independent growth, and matrix invasion in vitro. Antisense suppression of TIMP-3 was associated with a scattered, fibroblastic cell morphology, as well as enhanced proliferation, anchorage-independent growth, and matrix invasion. A survey of tumor cell lines revealed an inverse relationship between metastatic potential and TIMP-3 expression level. These data suggest that early, transient TIMP-3 expression mediates specific HGF-induced phenotypic changes, and that loss of TIMP-3 expression may enhance the invasion potential of certain tumors.

A novel human ERK phosphatase regulates H-ras and v-raf signal transduction.

A cDNA encoding a novel human extracellularly-regulated kinase (ERK) phosphatase, designated B59, was isolated from a B5/589 human mammary epithelial cell cDNA library. The 1104 nucleotide open reading frame encodes 368 amino acids including the highly conserved catalytic site sequence of protein phosphotyrosine phosphatases (PTPs), VXVHCXXGXXR, at amino acid position 276-287. The predicted 70 amino acid stretch surrounding the HC motif shares significant sequence identity with other human dual specificity PTPs (dsPTPs), including the known ERK PTPs CL100, PAC1, B23, as well as the dsPTPs VH-1 and VHR. B59 protein synthesized in vitro in a rabbit reticulocyte lysate dephosphorylated rat ERK1 and ERK2 proteins whose phosphorylation had been stimulated by v-mos kinase added to the lysate. Ectopic expression of B59 in NIH3T3 fibroblasts inhibited the induction of an oncogene-responsive promoter by the dominant-activating raf mutant, raf-BXB. Moreover, cotransfection of NIH3T3 cells with B59 inhibited morphological transformation by H-ras and v-raf oncogenes. These results suggest that B59 suppresses the transforming activity of H-ras or v-raf oncogenes through ERK dephosphorylation and inactivation.

Neu differentiation factor/heregulin induction by hepatocyte and keratinocyte growth factors.

Hepatocyte growth-factor (HGF) is a potent, widely produced, pleiotropic mediator of mesenchymal-epithelial interaction. In a study of changes in gene expression initiated by HGF in Balb/MK keratinocytes, we observed the induction of Neu-differentiation factor (NDF) mRNA (also known as heregulin, or HRG). Further characterization of the regulation of NDF expression in Balb/MK keratinocytes revealed potent induction by keratinocyte growth factor (KGF) and epidermal growth factor (EGF), but not by HGF/NK2, an alternative HGF isoform with motogenic but not mitogenic or morphogenic activities. Sustained treatment (8 h) of Balb/MK cells with KGF stimulated secretion of mature NDF protein into the culture medium, and Balb/ MK cells treated with purified recombinant NDF protein showed increased DNA synthesis. We also found evidence of NDF induction in two models of tissue repair in mice: in full-thickness skin wounds, following locally increased KGF production, and in kidney after partial hepatectomy, following elevation of circulating HGF levels. These results reveal that mesenchymally-derived HGF and KGF can activate autocrine NDF signaling in their epithelial targets, and suggest that this mechanism contributes to the coordination of stages of wound repair, and possibly development, where these growth factors act in concert to direct epithelial proliferation, morphogenesis and differentiation.

Transformation of NIH3T3 fibroblasts by an expression vector for the human epidermal growth factor precursor.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) bind to a common cell surface receptor that mediates their diverse biological activities. NIH3T3 fibroblasts transfected with either full-length EGF precursor (preproEGF) or proTGF alpha cDNA displayed distinct patterns of growth in culture. PreproEGF induced focal transformation, and transfectants grew in a chemically defined medium (CDM) at low cell density in the absence of added EGF. In contrast, TGF alpha failed to cause focal transformation, and transfectants grew in CDM in the absence of added growth factors only when seeded at high cell density. The 53 amino acid EGF portion of the preproEGF translation product was essential for its effects. These results indicate that constitutive expression of preproEGF is sufficient to establish autocrine growth of NIH3T3 expressing low levels of EGF receptors. At high cell density, where paracrine as well as autocrine effects of these growth factors would be evident, TGF alpha transfectants displayed at least as high or higher levels of EGF receptor (EGFR) tyrosine phosphorylation than preproEGF transfectants. Since quantitative levels of ligand expression did not account for differences in their transforming properties, preproEGF must be more efficient than proTGF alpha in binding and/or activating EGF receptors in an autocrine manner.

Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta.

The stabilization of endothelial cell (EC) barrier function within newly formed capillaries is a critical feature of angiogenesis. We examined human lung EC barrier regulation elicited by hepatocyte growth factor (HGF), a recognized angiogenic factor and EC chemoattractant. HGF rapidly and dose-dependently elevated transendothelial electrical resistance (TER) of EC monolayers (>50% increase at 100 ng/ml), with immunofluorescence microscopic evidence of both cytoplasmic actin stress fiber dissolution and strong augmentation of the cortical actin ring. HGF rapidly stimulated phosphatidylinositol 3'-kinase, ERK, p38 mitogen-activated protein kinase, and protein kinase C activities. Pharmacological inhibitor studies demonstrated each pathway to be intimately involved in HGF-induced increases in TER, cortical actin thickening, and phosphorylation of the Ser/Thr glycogen synthase kinase-3beta (GSK-3beta), a potential target for the HGF barrier-promoting response. GSK-3beta phosphorylation was strongly correlated with reductions in both HGF-induced TER and enhanced beta-catenin immunoreactivity observed at cell-cell junctions. Our data suggest a model in which HGF-mediated EC cytoskeletal rearrangement and barrier enhancement depend critically on the activation of a complex kinase cascade that converges at GSK-3beta to increase the availability of beta-catenin, thereby enhancing endothelial junctional integrity and vascular barrier function.

The 18 kDa cytosolic acid phosphatase from bovine live has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor.

In this paper we demonstrate that the cytosolic low-Mr acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase activity on 32P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent Km for 32P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not 32P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-Mr acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling.

Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction.

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, though from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and analysis of agents regulating KGF expression reinforced the idea that KGF acts predominantly on epithelial cells. While the data do not implicate a KGF autocrine loop in neoplasia, paracrine sources of factor or ligand-independent signaling by the KGFR might contribute to malignancy. Alternatively, because of its differentiation-promoting effects, KGF may retard processes that culminate in uncontrolled cell growth.

A cell-permeable peptide inhibits activation of PKR and enhances cell proliferation.

The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation.