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.

Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-met interaction facilitates signaling.

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Hepatocyte growth factor induction of collagenase 3 production in human osteoarthritic cartilage: involvement of the stress-activated protein kinase/c-Jun N-terminal kinase pathway and a sensitive p38 mitogen-activated protein kinase inhibitor cascade.

OBJECTIVE: Osteoarthritis (OA) involves both a decreased reparative process and an increased degradative phenomenon. Several cytokines and growth factors are known to facilitate the repair of articular cartilage defects. The hepatocyte growth factor (HGF) present in OA cartilage is suggested to be involved in the cartilage repair process as well as in matrix remodeling and chondrocyte migration, leading to partial reconstruction of articular cartilage. Since cell migration is often correlated with metalloprotease activity, the effect of HGF on collagenase 3 production was studied because of its possible implication in OA cartilage remodeling. METHODS: We examined HGF-stimulated collagenase 3 production in human OA chondrocytes by Western and Northern blotting. Furthermore, we explored the intracellular signaling pathways through which HGF induced collagenase 3 production. RESULTS: This study showed that HGF stimulated collagenase 3 production in human OA chondrocytes at the transcriptional level, and this induction was mediated by activation of the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway, but not the p38 mitogen-activated protein kinase (MAPK). The p44/42 MAPKs were also phosphorylated and the use of their specific inhibitor (PD 98059) did not affect HGF-induced collagenase 3 production in OA chondrocytes. Induced collagenase 3 production via the SAPK/JNK pathway was mediated, at least in part, by the TRE site in the promoter, and in the activator protein 1 complex, c-Jun, JunD, and Fra-1 were activated. Surprisingly, further experiments revealed that the specific p38 MAPK inhibitor SB 202190 also inhibited collagenase 3 production early in the HGF-induced process. The 50% inhibitory concentration was as low as 50 nM, which is unlikely to be related to p38 MAPK inhibition (which is usually in the microM range), suggesting the involvement of another kinase sensitive to SB 202190. CONCLUSION: This is the first study to show that HGF has the ability to induce both the expression and synthesis of collagenase 3 in OA chondrocytes. The effect is mediated by kinase cascades involving SAPK/JNK and another, unidentified kinase. This study provides novel information implicating a role for HGF in the pathophysiology of OA through its effect on the production of collagenase 3, which is an enzyme that is possibly involved in OA cartilage remodeling.

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.

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

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.

Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling.

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

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.

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.

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.

Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling.

In an effort to isolate novel growth factors, we identified a human protein, designated Sk, that co-eluted with Neuregulin during chromatographic separation of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell line. Degenerate oligonucleotides based on amino-terminal sequence analysis of the purified protein were used to isolate the corresponding cDNA from a library generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase transcript was expressed in several embryonic tissues. Transfection of mammalian cells demonstrated that the protein was secreted and expressed as a doublet of approximately 35 kDa. In vitro translation and endoglycosylase analysis indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A search of the GenBankTM data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, human Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations and inhibited the Wnt-2 induced increase in uncomplexed beta-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on beta-catenin regulation.

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.

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor.

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.

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.

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.

Differential effects of hepatocyte growth factor isoforms on epithelial and endothelial tubulogenesis.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a pleiotropic cytokine that acts as a mitogen, motogen, and morphogen for a variety of cell types. HGF/NK1 and HGF/NK2 are two naturally occurring truncated variants of HGF/SF, which extend from the NH2 terminus through the first and second kringle domain, respectively. Although these variants have been reported to have agonistic or antagonistic activity relative to HGF/SF in assays of cell proliferation and motility, their potential morphogenic activity has not been investigated. To address this issue, we assessed the ability of HGF/NK1 and HGF/NK2 to induce tube formation by (a) MCF-10A mammary epithelial cells grown within collagen gels and (b) human umbilical vein endothelial (HUVE) cells grown on Matrigel. We found that HGF/NK1 stimulated tubulogenesis by both MCF-10A and HUVE cells, whereas HGF/NK2 did not stimulate tubulogenesis, but efficiently antagonized the morphogenic effect of full-length HGF/SF. HGF/NK1 and HGF/NK2 also had agonistic and antagonistic effects, respectively, on MCF-10A cell proliferation and HUVE cell migration. These results demonstrate that HGF/NK1, which only consists of the NH2-terminal hairpin and first kringle domain, is sufficient to activate the intracellular signaling pathways required to induce morphogenic responses in epithelial and endothelial cells. In contrast, HGF/NK2, which differs from HGF/ NK1 by the presence of the second kringle domain, is devoid of intrinsic activity but opposes the effects of HGF/SF. The differential properties of the two HGF/SF isoforms provide a basis for the design of more potent HGF/SF agonists and antagonists.

Colocalization of heparin and receptor binding sites on keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. FGFs are also known as heparin-binding growth factors because they bind to heparin and their physical and biological properties are modulated by heparin. Consistent with a role as a paracrine effector, KGF is produced by cells of mesenchymal origin but is active primarily, if not exclusively, on epithelial cells. KGF is involved in a variety of physiological processes, including proliferation, differentiation, wound healing, and cytoprotection. To identify regions in KGF that contribute to heparin and tyrosine kinase receptor interactions, nine peptides spanning defined motifs in the predicted structure of KGF were synthesized, and their heparin and receptor binding properties were analyzed. Peptides at the amino and carboxyl termini bound heparin, and one peptide showed relative binding comparable to that of KGF. Competitive binding studies showed that this peptide along with two other overlapping peptides specifically displaced KGF bound to the KGF receptor. These three peptides were also selectively recognized by a neutralizing monoclonal antibody against KGF, though only in the presence of heparin. Together, these data suggest that the sites for heparin and receptor binding both reside in the amino and carboxyl termini of KGF, which are spatially juxtaposed in the predicted three-dimensional structure of this molecule.

Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases.

SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.