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REVIEW |
a Division of Molecular Oncology, IRCC, Institute for Cancer Research, University of Torino School of Medicine, 10060 Candiolo, Torino, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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Key Words: hepatocyte growth factor/scatter factor • macrophage stimulating protein • c-MET • RON • tyrosine kinases • growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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In epithelia, HGF is a potent survival and regeneration factor after severe tissue damage. Its ability to accelerate organ reconstruction resides in both enhancement of cell growth and modulation of complex architectural events that contribute to the reestablishment of normal tissue patterning. Indeed, HGF promotes remodeling of epithelial cells cultured in 3-dimensional collagen gels (10–13), and induces the formation of branching tubular structures in mammary gland (14) and metanephric organ cultures (15). More generally, a vast number of studies following its identification demonstrated that HGF functions in virtually every tissue of the body (16): hematopoiesis (17, 18), bone formation and resorption (19), chondrogenesis (20), angiogenesis (21, 22), and axonal chemoattraction (23, 24) are all critically controlled by HGF. Together, these features make HGF a powerful mitogenic and morphogenic molecule endowed with crucial functions during embryonic development and tissue regeneration.
During embryogenesis, the HGF receptor is expressed in the epithelial component of various organs, whereas HGF is expressed in the adjacent mesenchyme (25). Genetic inactivation of HGF or its receptor in the mouse causes embryonal lethality between E12.5 and E15.5 because of abnormal development of the placenta (26, 27). In homozygous mutant embryos, the liver is reduced in size and shows extensive loss of parenchymal cells (26). Furthermore, consistent with the scattering activity of HGF, receptor-deficient embryos lack muscles of the limbs, diaphragm, and tip of the tongue, all deriving from migratory precursors (28).
HGF-dependent epithelial morphogenesis is based on a finely tuned interplay between related phenomena including cell proliferation, motility, extracellular matrix degradation, and survival (2, 3, 6, 29, 30). In transformed epithelia, this interplay is responsible for invasive growth, a process by which cancer cells weaken tissue constraints and invade foreign districts, where they may migrate, proliferate, and survive (31). The xenophilic tendency of carcinomas is fostered by the very same events that, under physiological conditions, account for the generation and maintenance of tissue complexity.
Macrophage stimulating protein (MSP), although originally identified for its ability to make resident peritoneal macrophages responsive to chemoattractants (32), is actually capable of inducing multiple biological effects similar to those described for HGF (13, 33). Specifically, MSP can induce DNA synthesis and trigger cell scattering, invasive growth, and tubulogenesis in specific cell types (13, 34, 35).
The HGF and MSP receptors are encoded respectively by the MET (36, 37) and RON (33, 38) proto-oncogenes. The protein products of these oncogenes (39–41) are single-pass, disulfide-linked 
/ß heterodimers arising by proteolytic processing of a common precursor in the post-Golgi compartment (42–45). In both receptors, the chains are extracellular glycoproteins whereas the ß chains are transmembrane subunits responsible for the tyrosine kinase activity (
Fig. 1).
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The intracellular domains of these receptors include well-conserved tyrosine kinase catalytic sites flanked by distinctive juxtamembrane and carboxy-terminal sequences. In the Met protein, phosphorylation of the tyrosine residues in positions 1234–1235 has a positive regulatory effect on the enzyme activity (46–48), whereas phosphorylation of a serine residue in the juxtamembrane domain negatively regulates the kinase (49, 50). The carboxy-terminal domains of both receptors include two critical tyrosine residues that, when phosphorylated, together form a specific docking site for multiple signal transducers and adaptors (51–61; see 
Fig. 1). In the Met receptor, this multifunctional docking site is essential to transduce the HGF signal during mouse embryonal development (62): Tyr to Phe substitutions result in a severe loss-of-function mutant displaying a phenotype remarkably superimposable to that of Met homozygous null mice (62). Interfering with the ability of Met to bind the adaptor protein Grb2 by disrupting its optimal consensus allows development to proceed to term, sparing placenta and liver, which in these mice appear to be normal. In contrast to placenta and liver, muscles deriving from migratory precursors are heavily affected by the Grb2-directed mutation, indicating a requirement for Grb2-mediated signaling in migratory myoblasts (62).
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STRUCTURE–FUNCTION ANALYSIS OF SCATTER FACTORS: NATURAL AND RECOMBINANT VARIANTS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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family of growth factors, but also in a number of enzymes involved in the coagulation, fibrinolytic, and complement cascades, in the matrix protein thrombospondin, in adhesion molecules belonging to the selectin family, and in the low-density lipoprotein receptor (63). Whereas such domains are clearly associated with exon shuffling and duplication (64), scatter factors use an original evolutionary strategy for the modulation of their several activities. From a structural viewpoint, the overall domain organization of both HGF and MSP is remarkably similar to that of the blood protease plasminogen. During evolution, HGF and MSP have lost protease activity but have retained the proteolytic mechanism of activation of the proteases (65–68). Therefore, the activity of these growth factors does not only rely on transcriptional and translational control, but also on proteolytic events that occur in the extracellular milieu, and are the same ones that initiate blot clotting and fibrinolysis. This indicates that one possible way to inhibit scatter factor activity is to interfere with their activation mechanisms either at the site of production or at the level of target cells.
HGF, MSP, and plasminogen belong to a family of proteins defined by the presence of at least one peculiar domain known as kringle (an 80 amino acid double-looped structure formed by three internal disulfide bridges), a serine protease domain, and an activation segment located between the kringle and the protease domains. The other members of this family include apolipoprotein(a), urokinase-type and tissue-type plasminogen activators, prothrombin, factor XII, and the HGF activator protein, which has a domain organization essentially identical to that of factor XII (67; see below). In these proteases, the kringle domains and other modules contained within one subunit bind to specific sites (e.g., to fibrin or to plasminogen activator receptor), whereas the second subunit, containing the serine proteolytic activity, cleaves the specific substrate (69, 70). Among the members of this family, HGF and MSP are unique in that they do not possess intrinsic enzymatic activity and are ligands for transmembrane tyrosine kinases.
HGF is secreted as a single-chain, biologically inert glycoprotein precursor (pro-HGF). Under appropriate conditions, pro-HGF is converted into its bioactive form by proteolytic digestion within two positively charged amino acids (the so-called dibasic site Arg494-Val495). Mature HGF is a heterodimer consisting of a 62 kDa chain and a 32/34 kDa ß chain held together by a disulfide bond ( Fig. 2).
The nucleotide sequences of human, rat, and mouse HGF cDNA predict that both chains are encoded by a single open reading frame resulting in a 728 amino acid polypeptide. Starting from the amino terminus, the chain of HGF contains a hairpin loop of about 27 amino acids (homologous to the preactivation peptide of plasminogen), followed by four kringles, whereas the ß chain contains the serine protease-like structure (8).
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The lack of proteolytic activity in the HGF molecule is due to the replacement of the histidine and serine residues contained within the catalytic site of serine proteases with glutamine and tyrosine, respectively; however, the residues surrounding the active site are quite well conserved in the HGF ß chain. From a structural viewpoint, analysis of the alignment of HGF with conventional serine proteases ( Fig. 3)
points out a deletion in the HGF molecule at residue Glu575, corresponding to a loop placed at the entrance of the catalytic site. Lack of this loop, together with the larger size of the side chains within the amino acids replacing the catalytic triad, makes these residues more exposed and available for interactions with other molecules (L. Pugliese, unpublished observations).
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The HGF gene is composed of 18 exons interrupted by 17 introns and spans in about 70 kb (71). The overall genomic organization of the HGF gene is closely related to that of plasminogen (72), as expected from the relatively high homology in amino acid sequence between the two gene products.
By exploiting the well-defined modular organization of HGF, several structure–activity relationship studies have been performed (73–75). Mutants in which cleavage of pro-HGF into the two-chain form is blocked are completely defective for mitogenic and motogenic activity and are unable to activate the receptor tyrosine kinase, but retain substantial binding capacity (74, 75); moreover, the separately expressed ß chain in its wild-type form is devoid of any activity (74). By progressive deletions of the HGF molecule through sequential carboxy-terminal truncations, several variants have been generated and tested for receptor binding capacity and mitogenic effect (75, 76). All variants containing either the chain alone or proteins truncated after the third or second kringle are still capable of binding to the receptor but do not induce mitogenic activity.
The mutant consisting of the amino-terminal region and the first two kringle domains of HGF (HGF-NK2), although unable to enhance DNA synthesis, can efficiently stimulate the receptor autocatalytic activity and dissociate epithelial cell monolayers (74). The fact that HGF-NK2 retains the ability to bind to the receptor, stimulates its tyrosine phosphorylation, and activates the scattering machinery, yet displays antagonistic activity toward native HGF for DNA synthesis, indicates that this variant behaves as a partial agonist (74). The HGF-NK2 molecule can also be found as a naturally occurring variant resulting from an alternative RNA splicing event. The sequence is identical to that of HGF cDNA, including the 5' untranslated region, and diverges precisely at the end of the K2 domain, where the open reading frame continues for two additional amino acids followed by an in-frame stop codon (77); identical to the recombinant form, the natural truncated ligand behaves as a partial agonist.
A second alternatively spliced variant consisting of the HGF amino-terminal sequence and the first kringle domain (HGF-NK1) has recently been identified (78). In contrast to what has been observed for HGF-NK2, the biological activity of the natural HGF-NK1 variant differs from that displayed by its artificially engineered version: whereas the natural form possesses mitogenic and scattering activity, although with 50-fold lower specific activity, the recombinant variant retains receptor binding activity but does not induce receptor tyrosine autophosphorylation or enhancement of DNA synthesis (76, 78); this could be due to compromised protein refolding or lack of fidelity in disulfide bond formation in the bacterial expression systems used for production of the engineered mutant.
Finally, a third natural variant lacks a stretch of 15 nucleotides at the 5' end of the fifth exon (nucleotides 483–497 of the coding sequence), corresponding to an in-frame deletion of 5 amino acids in a loop of the first kringle domain (71, 74, 79, 80). When tested for its ability to induce tyrosine autophosphorylation of the receptor, mitogenesis, and scattering of epithelial cells, this variant was found to be as highly active as the wild-type molecule (74), implicating that the loop formed by these residues is not involved in receptor recognition.
To characterize further the receptor binding domain of HGF, individual deletions of each kringle have been carried out (75). According to this study, only deletion of the first kringle is accompanied by a substantial reduction in receptor binding capacity, suggesting that the primary receptor binding site is located within this region. The first kringle was subjected to a combined approach of mutational analysis and computer modeling (81): two patches of residues presumably involved in receptor binding were identified. These patches, predicted to be surface-exposed, are placed at opposite sides of the kringle: the first one includes residues Glu159, Ser161, Glu195, and Arg197; the second comprises Asp171 and Gln173. Alanine substitution of Glu159 and Arg197 generates variants endowed with 50-fold reduced activity and affinity for the receptor. Likewise, site-directed mutagenesis of the residues lying in the second patch results in nearly 10-fold reduction of ligand specific activity. These data suggest that the two patches may define different contact points for the receptor.
Together, these extensive mutational analysis experiments can be summarized as follows. 1) The receptor binding domain is contained within the first kringle domain. 2) The functional domain responsible for activation of the motogenic response resides within the amino terminus and the first two kringle domains. 3) The third and fourth kringle domains are not sufficient per se for the induction of mitogenesis and motogenesis. 4) The ß chain, although not required for receptor binding, contributes to receptor activation. 5) Full biological response following stimulation at physiological doses is exerted only by the wild-type molecule. 6) Cleavage of HGF into the two subunits is required for proper induction of the biological response.
The synthesis and modular structure of MSP is remarkably similar to that of HGF. Even in the case of MSP, the molecule is secreted as a biologically inactive single-chain precursor, which is then converted into the active heterodimer by endoproteolytic cleavage. The translated amino acid sequence of the cDNA predicts a 711 amino acid polypeptide (82–84). Similar to HGF, the 53 kDa chain consists of an amino-terminal hairpin loop, followed by four kringle domains; the 25 kDa ß chain contains the serine protease-like domain, but is devoid of enzymatic activity due to amino acid substitutions in the catalytic triad ( Fig. 2). Again, the strict structural connection with HGF is also evident at the genomic level: like HGF, MSP is composed of 18 exons separated by 17 intervening sequences (83, 84).
Recently, partial structure–function analyses of the MSP molecule have been carried out (85, 86). Several variants were used, including pro-MSP, mature MSP, MSP and ß chains, and an amino-terminal segment of recombinant MSP comprising the first two kringles (MSP-NK2); in addition, deletion mutants lacking each individual kringle were generated. Unexpectedly, these studies show that the ß, serine protease-like chain of MSP contains a binding site for the receptor. However, binding of the isolated ß chain is not followed by activation of the receptor kinase activity and does not induce any biological activity; the mature /ß disulfide-linked heterodimer is required to fully activate the receptor. The MSP/NK2 mutant appears to act in an agonistic fashion with respect to its ability to induce mitogenic and macrophage stimulating activities. This finding, together with the observation that MSP/NK2 triggers tyrosine autophosphorylation of the receptor (33), might indicate that two receptor binding sites are located within the same molecule, one along the NK2 region and the other on the ß chain. Saturation and competition binding assays with purified ligand variants are necessary to clarify this issue.
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ACTIVATION MECHANISMS OF THE SCATTER FACTORS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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Four proteases are reported to activate HGF in vitro by cleavage of the zymogen at the Arg494-Val495 dibasic site: urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (65, 66); a serine protease isolated from serum, homologous to coagulation factor XII (67); and coagulation factor XII itself (88).
The proteolytic maturation produced by uPA may take place either at the producer or target cell level. When pro-HGF is incubated with uPA for prolonged periods, the limiting step in the yield of active factor is the concentration of enzyme added and not the initial concentration of substrate (89). This indicates a stoichiometric reaction and not a catalytic mechanism. The stoichiometric reaction of uPA with pro-HGF has been explained with the formation of a stable complex between the reaction products either in the extracellular medium or on the membrane of target cells, where the molecules are bound to their specific receptors. Within the complex, pro-HGF is processed to the two-chain form, which binds with high affinity and triggers the receptor kinase. Such a stoichiometric reaction is supposed to provide tight control on the extent of pro-HGF extracellular activation. The levels of uPA activity would in fact titrate the yield of bioactive HGF within the tissue microenvironment; uPA activity would, in turn, be subjected to strict spatial and temporal regulation by the expression level of the protein and its interplay with activators, inhibitors, and cellular receptors (90).
The HGF precursor can also be processed by a serum-derived serine protease (67). This soluble glycoprotein, known as HGF activator, appears to be produced mainly by hepatocytes as an inactive 96 kDa zymogen and further processed into the bioactive 34 kDa form by remotion of the amino-terminal half and proteolytic cleavage of the carboxy terminus at the bonds between Arg372 and Val373 and between Arg407 and Ile408, giving rise to a disulfide-linked heterodimer. The overall amino acid sequence of the HGF activator precursor shows extensive sequence similarity with blood coagulation factor XII, a serine protease capable of initiating the blood coagulation cascade, the fibrinolytic system, and the production of kinins (91). Activation of the zymogen of HGF activator is generated by thrombin through cleavage of the bond between Arg407 and Ile408, which produces a fragment able to convert the HGF precursor into the two-chain active form (92). HGF is expressed in the inactive single chain form in normal tissues and is converted into the heterodimeric active form specifically in the injured tissues (93); the locally restricted generation of active HGF in the injured tissues is most likely mediated by HGF/SF1 activator, which, in turn, is activated by thrombin during blood coagulation at the injured site (88). In contrast to uPA, HGF activator works as a typical catalyst (67), thus possibly effecting quantitative activation of all-stored pro-HGF. According to these data, HGF activator may bring about quantitative activation of pro-HGF in response to the triggering of the blood coagulation cascade, as in the case of tissue injury, whereas uPA may effect a more restricted activation of the precursor in the tissues and on the membrane of target cells under conditions other than trauma or injury (84; Fig. 4).
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In vitro conversion of pro-MSP into the bioactive two-chain molecule can be operated by three contact enzymes of the intrinsic coagulation system: kallikrein, factor XIIa, and factor XIa (94). However, in contrast with the activities of pure coagulation enzymes, freshly shed venous blood does not cleave pro-MSP, and the protein found in serum or EDTA plasma is the zymogen, not the mature heterodimer (94). Recently, a membrane-associated pro-MSP convertase activity has been demonstrated in resident murine peritoneal macrophages (95), suggesting that macrophage pro-MSP proteases and serum protease inhibitors are involved in a pro-MSP/MSP control system. Under inflammatory conditions or within injured sites, increased vascular permeability may allow diffusion of pro-MSP from the circulation into tissues, where the macrophage converting enzyme can cleave pro-MSP to the biologically active form. Hence, the final concentration of mature MSP at tissue sites is probably determined by the cooperative activity of several factors, including other macrophage-bound enzymes that degrade either pro-MSP or MSP and the local presence of serum-derived inhibitors of different surface-associated proteases.
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SULFATED POLYSACCHARIDES AS MODULATORS OF SCATTER FACTOR ACTIVITIES |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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chain (6), by the elution of the site by excess heparin (9), and by direct binding of HGF to sulfoglycolipids (97) and heparan-sulfate (98, 99). The physiological role of heparin binding of HGF has been examined in detail. Extensive mutational analysis studies have provided evidence that the amino-terminal hairpin loop and the second kringle domain within the HGF molecule are essential for heparin binding of this growth factor (100, 101). Sequences of heparin binding sites of many polypeptide growth factors contain a cluster of basic amino acids and a high positive net charge, presumed to be important for interacting with anionic moieties (102); accordingly, both the hairpin loop and the second kringle display a positive net charge in the HGF molecule. This charge is lower in the hairpin loop of the MSP ligand and possibly accounts for the different elution properties showed by the two growth factors when loaded onto heparin columns (101); the strength of heparin binding is in fact much higher in HGF than in MSP. The polysaccharide structural determinants involved in heparan sulfate binding to HGF have been studied as well and found to require 6-O-sulfation and a carbohydrate module of at least six saccharides (99).
Recently, it has been demonstrated that sulfated polysaccharides such as heparin and heparan-sulfate can enhance the potency of HGF in terms of growth rate and receptor autophosphorylation (103). This enhancement is dependent on oligosaccharide unit size, specifically on the presence of at least six glucose units (see previous paragraph). Those oligosaccharide preparations that potentiate HGF activity are also able to promote the formation of stable HGF oligomers, thus leading to the hypothesis that the presentation of multivalent HGF molecules, induced by the binding of HGF to sulfated epitopes on heparan sulfate proteoglycan chains, may facilitate subsequent receptor dimerization and triggering of the biological response.
Glycosaminoglycans have also been found to stabilize HGF/NK1 and HGF/NK2 variants and to induce their oligomerization, thus activating their full biological effect (104). The authors claim that the antagonistic behavior previously ascribed to HGF/NK1 and HGF/NK2 is, in fact, cell type-specific and hypothesize that these variants can induce anti- or promitogenic effects depending on the heparin-like composition of the cells used. According to this assumption, one might explain the reason why HGF/NK2 retains scattering but not mitogenic activity (74) on the basis of the fact that the two biological responses have been examined in different cell types expressing different glycosaminoglycan moieties. The hypothesis that, under given conditions, HGF natural splice variants can recapitulate most of the biological functions of the parental ligand is further supported by in vivo models of HGF/NK1 transgenic mice (105). These animals display most of the phenotypic features associated with wild-type HGF transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors (105–107). Together, the above results suggest that changes in glycosaminoglycan structure on the surface of a target cell, either through modifications in the proteoglycan repertoire or through alterations produced by enzymes of the cell microenvironment, may provide a novel mechanism by which responsiveness to canonical and variant HGF isoforms can be locally modulated in several physiological and pathological conditions.
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THE OTHER SIDE OF THE COIN: THE MET AND RON RECEPTORS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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The extracellular domains of Met and Ron do not contain obvious protein patterns, but comprise a number of cysteines well aligned in their relative positions. An eight-cysteine motif identified by us and conventionally termed MRS (for Met-related sequence) is strictly maintained in a family of proteins displaying sequence similarities with scatter factor receptors, the so-called sex gene family, which is the human counterpart of the Xenopus and mouse plexin genes (31, 111–113; see Fig. 1).
Few data are available on the specific role of Met and Ron extracellular domains in scatter factor-mediated signal transduction pathways. By producing a soluble chimeric protein consisting of the /ß extracellular domain of Met fused with a portion of the human IgG
1 heavy chain, Mark and collaborators (114) have shown that Met extracellular domain is sufficient to mediate high affinity ligand binding and that a noncleavable mutant, in which generation of the mature /ß heterodimer is prevented, binds HGF with an affinity similar to that of wild-type Met. Therefore, the establishment of high affinity binding sites for Met is independent of the transmembrane and cytoplasmic domains and does not require correct processing of the uncleaved precursor. A natural carboxy-terminal truncated variant, lacking the cytoplasmic and transmembrane domains but with preserved heterodimeric structure, is released into the culture medium by proteolytic processing of the wild-type membrane form (115). This truncated form might conceivably interfere with the Met receptor signal transduction pathway by competing with the intact receptor for binding to the ligand.
It has been demonstrated that truncation of Met extracellular domain results in constitutive activation of the kinase (116). After translocation, replacement of both the extracellular and the juxtamembrane domains by a dimerization motif generates an oncoprotein that is highly transforming in vitro and tumorigenic in vivo (117, 118). By contrast, truncation of the extracellular domain alone (cyto-MET) creates a constitutively activated tyrosine kinase that is only weakly transforming (119). Unexpectedly, expression of cyto-MET in transgenic hepatocytes does not result in tumor formation, but rather renders hepatocytes permissive for immortalization (29), presumably as a consequence of a cyto-MET-mediated protection from programmed cell death.
In a colon carcinoma cell line, surface exposure of an uncleaved 190 kDa Met protein has been reported (120). Lack of proteolytic cleavage of the single-chain precursor is not due to genetic events such as amplification, rearrangement, mutations, or protein overexpression, but to the absence of a specific proteolytic enzyme of the trans-Golgi network. This uncleaved form of the Met receptor is constitutively tyrosine phosphorylated: it is likely that the uncleaved extracellular domain mimics the activating conformation reached by the canonical /ß heterodimer on binding to HGF (120).
A noncleaved variant has also been reported for the MSP receptor Ron (121). Unlike the Met uncleaved form, the Ron variant is not generated by defective posttranslational processing but by alternative splicing of a 147 bp cassette exon encoding for a short segment of the Ron extracellular domain (
-Ron). This in-frame deletion induces a critical change in the protein structure by creating an unbalance of the cysteine pairs that renders the uneven cysteines available for intermolecular disulfide bonding with other -Ron partners, creating oligomers. Oligomerization leads to segregation of the protein in the intracellular vesicular compartment and to its constitutive tyrosine phosphorylation, which in turn activates downstream motile invasive events independent of exogenous signaling.
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A POTENTIAL SCENARIO FOR SCATTER FACTOR RECEPTOR ACTIVATION |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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Several speculations can be inferred to draw a potential scenario for scatter factor receptor activation. Scatter factors seem not to homodimerize spontaneously; thus, receptor dimers probably are not stabilized by simple ligand bivalency, at least under standard conditions in vitro. Based on the observation that multimeric forms of the Met receptor can preexist in the absence of ligand (123), dimer stabilization might be accomplished by direct receptor–receptor interactions. In addition, local receptor concentration within the plane of the plasma membrane could be tightly controlled by ligand and/or receptor interaction with extracellular matrix or membrane molecules, including uPA, uPa receptor, and proteoglycans, which together could form a multimolecular complex. The findings that heparin is crucial in inducing HGF dimerization and that cellular responses to HGF depend on glycosaminoglycan composition of the cell membrane further support the hypothesis that receptor activation requires a cooperative participation by multiple surface and soluble components. According to the above data, one could speculate that scatter factors, weakly adsorbed onto matrix or cell surface proteoglycans and then assembled into oligomeric moieties, display multivalent interactions with preclustered receptors; proper growth factor activation at target cell sites would be accomplished by noncovalent interactions with uPA and uPA receptor.
Alternatively, the observation that there may be multiple contact points between the first kringle of HGF and the receptor (81) is consistent with a model of ligand multivalency under monomeric conditions, similar to growth hormone and tumor necrosis factor (124–126). This hypothesis is in accordance with the data obtained for the MSP molecule, where both the NK2 segment and the ß chain seem to interact with the Ron receptor.
No data are now available on whether the transmembrane and cytoplasmic portions of the scatter factor receptors may be positively involved in promoting dimerization, as in the case of EGF receptor and Neu protein (127–129), or in restricting dimer formation, in analogy with Kit (130). It has been reported that release of an extracellular proteolytic derivative of Met is up-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment (115). Because TPA-dependent activation of protein kinase C negatively regulates the Met receptor tyrosine kinase activity by increasing serine phosphorylation of the ß chain (49), one could argue that antagonizing of cellular responses to HGF could be elicited by TPA through a double mechanism consisting of biochemical down-modulation and receptor competition by the Met proteolytic form.
Detailed mutational analysis must now be integrated by high-resolution X-ray crystallographic and nuclear magnetic resonance studies to shed light on the 3-dimensional scaffold of the complexes at the cell membrane. This approach will provide additional clues about the structural organization of ligands and receptors and about their modular function, which could then be explored by substitutions of candidate residues.
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SCATTER FACTORS AS THERAPEUTIC TOOLS FOR ORGAN FAILURE AND CANCER |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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The structure–function relationship studies performed so far on the HGF and MSP molecules (73–76, 85, 86) have highlighted the importance of the intact, two-chain molecule for proper execution of full functional responses; they have also demonstrated that deletion of one or more critical domains generates variants able to exert selective biological effects or even to abrogate cellular responses, thus limiting the harmful potential of wild-type factors. Some of these recombinant variants exist as naturally occurring isoforms; such natural mutants behave as antagonists or partial agonists and, because they are smaller than parental factors, can have pharmacological applications.
Recent work (103, 104) has established there are important differences in scatter factor activity that depend on the glycosaminoglycan composition of the cells used. This suggests that some effects of scatter factors can be discriminated, hence selectively controlled, according to the cell types used and, in vivo, on the basis of the proteoglycan repertoire of the tissues involved. Moreover, responsiveness to canonical and variant HGF isoforms may be locally modulated by chemically induced modifications of surface-associated sulfated oligosaccharides.
Modulation of scatter factor activity can also be obtained by interfering with their finely tuned activation mechanisms. The presence of both surface-associated convertases such as uPA, which act through stoichiometric reactions (89), and serum enzymes working as typical catalysts (92–95) suggests that the final concentration of scatter factors at tissue sites is subjected to strict spatial and temporal regulation by their interplay with activators, inhibitors, and cellular receptors, and that the yield of bioactive factors is titrated by either enzyme activity, enzyme concentration, or substrate concentration. This indicates that even slight perturbations of such a critical equilibrium within the tissue microenvironment can produce striking differences in the extent of scatter factor activation, and thence in their biological effects.
At the therapeutic level, scatter factors and their variants could be exploited for both organ regeneration and protection as well as inhibition of tumor invasive growth. For example, HGF is considered to be a good candidate for a renotrophic factor: it induces mitogenic and morphogenic responses in renal tubular cells (131) and is able to accelerate regeneration of tubular parenchymal components after acute renal injury in mice (132, 133). Furthermore, HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats as a trigger for compensatory growth (134, 135). With regard to chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction by sustaining tubular growth and impairing interstitial expansion (136). Finally, HGF has been shown to prevent severe renal dysfunction caused by cisplatin (132), possibly because of its anti-apoptotic activity. From this standpoint, scatter factors might prove powerful molecules to reduce kidney damage during cisplatin-based chemotherapy. Preliminary data from our laboratory indicate that HGF truncated variants, whose smaller size accounts for better pharmacological activity, can protect kidney cells from drug-induced programmed cell death at doses comparable to those of the parental factors.
HGF has also been shown to be a potential therapeutic agent for peptic ulcer disease (137). Indeed, HGF is the strongest mitogen for the gastric epithelium compared to insulin or EGF. Moreover, HGF is able to induce a motile response in gastric epithelial cells, thus facilitating the reestablishment of mucosal integrity through the rapid migration of epithelial cells across the wound margins. The observation that HGF is produced by gastric fibroblasts (137) further supports the issue that this growth factor plays a key role in the mesenchymal–epithelial interactions of the gastric mucosa during repair processes.
Partial hepatectomy, acute liver failure, and acute lung injury are also common pathologies where the use of HGF as a mitogenic tool to repopulate damaged tissues can be potentially applied (138–146). As far as chronic diseases are concerned, HGF has been demonstrated recently to be effective in retarding progression of liver cirrhosis (147–149) or pulmonary fibrosis (150).
So far, no in vivo studies on potential therapeutic applications of the MSP molecule have been performed. The MSP receptor is localized at the apical surface of ciliated epithelia in the airways and the ligand is found in the bronchoalveolar space at biologically significant concentrations (151); similarly, MSP mRNA is expressed in the epithelium of epididymis and RON mRNA is expressed in sperm (152). The observation that activation of the Ron receptor by MSP leads to a significant increase in ciliary beat frequency of human nasal cilia suggests that the MSP-Ron signaling pathway could be a novel regulatory system of mucociliary function and might be involved in host defense and fertilization.
The use of scatter factor competitors or inhibitors to interfere with the onset of tumor metastases is also a crucial issue deserving intense research. Ideally, soluble peptides mimicking the ligand–receptor recognition sites should displace the binding of the growth factor to the kinase receptor, thus inhibiting the transduction of invasive signals. Similarly, interaction of scatter factors with their cognate receptors could be inhibited by ligand recombinant variants in which cleavage into the two-chain active form is blocked; these molecules retain high binding capacity but are devoid of any biological activity, thus acting as powerful competitors. Ongoing in vivo studies with transgenic models of multistep carcinogenesis will better define the role of scatter factors in tumor progression in order to counteract their effect with oriented therapeutic strategies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: HGF, hepatocyte growth factor; MSP, microphage stimulating hormone; EGF, epidermal growth factor; pro-HGF, HGF secreted as a single-chain, biologically inert glycoprotein precursor; : uPA, urokinase-type plasminogen activator.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSTRUCTURE–FUNCTION ANALYSIS...ACTIVATION MECHANISMS OF THE...SULFATED POLYSACCHARIDES AS...THE OTHER SIDE OF...A POTENTIAL SCENARIO FOR...SCATTER FACTORS AS THERAPEUTIC...REFERENCES |
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