Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Biochimica et Biophysica Acta, 1155 (1993) 357-371 357 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00

BBACAN 87280

Hepatocyte growth factor/scatter factor and its receptor, the c - m e t proto-oncogene product

Jeffrey S. Rubin *, Donald P. Bottaro and Stuart A. Aaronson 1

Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892 (USA)

(Received 27 September 1993)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

II. HGF/SF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 A. Purification and physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 B. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 C. cDNA cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

1. Gene structure and chromosomal localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 2. Alternative mRNA transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

D. Structure/function analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 E. Distribution and regulation of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

III. The HGF/SF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 A. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 B. The c-met proto-oncogene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 C. Regulation of c-Met activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 D. Post-receptor signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

IV. Broad perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 A. HGF/SF- and c-Met-related molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 B. Clinical relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 C. Summary and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

I. Introduct ion

Cellular prol iferat ion, migrat ion, d i f ferent ia t ion and p rog rammed cell dea th all par t ic ipate in the develop- men t and m a i n t e n a n c e of mult icel lular organisms.

These processes require coord ina ted in teract ions between mul t ip le cell types and are of ten media ted by polypept ide growth factors acting in ei ther an au-

tocrine or paracr ine fashion. The ident i f icat ion of these growth factors and the biochemical pathways responsi-

* Corresponding author. Fax: + 1 (301) 4968479. 1 Present address: Derald H. Runenberg Cancer Center, Mount

Sinai Medical Center, 1 Gustave L. Levy Place, P.O. Box 1130, New York, NY 10029.

ble for the t ransduc t ion and implementa t ion of their signals are essential first steps toward an unde r s t and- ing of growth control.

The specific target ing of growth factor action is accomplished by the expression of high-affinity receptors in responsive cells. High-affini ty b ind ing be tween growth factor and receptor improves the probabil i ty of interact ion, which is part icularly impor tan t as growth factors are f requent ly found at low concentra t ions . Growth factor receptors general ly possess three func- t ionally distinct domains: an extracellular domain which conta ins the growth factor b ind ing site, a t r ansmem- b rane region which anchors the receptor to the cell surface, and an intracel lular domain which can interact with other intracel lular molecules and thereby activate

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growth-related biochemical pathways. In eukaryotic cells, such pathways are regulated by protein phosphorylation, and the intracellular domains of many receptors contain an intrinsic tyrosine kinase activity that is stimulated upon growth factor binding [1-3]. Phospho- rylation of various protein substrates alters a remarkably complex array of intracellular enzymatic activities and structural components, triggering changes in gene expression and ultimately cellular functions [4-7]. Elu- cidation of these substrates and pathways is a major focus of research today.

A specific example of the exquisite regulation of growth in the adult mammal involves liver regeneration. Following injury or removal of as much as two thirds of the liver, the organ can undergo a repair process that restores its normal state (reviewed in Ref. 8). The mechanisms responsible for this phenomenon have long been the subject of inquiry. Using a parabi- otic model, several laboratories demonstrated that factor(s) in the circulation of a hepatectomized animal could stimulate hepatocyte proliferation in another animal with an intact liver [9-13]. This suggested that a hormone-like activity was present in the bloodstream of the hepatectomized animal and thus indicated a source from which it could be isolated. Purification was facilitated by the development of a convenient mitogenic assay involving primary cultures of rat hepatocytes [14-16].

While the isolation of this activity, designated hepatocyte growth factor (HGF) was underway, independent studies led to the identification of the same molecule as being responsible for a variety of other biological activities. It is now recognized that HGF is a growth factor for a number of cell types in addition to hepatocytes, as well as scatter factor (SF), a molecule capable of dispersing certain epithelial and endothelial cell colonies. Accordingly, the factor was renamed HGF/SF. Identification of its high-affinity receptor as the c-met proto-oncogene product has accelerated efforts to explore the potential relevance of these proteins to neoplasia. What follows is a summary of the rapidly expanding literature concerning H G F / S F signal transduction pathways, as well as the role of this molecule in normal and abnormal growth and repair processes.

lI. HGF / SF

II-A. Purification and physical properties

H G F / S F was originally purified to homogeneity from rat platelets [17], human plasma from patients with fulminant hepatic failure [18] and rabbit serum [19], consistent with the observation of hepatocyte growth-promoting activity in the systemic circulation. It was independently isolated from the conditioned

medium of cultured fibroblast cell lines based on its mitogenic activity for a wide range of cellular targets [20], or its cellular scatter activity [21,22]. Heparin-Sep- harose chromatography provided a major enrichment step in many of the purification schemes [17-24]. The strong affinity of H G F / S F for heparin, reflected in the requirement of ~ 1.0 M NaC1 for elution, may have physiological relevance as H G F / S F is reportedly se- questered in the extracellular matrix (ECM) of an organ culture system [25]. With an efficient recovery of activity and estimated enrichment of several thousand- fold, the yield of H G F / S F in these preparations was typically on the order of tens of micrograms from many liters of starting material or the platelets of thousands of rats. This is indicative of the relative scarcity of this potent molecule.

H G F / S F is acid- and heat-labile [26], and its mitogenic as well as motogenic activities are inhibited by heparin or its synthetic analog, suramin [19,20,27]. It was purified from platelets and serum or plasma as a disulfide-linked heterodimer consisting of a 55-60 kDa heavy (alpha) chain and a 32-34 kDa light (beta) chain [17-19]. There was evidence of microheterogeneity in both of these chains which may be attributable to proteolysis or differences in glycosylation. When isolated from fibroblast culture fluids, a ~90 kDa monomeric polypeptide co-purified with this heterodimer [20-22]. The monomeric and heterodimeric molecules co-migrated in non-reducing SDS-PAGE, and trypsin [22] and V8 [20] proteolytic peptide map- ping suggested that the two were structurally related. Subsequently, molecular cloning and recombinant expression of H G F / S F revealed that it was synthesized and secreted in the monomeric 90 kDa form [20,28]. Several laboratories have shown that proteolytic conversion of the monomeric precursor to the heterodimer, which can occur in situ, is required for biologic activity [29-33]. The hepatocyte growth-promoting activity detected in plasma or serum had an apparent molecular weight of 150-200 kDa based on molecular sieving chromatography [26,34,35]. Barring anomalous behavior, this raises the possibility that H G F / S F may associate with other molecules when in the circulation. If so, as in the case of insulin-like growth factors [36], interactions with binding proteins could modulate H G F / S F activity.

II-B. Biological activities

H G F / S F stimulates DNA synthesis and /o r the proliferation of rat and human hepatocytes [17-19,37] and biliary epithelial cells [38], human mammary epithelial cells [20], mouse [20,39] and human [40] keratinocytes, rat [39,41] and rabbit [42] renal tubular epithelial cells, human melanoeytes [20,43,44] and melanoma [39,44] cells, as well as human vascular

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endothelial cells [20,45,46]. H G F / S F also supports the proliferation and differentiation of myeloid progenitor cells from mouse bone marrow [47], and the differentiation of the promyelocyte leukemia cell, HL-60 into granulocyte-like cells [48].

A convergence of research efforts occurred with the discovery of an extraordinarily high amino-acid sequence homology of HGF and the protein known as scatter factor (SF) [49]. SF was first described by Stoker and colleagues [50,51] as a fibroblast-derived motility factor for epithelial cells that did not stimulate proliferation of Madin-Darby canine kidney (MDCK) cells, its prototypical target. When cells were seeded in plas- tic petri dishes or microtiter wells, the factor prevented or disrupted the formation of mutually adherent cell colonies. Moreover, the cells acquired a spiky, fibrob- lastoid phenotype that contrasted with the polygonal shape characteristic of epithelial cells. SF had a similar effect on human primary mammary epithelial ceils [50,51] and several carcinoma cell lines [22]. Subse- quently, vascular smooth muscle cells were identified as another source of SF [23] which was also shown to stimulate the motility of vascular endothelial cells [52]. The knowledge that HGF and SF were both produced by fibroblasts, active on epithelial and endothelial cells, purified by similar means, and had similar physical properties led to the idea that they were closely related. The efforts of a number of laboratories exchang- ing reagents and using a combination of molecular cloning, biological, biochemical and immunological analysis has established that HGF and SF are identical [49,53-56]. As exemplified by the MDCK line, some cells respond to H G F / S F solely with changes in cell shape and motility, while others undergo proliferation with or without a concomitant increase in motility [22,51,57]. The factors that determine which particular cellular response is manifested remain to be elucidated.

In addition to being a mitogen and motogen, H G F / SF can also function as a morphogen in the appropri- ate context. Montesano et al. [58] observed that MDCK cells formed organized, tubular structures when grown in collagen gels in the presence of fibroblast-conditioned medium or when grown with fibroblasts under conditions that prevented heterocellular contact. Using HGF/SF-specific neutralizing antiserum and recombinant growth factor, the same group further established that H G F / S F was responsible for this effect [59]. Subsequently, others reported a similar influence of H G F / S F on the formation of lumen-like structures by human epithelial carcinoma cell lines in vitro [60]. They observed activated H G F / S F receptors on the luminal surface of cells forming these structures both in vitro and in sections of whole breast tissue. More recently, H G F / S F was reported to induce non- parenchymal liver epithelial cells to form a network of

anastomosing tubules in a collagen matrix [61]. H G F / SF also stimulates the formation of tubular structures by vascular endothelial cells in culture [62] and angiogenesis in vivo [46,63].

Cell scattering and tubular morphogenesis observed in vitro may belie important activities that take place in vivo. As mentioned above, when cells disperse on plas- tic in response to H G F / S F their morphology also changes in a fashion that resembles an 'epithelial- mesenchymal transition' [64-66]. Conversion from an epithelial to mesenchymal cell phenotype occurs in several instances during development when cell migration is required [64-66]. Based on in vitro studies, H G F / S F as well as other factors [67,68] may be mediators of this process, The potential importance of H G F / S F in development was demonstrated in a chick embryo model in which exogenous H G F / S F stimulated the formation of multiple axial structures resembling the primitive streak and /o r neural plate [69]. It remains to be seen whether endogenous H G F / S F participates in these events.

In contrast to its growth-promoting effects, H G F / S F also has cytotoxic or growth-inhibitory properties in some situations [48,70,71]. It was purified from fibroblast culture fluid as a tumor cytotoxic factor (F-TCF) which was especially potent on certain mouse tumor cell lines such as Sarcoma 180, Meth A sarcoma and P388 (a lymphocytic leukemia line) and had modest effects on a few human carcinoma lines including KB, BG-1, MCF-7 and Hs913T [48,70]. It inhibits the proliferation of several gastric and hepatocellular carcinomas in culture, although there are other examples of these tumor cell types that respond positively to the factor [57,72,73]. While the mechanisms underlying these divergent effects is not understood, similar phe- nomena have been reported for other growth regulators such as transforming growth factor beta (TGF/3) [74].

II-C. cDNA cloning

II-C.1. Gene structure and chromosomal localization Using oligonucleotide probes based on amino-acid

sequence data obtained either from intact H G F / S F or peptide fragments, cDNA clones were isolated from placental [75], liver [76] or fibroblast libraries [20,53]. Analysis of these clones revealed that a single open reading frame encoded both chains of the H G F / S F heterodimer, and established that internal proteolytic cleavage is required to generate the two-chain structure. A secondary and perhaps incidental cleavage event may account for the discrepancy between the reported amino-terminal sequence of rat platelet-derived H G F / S F alpha chain [76] and that of human H G F / S F [77]. The H G F / S F open reading frame encodes a 728-amino-acid molecule containing a classical

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signal peptide sequence for secretion and four potential N-linked glycosylation sites, two in both the alpha (heavy) and beta (light) chains.

A striking sequence similarity to plasminogen was observed, with 38% identity at the amino-acid level and conservation of the kringle motif and serine proteinase-like domain [76]. A kringle is an approximately 80-amino-acid stretch containing a characteristic set of three internal disulfide bonds [78] and which, in the case of plasminogen, can include a protein binding site [79]. H G F / S F has four kringle domains while plasminogen has five; all of these kringles are roughly 40-50% identical to each other. The serine proteinase-like region of H G F / S F differs from that of plasminogen at two of the three amino acids required for catalysis [76]. Thus it is unlikely that H G F / S F has proteolytic activity, and none has been demonstrated. Moreover, neither plasminogen nor the processed and proteolytically active plasmin mimic the biological activities of H G F / S F ([76], and unpublished observations). Nonetheless, the cysteine residues in the proteinase-like domain of H G F / S F are conserved, imply- ing that this portion of the molecule probably exhibits a folding pattern resembling that of functional serine proteinases (see Fig. 1 for summary of H G F / S F structural features).

The similarities between H G F / S F and plasminogen may extend to aspects of biosynthetic processing and protein activation. The internal cleavage site that gives rise to the disulfide-linked H G F / S F heterodimer is identical to that of plasminogen [20,76]. It is known that plasminogen undergoes a significant conformational change when the proteolytically active plasmin is generated [80], and this may also be the case with HGF/SF. Not only is the internal cleavage site similar, but the catalytic mediators of this proteolytic processing may be as well. There is evidence that either the plasminogen activators [32] or plasmin itself [31] can perform this function with respect to HGF/SF. Con- sidering the postulated role of plasmin as a mediator of tissue remodelling, this raises the prospect of a con- certed regulatory scheme in which H G F / S F is activated in the same fashion to act as mitogen and /o r motogen along side plasmin in a variety of growth and repair-related contexts. Alternatively, a novel serine proteinase recently purified from serum also is capable of processing the H G F / S F monomer [81].

The H G F / S F gene spans approx. 70 kb and is comprised of 18 exons separated by 17 introns [82,83]. The pattern of exon-intron arrangement resembles that of plasminogen, reinforcing the view based on amino- acid sequence homology that the genes have a common origin. The first exon contains the 5'-untranslated sequence and signal peptide region, while the next five pairs of exons correspond to the amino-terminal and four successive kringle domains, respectively. The

K1 K4

COOH

Fig. 1. Schematic diagram of the H G F / S F molecule, adapted from [76]. The primary sequence of the complete translation product is depicted as a single, unbroken line extending from the amino (NH 2) to earboxyl (COOH) terminus. Disulfide bonds are based on alignments in plasminogen and typically indicated by short solid lines. The characteristic positioning of three disulfide bonds defines part of the essential structure of each of the four kringle domains (K1-K4). The disulfide linkage that bridges K2 and K3 is represented by a dashed line, while the bond that unites the heavy and light chains which form after internal proteolysis is highlighted as -S-S-. Note- worthy amino-acid residues are indicated by an open circle and position number in the primary sequence. Q32 becomes the amino- terminal residue following removal of the signal peptide; it cyclizes to form pyroglutamic acid [77] which is responsible for the blocked amino terminus of the monomeric H G F / S F polypeptide [20,77] and the heavy chain derived from it [75]. Additional proteolysis of rat H G F / S F yielded a biologically active molecule beginning at P55 [76], indicating that the intervening sequence is not required for mitogenic activity. FLPSS is the amino-acid segment absent from an H G F / S F isoform which has activities qualitatively similar to those of the full-length molecule [54,70]. Non-conservative substitutions of R494 at the internal cleavage site prevent activation of the molecule by blocking proteolytic processing to the heterodimeric form [29-31,90]. Q534 and Y673 are the two substitutions in H G F / S F which distin- guish it from serine proteinases possessing a catalytic triad and presumably account for its lack of proteolytic activity. Site-directed mutagenesis of the corresponding codons to introduce the amino acids present in active serine proteinases (Q534H and Y673S) was reported to result in diminished H G F / S F activity [29]. Mutagenesis of V692 also decreased activity [29]. D578 is the member of the

catalytic triad that is conserved in HGF/SF.

twelfth exon encodes the region that links the heavy and light chain, and the remaining six exons specify the serine-proteinase domain. In addition to containing the codons for the last 58-amino-acid residues of HGF/SF, exon 18 includes > 3 kb of 3'-untranslated sequence present in the 6 kb H G F / S F transcript. The major transcription initiation site was found to be 76 nu- cleotides upstream from the translation initiation site [82]. An interleukin 6 response element (CTGGGA) and a potential NF-IL6 binding site (TGAGGAAAG) were identified near the transcription initiation site [82]. It has been suggested that these sequences, which may have a role in the rapid induction of 'acute phase reactants' associated with inflammation, could account

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for the increase in expression of H G F / S F following injury [821.

The H G F / S F gene has been localized by in situ hybridization with a 3H-labeled probe to chromosome bands 7q11.2-21 [53]. Using a technique based on biotin-avidin interactions which affords a greater re- solving capacity, the gene was mapped to the proximal portion of the 7q21.1 band [84,85]. The gene encoding the H G F / S F receptor (c-Met) is also present on chromosome 7 [86]. Polysomy of this chromosome, which has been observed in human gliomas [87], could contribute to malignancy via an autocrine mechanism if it resulted in overexpression of both growth factor and receptor.

II-C.2. Alternative mRNA transcripts Comparison of different H G F / S F cDNA clones as

well as Northern blot analysis demonstrated the exis- tence of multiple H G F / S F mRNA transcripts with lengths of 6, 3, 2.2 and 1.3 kb [20,28]. Sequence analysis revealed that both the 6 and 3 kb forms encode variants either containing or lacking a 15 bp stretch in the coding region. This deletion is in flame and results in the loss of a 5-amino-acid sequence in the first kringle domain (FLPSS) [20]. Variants encoding both the 728- and 723-amino-acid forms of H G F / S F were detected in fibroblast and leukocyte cDNA libraries [20,53,88]. Because of their similar chromatographic properties, it is likely that they would co-purify from these and other sources that express both transcripts. Studies to date indicate that they exhibit comparable mitogenic, scattering and cytotoxic activities, although quantitative differences have been reported [54,70]. Whether these variants have any unique biologic functions remains to be elucidated.

The presence of the 1.3 kb transcript was associated with the expression of a smaller molecule, immunologi- cally cross-reactive with H G F / S F [89]. This transcript was recognized by oligonucleotide probes derived from H G F / S F heavy chain, but not light chain sequences. Based on this pattern of hybridization, cDNA clones corresponding to the 1.3 kb transcript were isolated from a human fibroblast cDNA library [89]. Nucleotide sequence analysis revealed that the cDNA encoded a protein, designated HGF/NK2, extending from the signal peptide sequence of H G F / S F to the end of the second kringle domain, followed by an additional three amino acids, a termination codon and a distinct 3'-untranslated sequence [89]. Further analysis established that this transcript resulted from an alternative splicing event which joined a kringle 2 exon with an exon having an in frame termination signal rather than a portion of the kringle 3 sequence [89]. Similar conclu- sions were reached independently by Miyazawa et al. [28]. The 2.2 kb transcript also encodes a truncated molecule [28] which extends only a few amino acids

beyond the first kringle domain (manuscript in preparation).

HGF/NK2 was purified from conditioned medium of the human cell line, SK-LMS-1, by a combination of heparin-Sepharose and sizing chromatography [89]. This truncated molecule lacked mitogenic activity on either the human mammary epithelial line, B5/589 or human melanocytes, but it specifically blocked H G F / SF mitogenic activity when present at a 10-fold molar excess [89]. It has been reported to have scattering activity, though with only ~ 3% the potency of full length H G F / S F [90]. Cross-linking experiments with [125I]HGF/NK2 demonstrated that it and H G F / S F bound specifically to the same high-affinity cell surface receptor [89,90]. A survey of different cell lines indicated a marked contrast in the relative levels of H G F / SF and HGF/NK2 expression [89]. These findings implied a novel mechanism for regulating the physiologic impact of H G F / S F in vivo by alternative splicing, which generates its own competitive antagonist. Re- cently, a similar observation was made concerning alternative splicing of transcripts from the acidic fibroblast growth factor gene [91].

II-D. Structure/function analysis of HGF / SF

The biological properties of HGF/NK2 define a portion of the H G F / S F molecule that is sufficient for high-affinity receptor binding. The absence of a mitogenic response after the smaller ligand binds to the H G F / S F receptor might be due to a lack of receptor oligomerization or appropriately directed tyrosine kinase activity, both of which have been implicated in the signal transduction pathways of tyrosine kinase receptors [3]. H G F / S F cDNA artificially truncated after the second or third kringle also resulted in the expression of polypeptides that lacked mitogenic activity but main- tained receptor binding [29]. Various other mutants have been generated to identify the structural features essential for biological activity. Expression of either heavy or light chains alone resulted in complete loss of mitogenic activity [29,92,93]. Deletion of any of the kringle domains or the hairpin structure in the amino- terminal region (amino acid residues 70-96) also caused a marked decline in activity, with the greatest reduction occurring when the first or second kringle domain was removed [29,92,93]. However, the apparent affinity of several kringle deletion mutants for the extracellular domain of the H G F / S F receptor in a soluble binding assay was not much lower than that of HGF/SF . Only the kringle 1 deletion mutant showed a substantial decrease in receptor binding affinity relative to H G F / SF, suggesting that at least one component of the receptor binding site(s) is localized to the first kringle domain [29] This interpretation is consistent with data obtained using truncated forms of H G F / S F extending

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from the amino-terminus through the first kringle ([94] and unpublished observations).

By using a combination of proteinase inhibitors and site-directed mutagenesis, several labs have shown that proteolytic processing of the monomeric H G F / S F precursor into the heterodimer is required for mitogenic [29-33] and scattering activity [31,32], although the monomer is capable of binding the H G F / S F receptor with high affinity [29]. As with plasminogen [80], proteolytic processing of the H G F / S F monomer presumably is associated with a conformational change which significantly alters H G F / S F activity.

Studies of site-directed mutants involving residues in the light chain corresponding to the catalytic triad of serine proteinases have yielded conflicting results. Ac- cording to one report [92], the replacement of tyrosine 673 with serine (Y673S), glutamine 534 with histidine (Q534H) or the simultaneous substitution at both posi- tions, did not result in any loss of biological activity (see Fig. 1). While Lokker et al. [29] noted full activity with the Q534H mutant, they observed approximately a 70% reduction of mitogenic activty with Y673S and > 97% decrease with the double mutant Q534H, Y673S. This reduction in activity occurred despite re- tention of high-affinity receptor binding. A sharp drop in activity without reduction in binding affinity also was reported for single or multiple substitution mutants involving a V692S replacement. Similar changes in the corresponding position of serine proteinases resulted in structural changes associated with formation of new hydrogen bonds involving the active site aspartic acid residue which is conserved in H G F / S F (D578, see Fig. 1) [95-97]. More work is required to establish a consensus in this area. Nonetheless, the knowledge that binding per se is not sufficient to trigger mitogenic signaling raises the likelihood that multiple binding sites or specific conformational changes in the ligand and /o r receptor molecules are crucial for biological activity.

II-E. Distribution and regulation of expression

H G F / S F is expressed in many organs throughout the body, as one might expect from the multiplicity of targets documented in vitro. Tissue extraction and im- munohistochemical staining of rabbit specimens ini- tially demonstrated H G F / S F in the pancreas, small intestine, salivary glands, thyroid and brain [98]. Subse- quent immunolocalization studies of human and rat tissues confirmed these findings and also detected a strong signal in surface epithelia, prostatic and seminal vesicle epithelia, distal renal tubules and collecting ducts, megakaryocytes, granulocytes and placental tissues [99-102]. Moderate staining was noted in respira- tory, gastrointestinal, biliary and uterine epithelium and in macrophages and endothelium, with weaker

staining elsewhere. In the case of a soluble growth factor, protein staining patterns may be more indicative of the factor's target cells than its site of synthesis. This presumably accounts for the strong immuno- staining of epithelia, as there is no evidence of H G F / SF expression by normal epithelial cells. Northern blot analysis of rat tissue specimens revealed a diverse pattern of expression generally consistent with protein staining results, with some differences in relative signal intensities [103]. In particular, the lung had the highest amount of H G F / S F transcript, though only a moderate signal on protein staining. This may imply that lung-derived H G F / S F is released into the circulation for systemic distribution, as has been suggested by reports of H G F / S F induction in the lung following injury in distant organs [104].

H G F / S F expression has been detected at the RNA or protein level in cultured fibroblasts derived from many organs, including the lung, stomach, colon, breast, prostate and skin ([20], and unpublished observations). The transcript also has been observed in other cells such as alveolar macrophages [99,104], peripheral leukocytes [88] and the HL-60 promyelocyte leukemic cell line [105], adding to the diverse pattern of expression. There is a consensus that H G F / S F is synthesized in the liver by non-parenchymal cells [106]. Based on in situ hybridization, Kupfer and endothelial cells contain the transcript [107]. However, cell fractionation followed by Northern blot analysis has indicated that the fat-storing, Ito cell is responsible for expression in the normal liver [108,109].

Several exogenous agents are capable of altering the magnitude of H G F / S F expression. Protein kinase C- activating phorbol esters stimulate H G F / S F secretion by fibroblasts in culture, and this effect is reversed by concomitant administration of dexamethasone [110]. Other tumor promoters which induce liver hyperplasia cause a rise in the plasma concentration of H G F / S F [111]. Hepatotoxins such as carbon tetrachloride and D-galactosamine induce a rapid and transient rise in H G F / S F transcript level in the liver and other tissues [104,112-114], accompanied by an elevation of circulating growth factor [113,114]. In some respects, these findings correlate with observations of patients with fulminant hepatic failure in whom high plasma levels of H G F / S F were detected [35,115]. Profound liver damage elicits a dramatic stimulation of H G F / S F expression, presumably as part of the repair process. Several labs have reported a similar induction of transcript following partial hepatectomy [104,113,114] or unilateral nephrectomy [116]. Of note, one study showed that after an initial rise in H G F / S F expression followed by a period of active cell proliferation, there was an increase in a ~ 1.5 kb transcript that may correspond to H G F / N K 2 [113]. Production of this competitive H G F / S F antagonist after a wave of hepa-

tocyte mitogenesis could provide a mechanism for dampening the growth stimulus as liver regeneration nears completion.

The induction of H G F / S F expression at locations distant from the site of injury suggested that systemic factor(s) might participate in this process [104,117]. Nakamura et al. have referred to such a factor as 'injurin' and proposed that its action serves to recruit production of H G F / S F by other tissues which release it into the circulation in response to injury. Hence a combination of endocrine and paracrine delivery sys- tems may be operative in repair processes involving H G F / S F . An apparently novel protein isolated from the serum of rats subjected to partial hepatectomy or ischemic insult has been reported to stimulate H G F / SF synthesis in other rats or in cell lines in vitro [118]. Interleukin 1 (IL-1), tumor necrosis factor-a and the phorbol ester, tetradecanoylphorbol 13-acetate (TPA) can independently increase H G F / S F expression, and the combination of IL-1 and TPA has a synergistic effect [119,120]. TGF-/3 and glucocorticoids can block H G F / S F induction elicited by IL-1 and TPA as well as other stimuli [109,110,119,121,122]. In addition, co- culturing of HGF/SF-producing MRC 5 fibroblasts with epithelial cells inhibits expression of the growth factor [123]. Thus, a variety of factors likely act locally or systemically to regulate H G F / S F production.

III. The H G F / S F receptor

I l i A . Identification

The observation that H G F / S F stimulated the rapid tyrosine phosphorylation of a 145 kDa protein provided an early clue concerning the identity of its receptor [20]. The molecular weight, rapid appearance and HGF/SF-specificity of this phosphoprotein suggested that it might represent autophosphorylation of a growth factor receptor kinase. Several 'orphan' receptors had been detected in activated forms as oncogenes in DNA transfection experiments or by reduced stringency hybridization as molecules structurally related to known receptor kinases [reviewed in [5]]. By using antibodies specific for different tyrosine kinases, we determined that the 145 kDa phosphoprotein was the/3 subunit of the c-met proto-oncogene product [124]. Cross-linking experiments with [125 I ]HGF/NK2 [124] and [125I]HGF/SF ([55], and unpublished observations) firmly established the c-Met protein as the H G F / S F receptor. Kinase activation and cross-linking to the c-Met protein was evident in a variety of cells which responded to H G F / S F with either an increase [124,125] or decrease [126] in proliferation, or by scattering [55,90]. Transfection of MDCK cells with a hy- brid cDNA encoding the ligand binding domain of trk, a nerve growth factor (NGF) receptor, and the trans-

363

membrane and cytoplasmic domains of c-Met con- ferred NGF-responsiveness in motility, proliferation and morphogenic assays [127]. This implied that stimulation of Met could elicit all of these diverse biological activities. Thus, the contrasting responses to H G F / S F likely reflect cell-specific differences in expression of other components of signal transduction pathways as well as variations in the extracellular milieu.

Competitive binding and Scatchard analysis with [125I]HGF/SF revealed two classes of binding sites on responsive cells: low capacity, high-affinity receptors (200-5000 per cell; 5-25 pM), and high-capacity, low- affinity receptors (~ 1000000 per cell; 0.2-5 nM) [55,126,128-130]. The former represented binding to c-Met protein(s), while the latter were attributed to binding to heparin-like proteoglycan [55,126,130], as reported for other heparin-binding growth factors [131-133]. Discrepancies in the literature regarding the estimated affinities of H G F / S F receptor binding may reflect differences in assay conditions or relative con- tributions of these two components amon~ cells ana- lyzed.

III-B. The c-met proto-oncogene

The met oncogene was originally identified following treatment of a human osteosarcoma line (HOS) with the chemical carcinogen, N-methy l -N ' -n i t ro -N- nitrosoguanidine (MNNG) [134]. Activated met was recovered from transformed foci obtained when DNA from the transformed line HOS-MNNG was trans- fected into NIH 3T3 ceils [134]. Subsequent work established that the met gene had been activated in HOS-MNNG cells by a rearrangement in which a truncated form of met from chromosome 7q21-31 was joined to a segment of chromosome 1, designated ' tpr ' (for 'translocated promoter region'), which resulted in the constitutive expression of a catalytically active tyrosine kinase molecule [86,135,136].

Molecular cloning of the c-met proto-oncogene revealed that it encoded a receptor-like protein for an undetermined ligand, with a classiCal signal peptide sequence, extracellular domain, putative transmembrane region and tyrosine kinase domain (Fig. 2) [137- 139]. Proteolytic processing of the extracellular domain at a site homologous to one present in the insulin receptor gives rise to a heterodimer consisting of a 45-50 kDa extracellular alpha (a) subunit linked via disulfide bond(s) to a 140-145 kDa /3 subunit that contains extracellular, transmembrane and intracellular catalytic domains (Fig. 2) [140,141].

Biosynthetic studies demonstrated that a precursor form (identified as p160 or p170) must undergo terminal glycosylation and disulfide bond formation prior to proteolytic conversion to the heterodimer [142]. This processing is not required for binding to the H G F / S F

364

p190 MET

c, S0 [~8

p 170 MET

c~50 1375 ~50

118aa

I 2 3 4 5

Fig. 2. Schematic diagram of the c-met proto-oncogene product, including isoforms resulting from post-translational modification or alternatively-spliced transcripts. The major transcript encodes a monomeric transmembrane (TM) polypeptide that undergoes glyco- sy]ation and disulfide bond formation to yield p190 MET (1). This protein is proteolytica]ly processed to generate a disulfide-linked heterodimer (2) consisting of an extracellular alpha subunit (a 5°) and a transmembrane beta subunit (/3145) with a tyrosine kinase domain (TK) [140,14]]. Carboxy-terminal truncation of the /3 chain [154], attributed to a PKC-dependent mechanism, results in smaller heterodimeric forms that are either anchored to the plasma membrane (3) or soluble (4). A minor transcript encodes another transmembrane polypeptide, p170 MET (5), having an additional 18- amino-acid residues in the extracellular domain downstream from the internal cleavage site. It is not processed to a heterodimeric form [145]. Note that this mature isoform derived from an alternative transcript differs from a biosynthetic precursor of p190 MET which has been labeled 'p170' and is characterized by incomplete glycosyla-

tion and disulfide bond formation [142].

ligand [143]. However, the importance of proteolytic processing was illustrated by work with a cell line deficient in the enzymatic activity responsible for this cleavage event. In this context the otherwise mature monomer, p190, was constitutively autophosphorylated [144]. In addition to these post-translational modifications, c-met transcripts are subject to alternative splicing which results in proteins that differ by 18-amino- acids in the extracellular domain [145]. The form containing the additional 18-amino-acids arises from a minor transcript (approx. 10% as abundant as the major transcript). It is present on the cell surface as a monomeric molecule with an apparent size of 170 kDa, and is autophosphorylated in an in vitro kinase assay (Fig. 2) [145]. Whether the c-Met proteins derived from alternative transcripts have distinct ligand binding properties and biological activities remains to be determined.

III-C. Regulation of c-Met activity

Like many other tyrosine kinases (reviewed in Refs. 1-7), autophosphorylation of the c-Met protein is associated with activation of its catalytic activity [146]. The major autophosphorylation site both in vitro and in

vivo (under steady-state conditions and at a high level of expression) is Y1235, which corresponds to the primary autophosphorylation site for several other tyrosine kinases [147]. Constitutive activation of the c-Met kinase can occur not only as a consequence of chromosomal rearrangement or failure to undergo post-translational processing, but also via overexpression. Gene amplification and overexpression of the major c-met transcript was documented in the human gastric carcinoma cell line GTL-16, where it was accompanied by high levels of the autophosphorylated c-Met protein [148,149]. No mutations in cDNAs derived from the c-met transcripts made by this line were found [149], nor was there any evidence of H G F / S F expression in this or certain other gastric carcinoma lines that over- express a constitutively active c-Met protein (unpublished observations). Barring the presence of an as yet unrecognized ligand, a high concentration of the receptor may therefore be sufficient for its activation. Given the tendency for the c-Met protein to form dimers and oligomers under these conditions [150], and the consensus view that dimerization is involved in ligand-in- duced receptor activation [3], ligand-independent den- sity-driven receptor-receptor interactions may account for kinase activation in these tumor cell lines. Amplifi- cation of c-met also has been detected, at a remarkably high frequency, in spontaneous transformants of mouse NIH/3T3 cells [151]. Because these ceils also produce HGF/SF, Met activation in this case appears to be due to an autocrine loop. Similarly, recombinant co-expression of human H G F / S F and Met correlated with constitutive Met autophosphorylation and a transformed phenotype [152].

In contrast to the above mechanisms of c-Met activation, stimulation of protein kinase-C (PKC) by tumor-promoting phorbol esters results in reduced tyrosine phosphorylation of the c-Met /3 chain with a concomitant rise in serine phosphorylation of the protein [153]. These observations suggest that certain protein kinases may act as negative regulators of c-Met signal transduction. Subsequent work indicated that a significant fraction of c-Met protein at the cell surface undergoes carboxy-terminal truncation through a PKC-dependent mechanism (Fig. 2) [154]. A similar PKC-dependent mechanism for receptor inactivation has been reported for colony stimulating factor-1 (c-fms gene product) [155]. One truncated form of the c-Met protein was released into the medium, while another lacking the tyrosine kinase domain remained anchored to the cell membrane [154]. Another study indicated that H G F / S F could be covalently cross-linked to a smaller molecule on the cell surface that appeared to be structurally related to the larger binding protein [126]. Together, these results suggest that signal transduction through the HGF/SF-c-Met pathway can be attenuated as a consequence of PKC-dependent gener-

ation of truncated forms of the receptor molecule which may bind the ligand but fail to transmit a functional signal. In support of this hypothesis, an earlier report showed that inhibitors of PKC potentiated the effect of scatter factor on MDCK cells [156]. Finally, elevation of intracellular calcium levels also has an inhibitory effect on c-Met autophosphorylation in a met-overexpressing cell line. This effect is independent of PKC and apparently involves a different serine kinase [157].

III-D. Post-receptor signal transduction

Several molecules have been implicated in the signaling pathways that operate downstream from activated tyrosine kinases [4-7]. For H G F / S F and c-Met, the issue is further complicated by the divergent responses of different cell types following the same lig- and-receptor interaction. Thus, one should expect to find distinct features in the signal transduction schemes associated with the different cell types. Identifying these particular components may provide insight into the mechanisms responsible for specific cellular responses.

Proteins associated with the HGF/SF-c-Met pathway to date are PI 3-kinase and MAP2 kinase (ERK2). The c-Met kinase, when activated either constitutively or via ligand-binding, couples with the 85 kDa PI 3-kinase subunit and co-precipitates with this enzymatic activity [158]. While the products generated by the action of PI 3-kinase are elevated in proliferating cells and are presumed to participate in mitogenesis, their effects are unknown [4]. Activation of MAP2 kinase was observed in human keratinocytes [159] and melanocytes [44] under conditions in which they proliferate in response to HGF/SF . There appeared to be an absence of MAP2 kinase tyrosine phosphorylation in MDCK cells, which scatter rather than proliferate following treatment with H G F / S F [159]. It will be of interest to determine whether this is a consistent dif- ference associated with these distinct biological responses. Activated c-Met can bind in vitro to many potential substrates containing SH2 (src homology) domains, including the GTPase activating protein for Ras (rasGAP) [160]. However, in whole cells phosphorylation of ras GAP by activated c-Met is only modest [44]. Thus, it is not known whether such interactions are relevant in vivo. Alternatively, H G F / S F has been reported to increase the proportion of GTP-bound Ras protein in A549 cells by stimulating the activity of a Ras-guanine nucleotide exchanger [161]. This suggests that activation of Ras may contribute to H G F / S F signaling.

H G F / S F also has been reported to stimulate a rise in the second messenger molecules, inositol 1,4,5-tri- sphosphate [162] and diacylglycerol, the latter derived

365

from both phosphoinositides and phosphatidylcholine, via activation of phospholipase C [163]. Oscillations of intracellular calcium concentrations also have been documented [164]. Elevation in calcium levels and activation of PKC resulting from the release of diacylglycerol may serve to attenuate the HGF/SF-c-Met signal by mechanisms that degrade and /o r inactivate the c-Met kinase.

A role for proteinases in the action of H G F / S F was suggested by a recent report indicating that H G F / S F increased the expression of urokinase (u-PA) and the u-PA receptor in MDCK cells [165]. A resulting increase in plasmin activity could in turn activate a variety of proteinases capable of modifying cell-cell and cell-substratum contacts, thereby facilitating cell migration. Moreover, the induction of plasmin and urokinase may augment the processing of H G F / S F from monomeric precursor to the active heterodimeric form [31,32].

Consistent with its dramatic effect on cellular morphology in certain situations, changes in cytoskeletal elements have been documented in response to H G F / SF. Early in the scattering process, there was an increase in the number of F-actin stress fibers corresponding to cell spreading. Subsequently, the cells showed a marked reduction in the number of stress fibers associated with a decline in cell attachment [166]. The highly motile character of these cells w, as explained in part by a decrease in focal contacts along the cellular processes, although the signaling pathways responsible for these changes remain to be determined [166].

IV. Broad perspectives

A. HGF / SF- and Met-related molecules

A HGF-like sequence was cloned from a human genomic library using conditions of reduced stringency with a cDNA probe encoding the kringle domains of bovine prothrombin [167]. The predicted protein sequence was comprised of 711 amino acids, including a classical signal peptide and putative internal proteolytic processing site which would yield a disulfide-linked heterodimer. As with HGF/SF , the amino-terminal chain contained four kringle domains while the carboxy-terminal portion had the serine proteinase structure but lacked an intact catalytic triad. Overall the amino-acid sequence was approximately 50% identical to that of HGF/SF . It is not yet known whether the HGF-like protein is a ligand for c-Met or a related receptor. This gene mapped to the DNF15S2 locus on chromosome 3 (3p21), the site of a suspected tumor suppressor gene [167,168]. Moreover, a genomic frag- ment of the HGF/SF-like sequence also hybridized to

366

HGFlike 147 T R N E F C R N MSP; B U l l F D D C R N P D G I1

WoW r.i HGFISF I~r7 Y R G Q "C R N P E

HGFISF 205 S T N G E 219

HGF-Iike 288 G E G Y R G T A N T T T A G V P C Q R W D A Q 310

MSP; BU'7 310 E G Y~E|a:G T A -- T T T A G -- P~E]:O D A Q .QF~S~ e (3 v ,= T N r I " ~ , e o

HGF-IIke ~ i L R E N F C R N P D G S E AA~ppP 341 __ MSP; BU12 L R E N F -- R N P D G IS| E A P HGF/SF 348 L R E N IY1 c R N P D G S E 363

HGFI 'k . =as log W Q _C QQ : WV G V O C Q R W S A E T P H K P Q F'F, 404 MSP;I IU*I / O V Q - Q R W [ S ] A E T I P ] - - - Q HGFISF 4011 ~ D K N M E D L H R 425

HGF like 413 ~ G~SDI Q L E E N F C R N P D G D S 428 MSP: BU13 O L - E N F -- - N P D E HGFISF 434 K C R N P D ~ D I D J A 447

HGF-IIke 484 V v G G H P G N S P W T • . V $ L N R Q| 592 R N R Q MSP; BU 9 V V G G H P G N I S ] P W ( T ] V - L - Nrrl~l]Q MSP:Bchain V V G G - P G N S P - ( T ] V I S I L R N~I~ HGF/SF 498 T R T N I G W M I V S L R 515

HGF I ,k. S30 P L T G Y E V W L G T L F Q N P Q H I ~ 562 _ _ G E P MSP; CB5 P L T ~ G Y E V - L G T L F Q N P Q - G E HGFISF 540 Y E J ~ W L Gil H D V H G R G D E ~ K C K 562

HGFI ' k . 574 ILL EE : SS vV TT ~ NN QO : W : LL , C L E R S V T L N O R V A L L P P ~ ~Y V [ V ~ PP E I W ~ ' ~ " hASP: B U l l l L E R S V T L N Q R V A L I C L P E] HGFISF 584 L A R P A V L D D F V S T I D L P I N Y G C T I 606

HGFlike a02 IC ~ I AAGGwWGG ~ T : ] C E I A G W G E T K I l l l MSP: BUff C E I A G W G E T K HGF ISF 612 C S V Y G W G Y T O 821

104

184

Fig. 3. Amino-acid sequence comparison of the HGF-like protein, macrophage stimulting protein (MSP) and HGF. The sequences of HGF-like protein and HGF were deduced from their respective eDNA coding sequences [75,167], while that of MSP was based on protein sequence analysis of isolated peptide fragments (designated BU-5, BU-7, etc.) [169]. Where two amino-acid derivatives were identified at a single cycle of Edman degradation, both are listed. Brackets are included where assignments were tentative. Dashes in the MSP peptide sequences are present where a positive amino-acid identification could not be made. Dots indicate where small gaps were introduced to optimize alignments. Identical residues in the different protein sequences are enclosed by solid lines, broken lines

exclude non-identical residues from regions of identity.

two loci on chromosome 1, raising the likelihood of additional HGF/SF-like genes or pseudogenes [167].

Although no function has yet been established for the HGF-like protein, we can infer a likely role based on a comparison of its predicted sequence with the partial amino-acid sequence of a macrophage stimulating protein (MSP) [169]. Such an analysis revealed that these molecules are very closely related (Fig. 3). MSP is a heterodimeric molecule purified from human plasma and shown to promote the responsiveness to chembattractants and phagocytotic activity of resident peritoneal macrophages [169]. Thus, it is probable that, among its potential physiologic roles, the HGF-like protein functions to stimulate macrophages during the immune response. The identity of MSP and the HGF- like protein was established in a report describing the molecular cloning and sequencing of MSP [170].

Molecules resembling the c - m e t proto-oncogene product also have been identified. An oncogene iso-

lated from the $13 avian erythroblastosis retrovirus was found to encode a tyrosine kinase which was most closely related to c-Met [171,172]. It was approx. 72% identical to the human c-Met sequence in the tyrosine kinase domain, with little homology outside of this region. Because the virus produces a syndrome characterized by sarcomas, erytroblastosis and anemias, the oncogene was named v-sea. This gene was fused in frame with the viral envelope gene, resulting in the expression of a cell surface tyrosine kinase molecule resembling a growth factor receptor. Molecular cloning of the avian c-sea counterpart demonstrated that it encodes a membrane spanning, putative tyrosine kinase receptor [173]. Using specific cDNA probes, transcripts corresponding to c-sea or related molecules were detected in chicken macrophage, B cell and liver cell lines as well as in primary chick embryo fibroblasts and peripheral white blood cells [172,173]. The human c-sea gene was cloned independently, although its identity was not recognized because the report was submit- ted prior to publication of the avian c-sea sequence [174]. The human gene was mapped to the chromosomal locus of HGF-like protein/MSP, 3p21 [174]. Whether any functional relationship exists between the corresponding gene products remains to be determined. Another tyrosine kinase-related molecule which has a TK domain more similar to that of c - m e t and v-sea than other tyrosine kinases (~ 64% identity to the TK domain of both c - m e t and v-sea) has been reported [175]. It has a putative transmembrane region and extracellular domain that bears no apparent homology to c - m e t [175], raising doubt that it would interact with an HGF/SF-related ligand.

IV -B . Cl in ica l re l evance

While it is premature to make any definitive state- ments about the clinical importance of H G F / S F and the c-Met protein, accumulating evidence suggests ar- eas of potential relevance. The expression of the ligand [98,99,101,103,176] and its receptor [176-180] in a variety of organs both during development and in the adult suggests a wide range of effects and possible applications. Participation of H G F / S F in liver regeneration appears likely in view of its induction in models of liver injury [104,112-114] and elevated circulating levels in conditions characterized by active liver regeneration [35,115,181]. Systemic administration of H G F / S F may augment the rate of hepatocyte proliferation and re- duce liver damage in some instances of liver injury [182,183]. In addition, reports indicate that serum H G F / S F concentrations have prognostic value in monitoring fulminant hepatic failure [184]. H G F / S F also may contribute to repair in other organs such as the kidney where its induction correlated with the compensatory response to unilateral nephrectomy [116]

367

and acute renal injury [185]. Besides stimulating proliferation of parenchymal cells, H G F / S F may foster wound healing by promoting angiogenesis [46,63]. For example, its effects on endothelial cell migration and proliferation might be of particular utility when angiogenesis is compromised by an underlying disease process. Alternatively, if H G F / S F were responsible for neovascularization in pathological conditions like dia- betic retinopathy, then H G F / S F antagonists, such as HGF/NK2 , may be useful.

Several observations suggest the possibility that modulation of H G F / S F and c-Met might have a place in cancer therapy. Met overexpression has been documented in carcinomas of the lung, pancreas, thyroid, colon and stomach [149,179,186]. Approximately 50% of thyroid follicular carcinomas exhibited as much as 100-fold increase in c-Met protein, and were associated with a poor prognosis [187]. A high frequency of gene amplification was observed in scirrous type stomach cancer [188], while the tpr-met oncogenic rearrangement was identified in cell lines derived from other gastric carcinomas and in premalignant gastric lesions [189]. In an experimental model, co-expression of H G F / S F and Met was associated with tumorigenicity [152].

Because H G F / S F acts as a mitogen for a variety of cells and can stimulate certain epithelial cells to invade collagen gels [22] or grow in soft agar [190,191], it is likely that in selected instances H G F / S F promotes tumor expansion and metastasis. Detection of H G F / SF in malignant pleural effusions associated with primary lung, mesothelial and breast malignancies is consistent with this idea [192]. Suramin, which nonspecifi- cally blocks HGF/SF-c-Met interactions, reversed part of the aberrant differentiation pattern displayed by a human nondifferentiating keratinocyte (ndk) cell line that produces H G F / S F [27]. Neutralizing the action of H G F / S F in vivo with this or more specific reagents may be of therapeutic benefit. Paradoxically, because H G F / S F was cytotoxic for certain tumor cells in vitro, it may be possible to identify specific tumors that would regress following treatment with the factor.

IV-C. Summary and future directions

H G F / S F is a protein with diverse biological effects on a broad spectrum of cellular targets. It can function as a mitogen, motogen or morphogen as well as a cytotoxic or growth inhibitory agent. Clearly the tem- poral, spatial and cellular context are likely to be crucial in determining its particular effect. The pattern of mesenchymally-derived H G F / S F sources adjacent to epithelial and endothelial targets suggests a paracrine mode of delivery, although the presence of circulating H G F / S F and induction of transcript at distant sites support the possibility of an endocrine

mechanism as well. Moreover, autocrine expression has been observed in vitro, and based on its effects in this setting, might be expected to contribute to aberrant differentiation, tissue organization or cellular motility in vivo. Several levels of regulation govern the activity of both H G F / S F and its receptor. Modulation of gene expression, alternative splicing and post-translational modifications involving proteolytic processing and phosphorylation have a major impact on ligand-receptor signaling, ranging from constitutive activation to creation of antagonists. Gaining a better understanding of these regulatory mechanisms, the signal transduction process and the structure-function relationships that govern H G F / S F agonist and antagonist action are important goals. Increasing our knowledge of the roles of H G F / S F , c-Met and related molecules in normal development, differentiation and repair as well as in pathological conditions such as cancer may ultimately lead to clinical applications.

Acknowledgement

We thank Rose Windle for assistance in preparation of the manuscript.

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