Summary Hepatocyte growth factor, a potent mitogen for epithelial and other cell types, and scatter factor, a stimulant of epithelial cell motility are identical. In addition to these mitogenic and motogenic functions, the factor has been shown to be an epithelial morphogen and also has antiproliferative effects in some cancer cell lines. The mernbrane receptor for hepatocyte growth factor1 scatter factor has been identified as the c-met proto-oncogene product. Introduction The multifunctional nature of peptide growth factors is well documented('). Their actions include the stimulation and inhibition of cell proliferation as well as cffccts unrclated to the control of growth('). In addition. many cytohnes may stimulate cell motility: a response which has been termed 'motogenic'(2'. For example, platelet-derived growth factor (PDGF) is motogenic. as well as mitogenic, for fibroblasts and smooth muscle cell^(^.^). Recently several cytokines have bcen reported that appear to function solely as motogens. These include autocrine motility factor (AMF), a protein released by melanoma and librosarcoma cell lines which stimulates motility in the producer and migration stimulating factor (MSF) which is produced by foetal and breast cancer patient fibroblasts and similarly acts in an autocrine F a ~ h i o n ( ~Although .~). initial reports suggested that scatter factor, a stimulant of epithelial cell motility, was primarily a motogenic cytokine, it is now clear that this is not so. The motogenic function of scatter factor is instead part of the functional repertoire of a pleiotropic polypeptide termed hepatocyte growth factor/scatter factor. In this article, the biological activities including mitogenic. motogenic, morphogenic and anti-proliferative effects of this factor are reviewed. Hepatocyte growth factor (HGF) which is also known as Hepatopoeitin A, was first demonstrated as a hepatotropic factor present in rat plateletd8) and in the serum of rats after partial h e p a t e ~ t o m y ( ~ JHGF ~ ) . purified from rat platelets(' I ) or huinan serum('?), potently stimulated DNA synthesis of adult rat hepatocytes in primary culture at concentrations as low as 1 ng/ml. Purified HGF was found to be a disulphidelinked heterodimcric protcin with a relative molecular mass

(M,) of 82,000, which when reduced. yielded two polypeptide chains of Mr 69,000 and 34,000(111.Molecular cloning showed that human HGF is encoded as a pre-pro precursor protein of 728 amino acids which is processed to the heterodimeric f ~ r m ( ~ ~ Interestingly, .'~). there is significant amino acid sequence identity (38%) of pro-HGF and plasminogen. Both proteins contain intra-chain disulphide loops tcrmed 'kringles.' HGF has four kringle structures (Fig. 1 ) whilst plasminogen has five. HGF however. is unlikely to possess protease activity as the histidine and serine residues of the serine protease domain of plasminogen, are replaced by glutamine and tyrosine respcctivcly. in EIGF(' 31. Naturally occuring variants of HGF have been described. The first, in which 5 amino acids ofkringle 1 are absent, reflecting alternative RNA processing, was isolated from human leucocyte and huinan fibroblast cDNA libraries(1s.'6). Both the 723 amino acid HGF variant and the 728 amino acid form were represented in each of thesc libraries, and are biologically a ~ t i v e ( ' ~ In , ~ ~addition, ). a truncated form of HGF, which also results from alternative mRNA splicing, has been isolated from human fibroblasts(18). This Mr 28.000 protein comprising the amino terminus and kringles 1 and 2 of HGF, and thus tcrmed HGF/NK2, was found to compete with HGF for its receptor (see below), thereby specifically inhibiting HGF induced mitogenesisII8). Scatter factor was initially described as an activity secretcd by human embryo fibroblasts thal caused epithelial cell colonies to dissociate or scatter(19) (Fig. 2). A direct effect of scatter factor on the motility of epithelial cells was

a-chain

P-chain

Fig. 1. Schematic diagram of Hcpatocyte Growth Factor. Reprinted by kind permission of the authors from Nature 342, pp 440-443. Copyright 0 1989 MacMillan Magazines Ltd.

Pig. 2. Effect of partially purificd mouse scatter factor on Madin-Darby Canine Kidney (MDCK) cells: (a) control; (b) addition of 32 units of scattcr factor. Reprinted by kind permission of the authors from Nurure 327, pp 239-242. Copyright 0 19x7 MacMillan Magazines Ltd.

shown by time-lapse photography(20),and a Boyden chambcr assay(*') in which chemotaxis and chemokinesis can be measured. Scatter factor also stimulates the invasion of

Madin-Darby Canine Kidncy (MDCK) epithelial cells and some human carcinoma cell lines into collagen gels(22).In addition to fibroblasts, smooth muscle cells and also certain

epithelial cells such as ndk, a non-differentiated keratinocyte cell line, releasc scatter f a c t ~ r ( ~ Vascular ~ . ~ ~ ) . endothelial cells. as well as epithelial cells, respond to scatter factor by an increase in cell migration(25).Thus as originally suggested by Stoker and colleagues(20),scatter factor generally appears to act as a paracrine agent, tnediating the interaction between fibroblast and epithelial cclls, as wcll as smooth muscleendothelial cell interactions. Purification of scatter factor from mouie or human fibroblast conditioned medium showed that scatter factor, in common with HGF, was a heterodimer(22,26). Estimation of the molecular weight of either chain by reducing polyacrylamide gel electrophoresis gave values of 57-62,000 and 30-34.000(22~26~,, in broad agreement with those for HGF.

Identity of Hepatocyte Growth Factor and Scatter Factor Thus any possible relationship between HGF and scatter factor lay in the behaviour of these proteins on polyacrylamidc electrophoretic gels. Analysis of partial protein sequence data from purified scatter factor, however, suggested that scatter factor and HGF were, at least, highly related(22.27). Similarly analysis of scatter [actor human fibroblast cDNA clones showed no major difference between scatter factor and HGF: the predicted 728 amino acid polypeptide was identical to human HGF(28).In addition, one cDNA clone encoded a variant of scatter factor with a 5 amino acid deletion in the first kringle domain and is thus identical to the previously described HGF variant(lS,l6).Further evidence for the identity of these cytokines was provided by biological and imInunochemical studies. In cross biological experiments, recombinant HGF potently scattered MDCK epithelial cells, and induced the invasion of MDCK cells into collagen gels in the same concentration range as purified scatter Conversely, purified scatter factor stimulated DNA synthesis in hepatocytes and other epithelial cell Furthermore. the mitogenic and motogenic activities of scatter factor and HGF‘ were neutralized by antibodies raised against scatter factor‘29).Reaction of these antibodies, and also monoclonal antibodies to HCF, with scatter factor and HGF in Western blotting gave immunoreactive bands of the same relative molecular The final line of evidence for the identity of HGF and scatter factor concerns the specific binding of both species to the c-met proto oncogene product and is considered below. c-met is the HGF/SF Receptor The product of the c-met proto oncogene is a heterodimcric M, 190,000 transmembrane protein possessing tyrocinc kinase activity. The tyrosine kinase domain is located on the Mr 145.000 p polypeptide which spans the cell membrane, and which is linked by disulphide bonding, to the extracellular Mr 50,000 a chain(31’.The demonstration that HGF was the ligand for this putative growth factor receptor was independently reported by two groups. Highly purified HGF stimulated the phosphorylation of tyrosine on the c-met receptor present on two cell lines: a human mammary epithe-

lial cell line which is sensitive to the mitogenic effects of HGF(32)and a gastric carcinoma cell linc GTL16. which over-expresses the c-met receptor(33).Similarly highly purified scatter factor bound to GTL16 cells with a high affinity equal to that of HGF(30).c-met was identified as the receptor for either ligand using a variety of techniques. Tmmunostaining with monoclonal antibodies to c-met following ligandinduced tyrosine phosphorylation of the r e ~ e p t o r ( ~ ~ . ~ ~ ) , together with chemical cross linking of radiolabelled ligand to ~ - r n e t ( ~ ~immunoprecipitation ,~~)), with antibodies to cnzet(30,32),and transfer of binding activity to insect cells transfected with c-met DNA(30).demonstrated that c-met was the receptor for hepatocyte growth factor/scatler factor.

Functional Activities of Hepatocyte Growth Factor/ Scatter Factor (HGF/SF) The effects of HGF/SF in vitro are wide ranging. In addition to the mitogenic effects on primary rat hepatocytcs(”lO), HCF/SI; also stimulated thynlidine incorporation, reflecting DNA synthesis, and/or an increase in cell number in a variety of other cell types. The factor was mitogenic for epithelial cells(’6,34,35), m e l a n o c y t e ~ ‘ ~ and ~ ? ~endothelial ~), cell~(l~,~~) but not fibroblasts(’6.26).Anti-proliferative effects on several cell lines have also been described for HGF/SF. A factor initially termed ‘tumor cytotoxic factor’, due to its inhibitory cffcct on the growth of sarcoma and epidermoid carcinoma cell lines, was subsequently identified as HGF/SF(”>”). In addition, a cytostatic effect of HGF/SF, notably on hepatocellular carcinoma cell lines was also shown; at concentrations of HGF/SF that stimulated mitogenesis in primary rat hepatocytes‘40,4’). A motogenic responsc to HGF/SF has been reported for epithelia1(20-22)and endothelial cell^(^^^^^). It is at present unknown, however, whether hepatocytes respond motogenically to the factor. Thus some cell types, for example endothelial cells and epithelial cells such as keratinocytes, show both a mitogenic and a motogenic response to HGF/SF(’6.21.29),while others, for example MDCK and A549 epithelial cell lines, respond by an increase in cell motility but not an increase in cell n ~ m b e r ( ~ ~These , ~ * ) .differences must be mediated downstream from the receptor, as HGF/SF has been shown to stimulate tyrosine phosphorylation on the c-met receptor of A549 cells(”). In addition, the interaction of HGF/SF with other growth factors and/or the extracellular matrix may be important in regulating the extent of each response. For example a synergistic effect of HGF/SF with insulin on the mitogenic response of keratinocytes has been described(29)although the interaction of these factors on the motogenic response was not studied. The interaction of HGF/SF with the extracellular matrix was recently illustrated when a fibroblast-derived epithelial morphogen was identified as HCF/SF. MDCK epithelial cells when culturcd in collagen gels in the presence of fibrob1ast conditioned medium or HGF/SF formed branching epithelial tubules instead of spherical cysts, which developed under control conditions or in the presence of HGF/SF and neutralizing HGF antibodies. These results suggest a paracrine role for HGF/SF in epithelial morphogencsis, poss-

ibly functioning in the formation of parenchymal organs in lhe developing embryo("). A role of HGF/SF in embryogenesis concerning the development of the embryonic axis, has been suggested by experiments in the chick in which HGF/SF applied locally to early chick embryos, produced characteristic axial malforma~ions(~~). What is the role of HGF/SF in viva? A key role of HGF/SF in liver regeneration is supported by a rise in HGF/SF activity in plasma with patients with fulminant hepatic failure(I2)and a marked increase in HGF/Sf; m R N A in liver following hepatic insult such as administration of hepatotoxins(44) or partial hepatectomy'"). Increases in hepatic HGF/SF mRNA following hepatic insult suggest a paracrine mode of action with Kupffer cells and hepatic endothelial cells being the probable source o ~ H G F / S F ( ~There ~ ) . is some evidence from studies in the rat that the lung may have an endocrine function in HGF/SF production: HGF/SF inRNA in the lung markedly increased following partial hepatect ~ r n y i ~Recent ~ ) . results also suggest a renotropic function for HGF/SF. Following unilateral ncphrectomy in the rat, there was a markcd increase of HGF/SF mRNA and HGF/SF mitogenic activity in the intact kidney undergoing compensatory renal regeneratiod4*). Endocrine secretion of HGF/SF by endothelial cells in the lung(") may account for these results although at present the humoral stimulus for regulation of HGF/SF secretion is unknown. One clue to the physiological regulation of HGF/SF at thc producer cell level has come from recent in vitro studies. Down-regulation of HGF/SF expression by MRC-5 fibroblasts has bccn shown by co-culture of these cells with a SV40-transformed human keratinocyte cell line or other epithelial cells(39).Significant reduction of HGF/SF scatter activity was accompanied by decreased HGF/SF mRNA levels which may be regulated by intimate ccll-cell (epithelialfibroblast) contact. The potential for HGF/SF responses to also be regulated at the receptor lcvcl is evident. Reference has already been made in this article to the inhibition of mitogenesis by the truncated form of HGF/SF('*J. which as well as the mature form of HGF/SF was secreted by a fibroblast cell line. In addition, C-terminal truncated forms of the c-met receptor which lack the tyrosine kinase domain have been described(50).Binding of HGF/SF lo inactive c-met variants would thus reduce the effective concentration of free ligand. Thus regulation of the relative levels of mature HGF/SF and truncated HGF, or mature c-met and truncated c-met would determine the extent of a functional response to HGF/SF.

A New Family of Cytokines? HGF/SF maps to the long arm of chromosome 7, 7q2 1. I (28.51) but recently a gene mapping lo lhc short arm of chromosome 3 ( 3 ~ 2 1with ) about 50% identity to HGF was described("). The gene encodes a 7 1 1 amino acid polypeptide with 4 kringle domains and a serine protease-like domain. The protein, however, in common with HGF/SF, should be devoid of protease activity as thrcc critical amino acids that are found in the active site of plasminogen and other serine proteases have been changed in 'HGF-like protcin'(5'). Indeed, at present, there is no known biological

function of this protein. It is, however, tempting to suggest that HGF/SF and HGF-like protein may be part of a new family ofcytokines.

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