Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects

A sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-hepatocyte growth factor (HGF) IgG for human HGF, also known as the scatter factor, has previously been developed for determining increases in serum HGF levels in various liver diseases. The sensitivity limit of the ELISA is, however, approximately 0.2 ng/ml sample, and HGF concentrations in about 50% of normal subjects are not accurately measurable by this method, because the mean level of HGF in normal serum is close to the sensitivity limit. In the present study, chicken Fab' from egg yolk anti-HGF immunoglobulin Y and rabbit Fab' from rabbit anti-HGF IgG were conjugated with beta-D-galactosidase. With these conjugates as the second antibodies, we developed two sandwich ELISAs for human HGF and found that the sensitivities were about 20 pg/ml with the former conjugate and 2 pg/ml with the latter. The HGF concentration in sera from 138 normal subjects determined by the ELISA with the rabbit conjugate was 244+/-65 (SD) pg/ml serum, and it correlated very well with the number of leukocytes. Moreover, the ELISA with the rabbit conjugate permitted the determination of HGF levels in urine from normal subjects without first concentrating the sample. The determination of HGF in various biological fluids other than blood and urine by these ELISAs may aid the diagnosis and prognosis of various diseases.     

Therapeutic angiogenesis using hepatocyte growth factor (HGF)

HGF is a mesenchyme-derived pleiotropic factor, which regulates cell growth, cell motility, and morphogenesis of various types of cells and is thus considered a humoral mediator of epithelial-mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis. Although HGF was originally identified as a potent mitogen for hepatocytes, it has also been identified as a member of angiogenic growth factors. Interestingly, the presence of its specific receptor, c-met, is observed in vascular cells and cardiac myocytes. In addition, among growth factors, the mitogenic action of HGF on human endothelial cells was most potent. Recent studies have demonstrated the potential application of HGF to treat cardiovascular diseases such as peripheral vascular disease, myocardial infarction and cerebrovascular disease. In this review, we will discuss a potential therapeutic strategy using HGF in cardiovascular disease.     

Rapid growth of invasive metastatic melanoma in carcinogen-treated hepatocyte growth factor/scatter factor-transgenic mice carrying an oncogenic CDK4 mutation

Currently, novel mouse models of melanoma are being generated that recapitulate the histopathology and molecular pathogenesis observed in human disease. Impaired cell-cycle control, which is a hallmark of both familial and sporadic melanoma, promotes slowly growing carcinogen-induced melanomas in the skin of mice carrying a mutated cyclin-dependent kinase 4 (CDK4(R24C)). Deregulated receptor tyrosine kinase signaling, which is another important feature of human melanoma, leads to spontaneous development of metastatic melanoma after a long latency period in mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF mice). Here we report that treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate induced metastatic melanomas in all HGF/SF mice on the C57BL/6 background, which histologically resemble human melanoma. Importantly, mutant CDK4 dramatically increased the number and the growth kinetics of carcinogen-induced primary melanomas in the skin and promoted the growth of spontaneous metastases in lymph nodes and lungs in all HGF/SF mice within the first 3 months of life. Apart from very few skin papillomas, we did not observe tumors of other histology in carcinogen-treated HGF/SF x CDK4(R24C) mice. This new experimental mouse model can now be exploited to study further the biology of melanoma and evaluate new treatment modalities.     

Exogenous expression of hepatocyte growth factor (HGF) in rat striatum by naked plasmid DNA

Hepatocyte growth factor (HGF) is a heterodimeric protein and shows mitogenic and morphogenic activities toward a variety of epithelial cells. There has been no immunohistochemical evidences for naked DNA mediated transgene expression of HGF into central nervous system. We initially demonstrated a naked plasmid mediated expression of HGF into rat striatum. The immunofluorescence staining revealed that exogenous protein of human HGF was expressed at 7 days after plasmid injection (300 microg). Exogenous HGF was mainly expressed in reactive astrocytes according to dual-labeling staining of HGF and glial fibrillary acidic protein or S100. It is also demonstrated that c-met, specific receptor of HGF, was expressed in the injection site. Intensive expression of c-met was found in the site to which HGF encoded plasmid was injected. These evidences for the exogenous expression of HGF and its receptor c-met may implicate an application of naked plasmid mediated HGF for neuronal disease as well as the other neurotrophic factors.     

Expression of c-Met receptor and hepatocyte growth factor/scatter factor in synovial sarcoma and epithelioid sarcoma

 Overexpression of c-Met receptor/hepatocyte growth factor (scatter factor) system (c-Met/HGF/SF) as a physiologically paracrine cellular signaling system is thought to be involved in the progression of malignant tumours. In 26 synovial sarcomas and epithelioid sarcomas, c-Met and HGF/SF expression was analysed immunohistochemically. There were 10 biphasic synovial sarcomas, 7 of which showed moderate to strong c-Met expression in epithelial areas compared with the fibrous component, with corresponding expression of HGF/SF. Six of 9 monophasic fibrous synovial sarcomas showed only very faint c-Met and corresponding HGF/SF expression. In 7 epithelioid sarcomas strong expression of c-Met and HGF/SF was observed within epithelioid tumour cells. Non-radioactive in situ hybridization demonstrated the synthesis of c-Met receptor in tumor cells by detecting c-met-mRNA. This analysis shows that in synovial sarcomas and epithelioid sarcomas, tumour entities with epithelial and mesenchymal structures, c-Met and HGF/SF overexpression can be detected, indicating a role of this signaling system in these subtypes of sarcoma, and especially in the more epithelioid tumour phenotype. An autocrine interaction between overexpressed c-Met receptor and HGF/SF may be hypothesized. Received: 21 July 1997 / Accepted: 19 November 1997     

Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis

Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule formation has many similarities to reported in vivo epithelial tubulogenesis.     

Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.     

Regulation of spreading and growth of colon cancer cells by hepatocyte growth factor

Hepatocyte growth factor (HGF), also known as scatter factor, regulates both cell motility and the growth of some cell types. We have determined the effects of HGF on the motility and growth of human colon cancer cell lines (HT115, HT29, HRT18 and HT55). Cell motility, as measured by dissociation from carrier beads or by scattering of cell colonies, was greatly increased in all cell lines. The effects were completely blocked by anti-HGF antibody. In contrast, cell growth of HT115, HT29 and HRT18 cells was inhibited by a wide range of concentrations of HGF. HT55 cell growth was also inhibited but needed a prolonged culture period (>5 days). The HGF receptor/Met protein is highly expressed in the membrane fraction of these cells as determined by Western blotting. It is concluded that HGF has an effect on both colon cancer cell motility and growth, which may be important in the control of the spread of colon cancer.     

肝细胞生长因子研究进展

查阅近二十年国外有关HGF/SF文献,基本弄清了HGF/SF的分子结构及组织分布,说明了HGF/SF的调节和cmet受体及两者的关系,揭示了HGF/SF的生物活性、HGF/SF和肿瘤扩散以及与肝再生的关系.因此,HGF/SF在肝再生中起主要作用;是肾再生的关键因子;可促进伤口愈合;通过增加恶性细胞的侵袭性,HGF/SF可能作为肿瘤转移的重要因素之一.     

Ulcerative proctitis, rectal prolapse, and intestinal pseudo-obstruction in transgenic mice overexpressing hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) can stimulate growth of gastrointestinal epithelial cells in vitro; however, the physiological role of HGF/SF in the digestive tract is poorly understood. To elucidate this in vivo function, mice were analyzed in which an HGF/SF transgene was overexpressed throughout the digestive tract. Nearly a third of all HGF/SF transgenic mice in this study (28 of 87) died by 6 months of age as a result of sporadic intestinal obstruction of unknown etiology. Enteric ganglia were not overtly affected, indicating that the pathogenesis of this intestinal lesion was different from that operating in Hirschsprung's disease. Transgenic mice also exhibited a rectal inflammatory bowel disease (IBD) with a high incidence of anorectal prolapse. Expression of interleukin-2 was decreased in the transgenic colon, indicating that HGF/SF may influence regulation of the local intestinal immune system within the colon. These results suggest that HGF/SF plays an important role in the development of gastrointestinal paresis and chronic intestinal inflammation. HGF/SF transgenic mice may represent a useful model for the study of molecular mechanisms associated with a subset of IBD and intestinal pseudo-obstruction. Moreover, our data identify previously unappreciated side effects that may be encountered when using HGF/SF as a therapeutic agent.     

Hepatocyte growth factor/scatter factor-induced intracellular signalling

Hepatocyte growth factor (HGF) identical to scatter factor (SF) is a glycoprotein involved in the development of a number of cellular phenotypes, including proliferation, mitogenesis, formation of branching tubules and, in the case of tumour cells, invasion and metastasis. This fascinating cytokine transduces its activities via its receptor encoded by the c-met oncogene, coupled to a number of transducers integrating the HGF/SF signal to the cytosol and the nucleus. The downstream transducers coupled to HGF/MET, most of which participate in overlapping pathways, determine the development of the cell's phenotype, which in most cell types is dual.     

Decreased maternal circulating hepatocyte growth factor (HGF) concentrations in pregnancies with small for gestational age infants

Our purpose was to evaluate whether maternal and fetal hepatocytegrowth factor (HGF) concentrations in pregnancies with smallfor gestational age (SGA) infants are different from those inpregnancies with appropriate for gestational age (AGA) infants.Maternal and fetal circulating HGF concentrations were comparedbetween 55 pregnancies with AGA infants and 16 pregnancies withSGA infants at birth. HGF concentrations were measured frommaternal and cord venous blood samples using an enzyme-linkedimmunosorbent assay. Umbilical artery blood pH and oxidativepressure (PO₂) were also measured. Maternal circulating HGFconcentrations (0.60 ± 0.35 ng/ml) in pregnancies withSGA infants were significantly lower than those (0.91 ±0.44 ng/ml) in pregnancies with AGA infants (P = 0.012). Therewere no significant differences in fetal circulating HGF concentrationsbetween both groups. No significant differences in umbilicalartery blood pH and PO₂ were found between both groups. Theseresults suggest that the maternal serum circulating HGF concentrationhas a significant role in fetal growth during pregnancy.     

New approaches for mapping the recognition sites in protein–GAG interactions: a study of GAG binding by hepatocyte growth factor/scatter factor

Introduction The minimal size and structure of GAG that binds to a protein, as well as the site of GAG interaction on the protein surface, are crucial recognition features for understanding, and potentially modulating, protein–GAG interactions. Methods that are easily and generally applicable for investigating such interactions, irrespective of the GAG species involved, are very limited. We have been developing such methodologies using the example of hepatocyte growth factor/scatter factor (HGF/SF). We have previously shown HGF/SF to have an unusual GAG‐binding specificity, in that it binds with high affinity to, and is activated by, both heparan and dermatan sulfates. There are two naturally occurring, highly truncated variants of HGF/SF. These comprise only the N‐terminus and either the first one (NK1) or two (NK2) of the four Kringle domains of the α‐chain of HGF/SF, and without a β‐chain. Some controversy presently exists as to whether GAG‐binding in HGF/SF requires the N‐terminus alone, or additionally the second Kringle domain. Materials and methods We have developed a protocol for analysing protein–GAG interactions utilizing fluorescently tagged GAG oligosaccharides run in a protein gel mobility shift assay under native associative conditions. The activity of oligosaccharides has been assessed by their ability to stimulate HGF/SF‐mediated activation of signals downstream of the Met receptor in sulfated GAG‐deficient CHO pgsA‐745 cells. Results HS tetrasaccharides and DS hexasaccharides are the minimal binding and activatory species for HGF/SF. The basis for this size difference between HS and DS species is not presently known. However, their ability to efficiently compete with each other does indicate a shared binding site. The three proteins HGF/SF, NK1 and NK2 all display identical GAG‐binding specificities and minimal oligosaccharide‐binding sizes. This suggests that NK1, the smallest variant, contains the entire minimal GAG‐binding functionality of full‐length HGF/SF, and thus the second Kringle domain is not required. Identification of NK1 and tetrasaccharides as the minimal binding partners has allowed us to directly target the binding site on NK1. Fluorescently tagged tetrasaccharides have been crosslinked into the binding site on NK1 using the zero‐length crosslinking methodology. After tryptic digestion, the specific fluorescently tagged peptides can be recovered for subsequent sequencing to locate the GAG‐binding site within the known amino acid sequence of the protein. Discussion Methods have been developed which can help to identify the respective domains on both GAG and protein involved in GAG‐protein recognition. These are relatively easy approaches, applicable to physiological solution conditions and having the advantage of being widely applicable to many protein interactions involving any uronate‐containing GAGs. In addition to HGF/SF, the gel mobility shift assay has also been applied by us to a variety of other diverse proteins.