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Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16INK4a in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27Kip1, a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. Implications: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells. Mol Cancer Res; 11(11); 1437–47. ©2013 AACR.

Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial–mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules. (Cancer Sci 2011; 102: 2164–2171)

Hepatocyte growth factor (HGF), an antimitogenic factor for HepG2 cells, increased mRNA and protein levels of UGT1A1 and CYP2B6, as well as the endogenous cyclin-dependent kinase (CDK) inhibitors p16, p21, and p27 in HepG2 cells but not in HuH6, Caco2, or MCF7 cells. Treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) (an extracellular signal-regulated kinase inhibitor) suppressed the HGF-induced expression of UGT1A1 and CYP2B6, as well as p16, p21, and p27 in HepG2 cells. The CDK inhibitor roscovitine also enhanced the expression of UGT1A1, CYP2B6, and CYP3A4. Transfection of anti-CDK2 siRNA led to elevated levels of UGT1A1, CYP2B6, and CYP3A4 in HepG2 and SW480 cells, whereas anti-CDK4 small interfering RNA (siRNA) did not significantly enhance the expression of these enzymes. In fact, CDK2 activity was decreased in HGF-treated HepG2 cells. In cells arrested in S phase by a thymidine block and then released into a synchronous cell cycle, there was a clear dissociation among the activation of CDK2 and the expression of UGT1A1, CYP2B6, and CYP3A4. Furthermore, the induction of CYP3A4 but not UGT1A1 or CYP2B6 mRNA expression by roscovitine was repressed in pregnane X receptor (PXR) siRNA-transfected HepG2 cells. Transfection with constitutive androstane receptor siRNA or PXR siRNA in HepG2 cells did not repress the HGF-stimulated expression of UGT1A1 mRNA. Taken together, our results show that the expression of UGT1A1 and CYP2B6 is negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 and SW480 cells, the mechanism of which may differ from that of CYP3A4 expression through PXR phosphorylated by CDK2.

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88)

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16INK4a, which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21CIP1, whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells. J. Cell. Biochem. 104: 176–188, 2008. © 2007 Wiley-Liss, Inc.

Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.

The association of hepatocyte growth factor (HGF) with its high-affinity receptor (c-Met) has been shown to induce mitogenesis, motogenesis and morphogenesis in a variety of cell types. Various point mutations in c-Met have been identified in hereditary and sporadic papillary renal carcinomas as well as in other carcinomas. In the present study, we examined the effects of c-Met point mutations on the morphology of a porcine aortic endothelial (PAE) cell line. When cultured in three-dimensional collagen gel, PAE cells formed branching tubule structures, and HGF treatment caused breakdown of the structures and induced a scattered morphology. The exogenous expression of c-Met point mutants inhibited the formation of tubules. HGF treatment induced the formation of tubules by PAE cells expressing some c-Met mutants, but it induced the scattering of PAE cells expressing other c-Met mutants. The presence of a low concentration of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor cancelled the inhibitory effect of the c-Met point mutations on the formation of tubules. These results suggest that c-Met point mutations affect the extracellular signal-regulated kinase (ERK) signaling required for the formation of tubules by PAE cells, and HGF binding changes the conformation of c-Met mutants, leading to the different signals required for formation of tubules and cell scattering.

Background & Aims: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. Methods: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Results: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. Conclusions: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.

c-Met is a high-affinity receptor for hepatocyte growth factor (HGF) and plays a crucial role in embryonic development, as well as in the process of tissue repair. Overexpression and amplification of c-Met are often observed in various cancer tissues, especially in gastric carcinoma. It has, however, been unclear whether the overexpression leads to activation of the c-Met receptor. To address this point, we prepared an antibody (anti-phospho-Met) which specifically recognizes c-Met that is phosphorylated at Y1235, a major phosphorylation site of c-Met. Normal as well as cancerous gastric tissue was positive for anti-total-Met staining, whereas only cancerous tissue was strongly positive for anti-phospho-Met staining; cells near the basal layer were moderately positive, and the proliferative zone in normal tissue was only weakly positive. Among cancerous tissues from seven patients examined in the present study, those from six patients were strongly positive for phospho-Met staining. These results indicate that c-Met is actually activated in gastric carcinoma tissue, and may trigger proliferation/anti-apoptotic signals.

Hepatocyte growth factor (HGF) plays a crucial role in the recovery of injured liver. Liver functions are mostly impaired in patients with liver diseases including cirrhosis. However, a significant amount of inactive HGF precursor (proHGF) is reported in the plasma of these patients. proHGF is proteolytically converted to an active form (mature HGF) by HGF-activator. Thus conversion of proHGF into mature HGF presumably contributes to the recovery of liver functions. In this study, rats with a partial hepatectomy were used, as proHGF is accumulated in the remnant liver. Recombinant human HGF-activator was administered via the portal vein to investigate the effect on molecular forms of HGF and its biological signaling. rhHGF-activator promptly converted proHGF into mature HGF, reaching maximal levels at 5–10 min after the injection, while the decreased proHGF was quickly recovered to the initial levels in the liver. The HGF receptor/c-Met was found to be autophosphorylated in the liver treated with rhHGF-activator. Further, the proliferating cell nuclear antigen labeling index and the liver regeneration rate were significantly higher in rhHGF-activator group than in control animals. These results indicate that exogenously administered HGF-activator produces a biologically active HGF from its precursor form and increases the potential for liver regeneration in vivo.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is a serine proteinase inhibitor containing two Kunitz-type inhibitor domains, initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). In a previous study (Biochem. Biophys. Res. Commun. 255, 740-748, 1999), we reported that a predominant transcript of mouse HAI-2 is a splicing variant lacking the first Kunitz domain (KD-1). Since KD-1 was reported to be responsible for the inhibition of HGFA in human HAI-2 and the second Kunitz domain (KD-2) of human HAI-2 was much less inhibitory against HGFA, it has been suggested that most of mouse HAI-2 may be ineffective in inhibiting HGFA. In this study, we have performed functional characterization of Kunitz domains in mouse HAI-2 by using recombinant proteins synthesized by Chinese hamster ovary cells without or with point mutation in the putative reactive site of each Kunitz domain. The results revealed that, unlike human HAI-2, KD-2 of mouse HAI-2 efficiently inhibits HGFA. Therefore, the major mouse HAI-2 protein that consists only of KD-2 can be a potent inhibitor of HGF activation in vivo.

A novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) was cloned and characterized in the process of the search for splicing variant forms of HAI-2 by 3′-rapid amplification of cDNA ends (RACE). The gene consisted of 4 exons spanning approximately 1 kb and was located in 11 kb downstream of HAI-2 gene (19q.13.11). The novel transcript identified by 3′-RACE was thought to be chimerically transcribed from both HAI-2 (exons 1–7) and H2RSP (exons 2–4) genes. Wild-type H2RSP mRNA (0.5 kb) was detected abundantly in various tissues including the gastrointestinal tract, whereas chimeric mRNA (1.5 kb) was found mainly in the kidney, prostate, and placenta by Northern blot analysis. The predicted amino acid sequence of H2RSP contained two unique domains, namely the serine-rich region (exon 3) and the lysine-rich region (exon 4). Transfection of deleted series of H2RSP cDNAs fused to enhanced green fluorescent protein (EGFP) into HeLa cells revealed that H2RSP has nuclear localization signal in the lysine-rich region. Immunohistochemical study using anti-H2RSP polyclonal antibody indeed revealed the nuclear localization of this peptide in vivo. These results suggest that H2RSP and H2RSP/HAI-2 chimeric peptides might function as a transcriptional regulatory peptide at the nucleus.

The mechanisms by which growth factors trigger signal transduction pathways leading to the regulation of c-Fos expression are of great interest. In this study we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on the expression of c-fos and its product, c-Fos, in human epithelial cell line MKN74. The expression level of c-Fos protein in HGF/SF-stimulated cells was 5--10-fold higher than that in EGF-stimulated cells, whereas the level of c-fos mRNA induced by HGF/SF was similar to that by EGF. The hyperphosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of an increased number of translation initiation complexes, was detected only in HGF/SF-induced MKN74 cells. Activation of phosphatidylinositol-3'-OH kinase and FKBP12-rapamycin associated mammalian target of rapamycin (FRAP/mTOR) was observed after the treatment with HGF/SF. Pretreatment with an inhibitor of either one, i.e. LY294002 for phosphatidylinositol-3'-OH kinase or rapamycin for FRAP/mTOR, completely inhibited 4E-BP1 phosphorylation and decreased the c-Fos synthesis induced by HGF/SF down to the level found in EGF-induced cells. These results suggest that the phosphorylation of 4E-BP1 is stimulated by HGF/SF in a manner requiring both phosphatidy-linositol-3'-OH kinase-dependent and FRAP/mTOR-dependent pathways, thereby stimulating c-fos mRNA translation. Regulation of the translation process of c-fos mRNA in addition to the immediate activation of c-fos transcription is necessary for the transient increase in the level of c-Fos protein to stimulate cell proliferation.

A cDNA encoding mouse hepatocyte growth factor activator (HGFA) has been cloned by RT-PCR, based on the screening result from the database of expressed sequence tags. Subsequently, its gene was cloned from a mouse genomic bacterial artificial chromosome library using the cDNA as a probe. Sequencing analysis revealed that mouse HGFA protein deduced from the cDNA, similar to its human and rat counterparts, has two epidermal growth factor-like domains, type 1 and 2 fibronectin homology domains, a single kringle domain and a catalytic domain of serine proteinase, and the gene consists of 14 exon spanning approximately 7.5 kb. Interestingly, mouse HGFA mRNA was detected not only in the liver but also in the gastrointestinal tract by RNA blot analysis. Since hepatocyte growth factor (HGF) is up-regulated in the damaged gastrointestinal mucosa, our present data suggest that HGFA might activate proHGF directly in the gastrointestinal mucosa and play an important role in wound repair throughout the gastrointestinal tract.

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.

Summary: While the targeted disruption of a gene is a powerful tool for investigating the physiological functions of that gene, disruption of a gene essential for embryogenesis leads to embryonic death. Rescue of the defect(s) causing embryonic death should promote survival, thus permitting further evaluation of the roles that the gene plays later in the developmental process. Disruption of the gene for mouse hepatocyte growth factor/scatter factor (HGF/SF) leads to middle-stage embryonic lethality because of a defect in placental development. Here we report that a single injection of HGF/SF at embryonic day 9.5 (E9.5) into the amniotic cavity of HGF/SF−/− embryos rescued the placental defect and resulted in the survival of the embryos until term. Histological analysis suggested that HGF/SF is also required at the late stage of development for tissue organogenesis. Thus, injection of a secreted factor can be a useful method to rescue the defects causing embryonic lethality. genesis 27:99–103, 2000. © 2000 Wiley-Liss, Inc.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor, which was identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human carcinoma cell line. HGF activator is a blood coagulation factor XII-like serine protease that is responsible for proteolytic activation of the inactive single chain precursor of HGF in injured tissues. The predicted sequence of the primary translation product of HAI-1, which has a hydrophobic sequence in its COOH-terminal region, suggested that HAI-1 is first produced in a membrane-associated form. In this study, we identified a transmembrane form of HAI-1 integrated in the plasma membrane of cultured cells using a monoclonal antibody against HAI-1. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGF activator. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of HAI-1.

We have reported that infusion of recombinant human hepatocyte growth factor (rhHGF) stimulates liver regeneration after hepatectomy in cirrhotic rats and increases the level of serum lipids and secretion of very-low density lipoprotein (VLDL). Studies were now performed to determine whether rhHGF directly influences lipid synthesis and its secretion in cultured rat hepatocytes. Isolated cells were cultured in the presence or absence of rhHGF (20 ng/mL) for 2 days. During the first 12 hours, rhHGF transiently inhibited the release of lipids (triacylglycerol, total cholesterol, and phospholipids), but stimulated their releases with maximal levels achieved at 36 hours. [3H]-glycerol experiment with the transcriptional and translational inhibitors revealed that rhHGF stimulated de novo synthesis of lipids by affecting activities of lipid metabolic gene. [35S]-Methionine experiment also revealed de novo synthesis of apolipoprotein B by rhHGF. Furthermore, lipid analysis of lipoprotein fractions in the conditioned medium showed that rhHGF enhanced levels of triacylglycerol, total cholesterol, and phospholipids by 50% to 200% in both VLDL and low-density lipoproteins (LDL)/high-density lipoprotein (HDL). Genistein, a tyrosine kinase inhibitor, blocked the secretion of VLDL, as well as synthesis of lipids and apolipoprotein B stimulated by rhHGF. These results indicate that HGF likely stimulates lipid biosynthesis and lipoprotein secretion in hepatocytes through its tyrosine kinase-associated receptor, c-met, and accelerates the progress of cell maturation in liver regeneration.

Hepatocyte growth factor (HGF) activator is a serine protease responsible for proteolytic activation of HGF in response to tissue injury and thus plays an important role in the regulation of biological functions of HGF in regenerating tissue. We previously purified an inhibitor of HGF activator (HGF activator inhibitor type 1, HAI-1) from the conditioned medium of a human stomach carcinoma cell line MKN45 and cloned its cDNA. HAI-1 is a novel member of the Kunitz family of serine protease inhibitors. In the present study, we purified a second type of HGF activator inhibitor (HAI-2) from the conditioned medium of MKN45 cells and molecularly cloned its cDNA. The cDNA sequence revealed that HAI-2 is derived from a precursor protein of 252 amino acids and contains two Kunitz domains, indicating that HAI-2 is also a member of the Kunitz family of serine protease inhibitors. The primary translation product of HAI-2 has a hydrophobic sequence in the COOH-terminal region, suggesting that, like HAI-1, HAI-2 is produced in a membrane-associated form and secreted in a proteolytically truncated form. Because HAI-2 and HAI-1 are potent inhibitors specific for HGF activator, they may be involved in regulation of proteolytic activation of HGF in injured tissues.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Hepatocyte growth factor activator (HGFA) is a serine protease that is responsible for localized activation of hepatocyte growth factor (HGF) in injured tissue. The activated HGF may be involved in regeneration of the injured tissue. HGFA is produced and secreted by the liver and circulates in the plasma as an inactive zymogen. In response to tissue injury, the HGFA zymogen is converted to the active form by limited proteolysis. In this study, we isolated a rat HGFA complementary DNA (cDNA) clone and analyzed the production of HGFA messenger RNA (mRNA) in response to tissue injury using this cDNA clone as a probe. The nucleotide sequence of the cDNA revealed that the amino acid sequences of rat and human HGFA showed a high degree of conservation in the regions of the characteristic domain structures, suggesting that rat and human HGFA are activated by a similar mechanism and have similar enzymatic activities in vivo. Tissue distribution analysis showed that the liver was the major site of rat HGFA mRNA synthesis. Moreover, the cells producing HGFA mRNA were identified as parenchymal liver cells. The level of HGFA mRNA increased in the liver after hepatotoxin or nephrotoxin treatment. This increase was also observed during acute inflammation induced by turpentine. These results suggest that the increase in production of HGFA mRNA in response to tissue injury is the result of an inflammatory response, and that HGFA is an acute phase protein.

The function of hepatocyte growth factor/scatter factor (HGF/SF) is to increase proliferation as well as to stimulate motility and disperse cell colonies of epithelial cells. In this study, we examined the motogenic and mitogenic responses of two human gastric carcinoma cell types, MKN7 and MKN74. Cell motility of both cell lines was markedly stimulated by HGF/SF. In contrast, HGF/SF stimulated cell growth of MKN74 cells, but did not stimulate growth of MKN7 cells. To address the cause of the difference in response of these cells, which may reflect some differences in signaling pathways downstream from the HGF/SF receptor, c-Met, we investigated the induction of the proto-oncogene c-fos. The level of c-fos mRNA increased and reached a maximum approximately 40 min after HGF/SF stimulation in MKN74 cells, and thereafter its level rapidly decreased. In contrast, the level of c-fos expression was very low irrespective of the stimulation in MKN7 cells. c-Fos protein was transiently induced only in MKN74 cells l h after treatment with HGF/SF, and its levels subsequently decreased. We subsequently examined the activation of mitogen-activated-protein kinase, which is a major mediator in the signaling pathway leading to the stimulation of c-fos transcription, after HGF/SF treatment in both cell lines. Mitogen-activated-protein kinase was markedly activated by this treatment in MKN74 cells, but was only slightly activated in MKN7 cells. These results suggest that although mitogen-activated-protein kinase activation and c-fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/SF.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain precursor from the producing cells, and normally remains in this form associated with the extracellular matrix. In response to tissue injury, the single-chain precursor is converted to a biologically active heterodimer by a serine protease, the activity of which is induced in the injured tissue. We have previously identified HGF activator, a serum serine protease that activates single-chain HGF. The sequence of HGF activator cDNA revealed that the HGF activator is homologous to blood-coagulation factor XIIa. In this study, we found that coagulation factor XIIa has an ability to activate single-chain HGF. Factor XIIa exhibited a significant level of HGF-converting activity in the presence of dextran sulfate, although the specific activity of factor XIIa was slightly lower than that of the HGF activator. Since factor XIIa is activated during the initiation of contact activation induced by tissue injury, factor XIIa may function as an HGF-converting enzyme together with HGF activator in the injured tissue. C1-inhibitor, antithrombin III and alpha 2-antiplasmin, that regulate the blood-clotting activity of factor XIIa, were also effective against the HGF-converting activity of factor XIIa. Furthermore, factor XIIa was not active in the HGF-converting activity in serum. Thus, the HGF-converting activity of factor XIIa may be regulated by these serum inhibitors.

HEPATOCYTE growth factor/scatter factor (HGF/SF) functions as a mitogen, motogen and morphogen for a variety of cultured cells1–7. The genes for HGF/SF and its receptor (the c-met protooncogene product8) are expressed in many tissues during the embryonic periods and in the adult9–14. HGF/SF is thought to mediate a signal exchange between the mesenchyme and epithelia during mouse development15. To examine the physiological role of HGF/SF, we generated mutant mice with a targeted disruption of the HGF/SF gene. Here we report that homozygous mutant embryos have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and die before birth. The growth of trophoblast cells was stimulated by HGF/SF in vitro, and the HGF/SF activity was released by allantois in primary culture of normal but not mutant embryos. These findings suggest that HGF/SF is an essential mediator of allantoic mesenchyme-trophoblastic epithelia interaction required for placental organogenesis.

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Ligand-induced tyrosine kinase activation of the scatter factor/hepatocyte growth factor receptor (c-Met) is thought to be essential for the biological responses of target cells. To assess the regulatory role of the major tyrosine autophosphorylation site (tyrosine 1233) of the mouse c-Met receptor in the tyrosine kinase activation of the receptor, we constructed a mutant receptor in which the tyrosine residue was replaced with phenylalanine. When the cells expressing the mutant receptor were incubated with the ligand, no biological responses were observed, and the level of tyrosine phosphorylation of the receptor was very low compared with that of the wild-type receptor. The in vitro kinase activity of the mutant receptor toward an exogenous substrate and the receptor itself was also low. Furthermore, tyrosine phosphorylation of the cellular proteins by ligand stimulation was not detected in intact cells expressing the mutant receptor. The low level of kinase activity and the lack of biological activity of the mutant receptor indicate that the major autophosphorylation site positively regulates the tyrosine kinase of the c-Met receptor and phosphorylation of cellular substrates in the scatter factor/hepatocyte growth factor signaling pathway.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and renal tubular epithelial cells and may play an important role in regeneration following hepatic and renal injury. HGF is first synthesized as a single chain precursor which is then converted to a heterodimeric form by proteolytic processing. This proteolytic conversion is required for HGF to function as a mitogen. In this study, we examined whether the proteolytic activation of HGF occurred in response to hepatic and renal injury. HGF remained as an inactive single chain form in the liver, kidney, lung, and spleen of normal rats. The production of HGF markedly increased in the liver after hepatotoxin treatment and in the kidney after nephrotoxin treatment. A significant portion of the increased HGF was converted to the active heterodimeric form, whereas in other tissues, conversion did not occur although the production of HGF increased in some of them. Furthermore, an enzymatic activity that converts the single chain form of HGF to the active heterodimeric form was detected in the injured liver but not in the normal liver. These results indicate that HGF is proteolytically activated in response to tissue injury, and this activation is mediated by an enzymatic activity which is induced exclusively in the injured tissues. Thus, the proteolytic activation system functions in vivo as a mechanism for localizing HGF activities to injured tissues.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Immunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, alpha- and beta-catenin and plakoglobin. beta-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver cells, epithelial cells, and endothelial cells and may play an important role in liver regeneration following hepatic injury. HGF is homologous to plasminogen and is first synthesized and secreted as an inactive single-chain precursor and then activated to a heterodimeric form by endoproteolytic processing. Recently, a novel serine protease responsible for this processing (HGF activator) has been purified from fetal bovine serum (Shimomura, T., Ochiai, M., Kondo, J., and Morimoto, Y. (1992) Cytotechnology 8, 219-229). In this study, we purified HGF activator from human serum and determined its partial amino acid sequence. Based on the amino acid sequence, we have molecularly cloned the cDNA for human HGF activator. The nucleotide sequence of the cDNA revealed that HGF activator is derived from the COOH-terminal half region of a precursor protein of 655 amino acids and that the precursor consists of multiple putative domains homologous to those observed in blood coagulation factor XII. These domains may be involved in the conversion of the precursor to the active form of HGF activator.

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for hepatocytes in primary culture. In response to liver damage, the levels of HGF/SF mRNA change in various tissues. In this study, we isolated a genomic DNA fragment containing the promoter region of the rat HGF/SF gene and analyzed transcription-initiation sites and their utilization in response to acute liver injury. Rat HGF/SF-mRNA synthesis starts from at least three sites in the liver, spleen and kidney. One of these sites is preferentially utilized in the liver and spleen in response to acute liver injury. In the 5' flanking region, several cytokine-related sequence elements that might be involved in the regulation of HGF/SF-gene expression are located near the transcription-initiation sites. The effects of cytokines related to these sequence elements on the production of HGF/SF mRNA were examined using a cell culture system. Transforming growth factor-beta 1 (TGF-beta 1) inhibits the production of HGF/SF mRNA by Shay granulocytic sarcoma-derived cells. The TGF-beta 1-inhibitory element, one of the sequence elements present in the promoter sequence, may mediate the inhibition of HGF/SF-gene expression by TGF-beta 1.

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.

The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.

Hepatocyte growth factor (HGF) induced scattering and cell migration of human gastric adenocarcinoma MKN-74. HGF also significantly promoted the growth of MKN-74 cells in a dose-dependent manner, although HGF is reported to be antiproliferative for the growth of tumor cell lines. This result indicates that HGF stimulates cell proliferation of not only normal epithelial cells but also certain carcinoma cells. Furthermore, transforming growth factor-beta (TGF-beta), which is recognized to inhibit the growth of most epithelial cells, additively enhanced both the cell proliferation and migration induced by HGF. These additive effects of HGF and TGF-beta may be responsible for the tumor invasiveness and uncontrolled growth of certain types of carcinoma.

Hepatocyte growth factor (HGF) has an anti-proliferative effect on many types of tumor cell lines and tumors in vivo. We found previously that inhibition of HGF-induced proliferation in HepG2 hepatoma cells is caused by cell cycle arrest at G1 through a high intensity ERK signal, which represses Cdk2 activity. To examine further the mechanisms of G1 arrest by HGF, we analyzed the Cdk inhibitor p16(INK4a), which has an anti-proliferative function through cell cycle arrest at G1. We found that HGF treatment drastically increased endogenous p16 levels. Knockdown of p16 with small interfering RNA reversed the arrest, indicating that the induction of p16 is required for G1 arrest by HGF. Analysis of the promoter of the human p16 gene identified the proximal Ets-binding site as a responsive element for HGF, and this responded to the high intensity ERK signal. HGF treatment of the cells led to a redistribution of p21(CIP1) and p27(KIP1) from Cdk4 to Cdk2. The redistribution was blocked by the knockdown of p16 with small interfering RNA, which restored the Cdk2 activity repressed by HGF, demonstrating the requirement of p16 induction for the redistribution and eventual repression of Cdk2 activity. Our results reveal a signaling pathway for G1 arrest induced by HGF.

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. We previously investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. The results suggested that the HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from the retinoblastoma tumor suppressor gene product pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal. In this study, we examined the molecular mechanism of the HGF-induced cell cycle arrest in HepG2 cells. Cyclin A/Cdk2 complexes phosphorylated serine residues on pRb crucial for the G1 to S phase transition in proliferating HepG2 cells, and HGF treatment inhibited the phosphorylation. The expression of cyclin A was decreased and the expression of a Cdk inhibitor p21 C i p 1 was increased in HGF-treated HepG2 cells, and these changes were prevented by pretreatment with a low concentration of a MEK inhibitor. These results suggest that the decrease in cyclin A expression and increase in p21 C i p 1 expression through a high-intensity ERK signal by HGF lead to suppression of the phosphorylation of pRb by Cdk2, which contributes to the cell cycle arrest at G1 in HepG2 cells by HGF. Furthermore, the expression of E2F-1, a member of the E2F transcription factor family, was decreased in HGF-treated HepG2 cells, suggesting that the decrease in E2F-1 expression may also contribute to the cell cycle arrest at G1.

Hepatocyte growth factor (HGF) is involved in the proliferation and migration of various types of epithelial cells. HGF is produced as a single-chain precursor (pro-HGF) and functions after activation by HGF activator (HGFA). In this study, we aimed to examine the activation of pro-HGF to mature HGF and the expressions of HGF-related molecules, such as HGFA and HGFA inhibitor type 1 (HAI-1), in a monkey model of gastric mucosal injury.Gastric mucosal injury was induced in Japanese monkeys by administration of HCl using nasal-gastric tubes. HGF activation was evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA), and the expression of HGF, HGFA, HAI-1, and c-Met were examined by Northern blotting and immunohistochemistry.Pro-HGF, but not mature HGF, was detected in normal gastric mucosa. When gastric mucosal injuries were induced, the expression of HGF significantly increased, and immunostaining of HGF was detected in fibroblasts in the gastric mucosa. In addition, conversion to mature HGF was observed in the injured gastric tissues, and mature HGF continued to increase for 48 h. HGFA was stably expressed in monkey livers, regardless of HCl administration. HAI-1 expression was detected in normal gastric mucosa, and its levels decreased after the induction of gastric mucosal injury.These results indicate that pro-HGF is stored in normal gastric tissues, and suggest that its conversion to mature HGF, associated with HGFA and HAI-1, is important for the repair of gastric mucosal injury.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor for hepatocyte growth factor activator (HGFA), is responsible for proteolytic activation of hepatocyte growth factor. We examined the expression of HGFA and HAI-1 in liver tissues of chronic liver diseases including hepatocellular carcinoma (HCC). HGFA expression was detected not only in the liver tissues of chronic hepatitis and cirrhosis and in the nontumorous liver tissues surrounding HCC, but also in HCC tissues. On the other hand, none of the liver tissues of hepatitis and cirrhosis and none of the nontumorous tissues surrounding HCC were stained with anti-HAI-1. However, 35% of HCC tissues were stained with anti-HAI-1, and HAI-1 positivity increased as the histological grade decreased and as serum alpha-fetoprotein increased. Transduction of antisense HAI-1 inhibited the growth of human hepatoma cells. These results suggest the possibility that HAI-1 plays an important role in the progression of HCC.

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.

Hepatocyte growth factor (HGF) is secreted as a single-chain form (proHGF) from mesenchymal cells of the liver, and proHGF is converted to heterodimeric HGF (mature HGF) by serine proteases, including hepatocyte growth factor activator (HGFA), in response to tissue injury. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) have recently been purified from the conditioned medium of a gastric cancer cell line, and their cDNAs have been successfully cloned. The hepatocytes of patients with chronic liver diseases including hepatocellular carcinoma (HCC) were stained with anti-HGFA. HAI-1 was detected in tumorous tissues of HCC, but not in liver tissues of patients with chronic liver diseases. Tumorous tissues in HCC were positive for HAI-1 mRNA. HAI-1 may play an important role in the development or invasiveness of HCC through the mechanism regulating HGF activation.

Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA.HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1beta accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA.HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.

The role played by the hepatocyte growth factor activator (HGFA) during morphogenesis of the gastrointestinal tract was investigated in fetal rats between days 16 and 21 of gestation. By our recently established method using chelation and dissecting microscope, samples could be separated into epithelium and mesenchyme, essentially without cross-contamination. The expression of the gene for HGFA together with those for hepatocyte growth factor (HGF) and its receptor, c-met, was investigated in each tissue element by RT-PCR. In the fetal rat gastrointestinal tract, mRNA signals for the HGFA gene were observed only in epithelia expressing c-met mRNA. In contrast, expression of HGF mRNA was limited to the mesenchymal elements, indicating the presence of a local HGF system in the gastrointestinal tract; an inactive form of HGF (proHGF) is secreted from the mesenchyme and then cleaved into the active form by HGFA secreted by the target epithelia. During the period of morphogenesis and histodifferentiation in the gastrointestinal tract, enhanced expression of the genes for HGF and its receptor/c-met was evident, with elevated HGFA mRNA level observed throughout the gastrointestinal tract except in the forestomach, where mRNA expression was barely detectable. These results strongly suggest the possibility that morphogenesis of the gastrointestinal tract is regulated not only by a local increase in production of HGF, but also by enhanced proteolytic activation of proHGF. Thus, it is probable that locally synthesized HGFA plays a significant role as a regulator of the morphogenic action of HGF during gastrointestinal tract development.

The organization and structure of the gene coding for hepatocyte growth factor activator (HGFA) have been determined by isolation of unique clones from a human genomic library. These clones were characterized by restriction mapping, Southern blotting and DNA sequencing. The complete sequence of the gene was determined and found to span about 7.5 kilobases of DNA and consist of 14 exons separated by 13 introns. The coding region of HGFA consists of multiple putative domains that are homologous to those observed in blood coagulation factor XII (FXII). These regions were found as separate exons in the gene, and the exon/intron arrangement was similar to that of FXII, suggesting that the genes for HGFA and FXII have arisen through gene duplication events from a common ancestral gene. The major transcription initiation site is located 75 bp upstream of the translational start codon. The gene was mapped to chromosome 4p16, using spot-blot hybridization on sorted chromosomes and fluorescence in situ hybridization on metaphase chromosome spreads. The phylogenetic and functional relationships between HGFA and FXII as well as urokinase and tissue-type plasminogen activator are discussed.

Gene expression of hepatocyte growth factor activator inhibitor (HAI), a recently identified Kunitz-type serine proteinase inhibitor, was analyzed in a series of human colorectal carcinoma cell lines and in human colorectal tissues. All of the 14 cell lines derived from adenocarcinoma of the colorectum expressed HAI in vitro, whereas a colon carcinoma cell line of neuroendocrine origin did not. In vivo, HAI was consistently expressed in the normal colorectal mucosa. Although the expression of HAI mRNA was conserved in adenocarcinoma tissues of the colorectum, the levels of expression were decreased in the adenocarcinoma tissues compared to the normal counterparts. There was a tendency towards an inverse correlation, albeit not well defined, between the amounts of HAI mRNA and the tumor progression. Immunohistochemical study indicated that HAI protein is present predominantly on the surface of epithelial cells of the colon and the immunoreactivity was decreased in the adenocarcinoma cells.

Background/Aims: Radical resection is accepted as one of the most curative treatments for hepatocellular carcinoma. However, most patients have coexisting cirrhosis and their liver function is usually impaired. It is therefore important to stimulate the regeneration and function of the remnant cirrhotic liver after hepatectomy. Hepatocyte growth factor is a potent mitogen that has been suggested to play a crucial role in liver regeneration. Methods: In this study, we performed 45% hepatectomy in rats with cirrhosis induced by thioacetamide, and administered recombinant human hepatocyte growth factor (rhHGF) with dextran sulfate by continuous infusion into the jugular vein with an infusion pump. Results: rhHGF stimulated an increase in the wet weight of the remnant liver compared with untreated control rats. The proliferating cell nuclear antigen labeling index showed that this increase resulted from the stimulation of DNA synthesis. Serum levels of liver enzymes increased after hepatectomy, but returned to the prehepatectomy level more rapidly in rhHGF-treated rats than in controls. rhHGF increased hepatic protein synthesis above prehepatectomy levels and also markedly increased the serum levels of hepatic lipid metabolites. Conclusions: These results demonstrate that continuous intravenous infusion of rhHGF enhances the growth and function of the remnant liver in rats with cirrhosis after partial hepatectomy. Therefore, rhHGF may be useful after hepatic resection in patients with cirrhosis.

The hepatocyte growth factor has been reported to be a potent mitogen of various epithelial cells, including gastric mucosal cells. Therefore, production and activation of hepatocyte growth factor in the gastric wall were investigated to speculate on the possible role of this factor in the healing of gastric ulcer in rats. Indomethacin-induced gastric mucosal lesions and acetic acid induced ulcers were employed as models of acute gastric lesions and chronic ulcer, respectively. Immunoblot and Northern blot analyses indicate that experimentally induced gastric mucosal lesions stimulate not only the production of hepatocyte growth factor, but also the conversion to its active form. This conversion was accompanied by increased gene expression of hepatocyte growth factor activator in the stomach. In rats with acute mucosal lesions, hepatocyte growth factor activator mRNA was most abundant 6 h after induction of mucosal lesions. On the other hand, hepatocyte growth factor and hepatocyte growth factor activator mRNA levels were elevated until 15 days after the induction of chronic ulcers. In summary, it has been clarified that not only production, but also activation of hepatocyte growth factor is stimulated during gastric ulcer healing.

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.

Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for epithelial cells in vitro. It appears likely that HGF participates in tissue regeneration following hepatic and renal injury in vivo. The activity of HGF is localized to the injured tissues by a proteolytic activation system; HGF remains as an inactive single-chain form in the normal state and is converted to an active heterodimeric form in response to tissue injury. A protease responsible for this conversion is induced in the injured liver, but it has not yet been identified. We have previously purified and characterized HGF activator (HGFA), a serum-derived serine protease that efficiently activates single-chain HGF in vitro. In this study, we found that the HGF-converting activity in the injured liver was inhibited by an anti-HGFA antibody. We also found that the active form of HGFA was generated exclusively in the injured tissues. Thus, it appears likely that HGFA is the key enzyme that regulates the activity of HGF in the injured tissues. We also analyzed the heparin binding properties of the precursor and mature forms of HGFA. HGFA had a weak affinity for heparin near the physiological salt concentration in its precursor form but acquired a strong affinity for heparin upon activation that is linked to blood coagulation. This property may ensure the local action of this enzyme at the site of tissue injury.