Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.     

TGF-beta is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.     

Vitronectin secretion by hepatic and non-hepatic human cancer cells

Summary Thirty-eight human cancer cell lines and subclones derived from 12 different organs were screened for vitronectin secretion in their culture media. By immunoblotting analysis we detected high secretion by three out of five hepatoma cell lines tested but no secretion by the others. In addition, significant secretion was observed in seven non-hepatic cancer cell lines and subclones derived from the cervix, lung, and pancreas. These vitronectin-secreting cells included PLC/PRF/5, HuH-6 #5, HuH-7, HeLa S3, HeLa · P3 #2, #3, #6, #8, A549, and MIAPaCa-2. The results were further confirmed by quantitative analysis using sandwich enzyme-linked immunoassay, and activity analysis of cell attachment promotion on Western blotted filters.     

Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.

The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.     

Effects of protein kinase inhibitors on the mitogenic activity of human hepatocyte growth factor on rat hepatocytes in primary culture.

To evaluate the role of protein phosphorylation reactions in signal transduction of human hepatocyte growth factor (hHGF), now known to be the same protein as the scatter factor and tumor cytotoxic factor, we examined the effects of various inhibitors of protein kinases on the mitogenic activity of hHGF on rat hepatocytes in primary culture. Genistein, a specific inhibitor of tyrosine kinase, dose-dependently inhibited the effect of hHGF in stimulating DNA synthesis of hepatocytes. By contrast, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine (H7), a specific inhibitor of protein kinase C, potentiated the stimulatory effect of hHGF on DNA synthesis of hepatocytes. H7 was effective at over 2 micrograms/ml and potentiated the effect of hHGF over 2-fold at 20 micrograms/ml. On the other hand, an inhibitor of Ca++/calmodulin-dependent protein kinase inhibited both the basal and hHGF-stimulated DNA synthesis in the cells, whereas an inhibitor of cyclic nucleotide-dependent protein kinases had little effect on the action of hHGF. These results suggest that tyrosine phosphorylation is required for stimulation of hepatocyte DNA synthesis by hHGF and that the action of hHGF is negatively regulated by protein kinase C activation.     

An alternatively processed mRNA generated from human hepatocyte growth factor gene.

We recently reported the isolation and sequencing of cDNA for human hepatocyte growth factor (hHGF) [Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. & Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967-973]. In the present study, we report the sequence of another cDNA clone for a shorter form of hHGF mRNA. Comparison of the sequence with that of the hHGF cDNA revealed that the two sequences are identical in their 5' ends up to 865 nucleotides downstream from the translation-initiation site, then completely diverge from each other. By Northern blot analysis, the hHGF-related 1.5-kb mRNA, which corresponded to the newly isolated cDNA variant, was identified in human placenta. Sequence analysis of a human genomic HGF clone showed that the diverged 3'-terminal portion of the mRNA is generated by alternative RNA-processing events utilizing a specific exon. The mRNA could encode a short hHGF molecule of 290 amino acids corresponding to the N-terminal portion of hHGF which consists of 728 amino acids. In order to examine the effect of the predicted translation product on hepatocyte growth, an expression plasmid for the cDNA variant was constructed and transfected into Cos cells. Immunoblotting analysis showed that the transfected Cos cells produced a protein of about 33 kDa. The protein product did not stimulate DNA synthesis by rat hepatocytes in primary culture.     

Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (tshr) phenotype. One tshr mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.     

Effects of various substrates on human hepatoblastoma and hepatoma cell culture

The effects on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines of collagen type I (CI), type IV (CIV), fibronectin (FN) and laminin (LAM) were investigated. CI and CIV promoted almost equally the attachment of both cell lines more strikingly than did FN or LAM. CI and CIV were also superior to FN or LAM in supporting the growth of HuH-6. These substrates, however, had no appreciable effect on the growth of HuH-7. The amount of alpha-fetoprotein (AFP) and albumin secreted in HuH-6 was found to be higher on FN- and LAM- coated substrates than on the other matrix material- coated ones. HuH-7 exhibited increased levels of AFP on LAM- coated substrates 4 days after plating. These results indicate that the ability of extracellular matrix materials to enhance attachment and/or growth is different from that to enhance AFP and albumin production in HuH-6 and probably in HuH-7.     

人胎肝中肝细胞生长因子生物活性的研究

人胎肝细胞裂解液经膜超滤,在分子量10～30kD组分中可检测出人肝细胞生长因子(hHGF)活性。hHGF为一热稳定的蛋白质或多肽类物质。它可特异地刺激肝来源细胞~3H-TdR掺入的增加,并且存在量效依赖关系,而对非肝来源细胞的DNA合成无刺激作用。hHGF的生物活性及理化性质与某些已知因子,如胰岛素、胰高血糖素、血小板来源的生长因子、表皮生长因子及增殖刺激因子等有所不同。     

Effects of serum and serum-derived factors on the growth and production of α-fetoprotein and albumin by human hepatoma cell lines

A serum-free culture system of human hepatoma cell lines (HuH-6 and HuH-7) was used to investigate the activity of bovine serum (BS) and of serum-derived factors on the growth and production of -fetoprotein (AFP) and albumin. At higher concentrations, dialyzed BS was inhibitory to the growth of HuH-6 and caused reduction of the level of AFP production by the cells. AFP and albumin levels in HuH-6 and HuH-7 were reduced or unchanged by fetuin, bovine serum albumin (BSA) and transferin (TF), although no cytotoxicity was shown by any of them. Commercial preparations of platelet-derived growth factor exhibited cytotoxicity to HuH-6 and HuH-7 and induced a decrease of AFP and albumin levels in a dose-dependent manner. Transforming growth factor (TGF-) exhibited no cytotoxicity to HuH-6. AFP levels in HuH-6 were unchanged with 1000 pg/ml TGF-, but albumin levels were decreased. TGF-7 at a concentration of 1000 pg/ml was cytotoxic to HuH-7 and AFP levels were a little increased. Albumin levels, however, were unchanged. Following exposure to cycloheximide, AFP and albumin levels in HuH-6 were inhibited.Abbreviations AFP -fetoprotein - BS bovine serum - BSA bovine serum albumin - EDTA ethylenediaminetetraaceticacid - ELISA enzyme-linked immunosorbent assay - HBSS Hank's balanced salt solution - PBS phosphate buffered saline - PDGF platelet-derived growth factor - TF transferrin - TGF-\ transforming growth factor beta     

Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.     

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.     

人肝细胞生长因子cDNA克隆在CHO细胞中的表达

肝细胞生长因子是一种由α、β链组成的杂合二聚体糖蛋白,能促进肝细胞、多种上皮细胞、内皮细胞和神经胶质细胞的有丝分裂,并对多种肿瘤细胞具有细胞毒性作用或者抑制其生长［１～４］,其作用无种属特异性,如人肝细胞生长因子能促进大鼠肝细胞增殖［５］。由于天然Ｈ...     

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.     

Natural occurrence of cancer-preventive geranylgeranoic acid in medicinal herbs

Geranylgeranoic acid (GGA; all-trans 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenoic acid) has been shown to induce apoptosis in a human hepatoma-derived cell line, HuH-7. We aimed not only to confirm the apoptogenic properties of GGA and its derivatives, but also to search for natural GGA in medicinal herbs. GGA induced apoptosis in human hepatoma-derived cell lines, HuH-7, PLC/PRF-5, and mouse transformed hepatocyte-derived cell line, MLE-10, in a dose- and time-dependent manner, but failed to induce cell death in human hepatoblastoma-derived HepG-2 and mouse primary hepatocytes in the same condition. Besides GGA, 4,5-didehydro GGA, 14,15-dihydro GGA, and 2,3-dihydro GGA were also active to induce cell death in HuH-7 cells, while 4,5-didehydro-10,11, 14,15-tetrahydro GGA, 4,5,8,9-tetrahydro GGA, farnesoic acid, and geranylgeraniol were inert. By using liquid chromatography/mass spectrometry, we found natural GGA as a negative ion of m/z 303.4 in a Chinese herb, Schisandra chinensis, and Schisandra GGA was identified by derivatization with both mild methylation and catalytic hydrogenation. Some other GGAs hydrogenated in the different degrees, including phytanic acid (perhydro GGA), were also found in S. chinensis. GGA and phytanic acid were detected in 24 out of 25 herbs tested. The present study is the first report of natural GGA in medicinal herbs.     

Transcriptional regulation of alpha-fetoprotein expression by dexamethasone in human hepatoma cells

The level of alpha-fetoprotein (AFP) mRNA in HuH-7 human hepatoma cells is elevated by the addition of dexamethasone to the culture medium. To locate the DNA region involved in hormonal regulation of the AFP gene, we constructed recombinant plasmids in which various lengths of the 5'-flanking sequence of the human AFP gene were fused to the CAT gene. Various cell lines were transfected with the recombinant plasmids, incubated with or without 3 x 10(-6) M dexamethasone, and then assayed for chloramphenicol acetyltransferase expression. In hepatoma cells that produce AFP, the dexamethasone treatment resulted in the stimulated chloramphenicol acetyltransferase expression when the transfected plasmids contained 169 base pairs (bp) or longer AFP 5'-flanking sequence. No dexamethasone effect was observed when the 5'-flanking sequence was less than 98 bp long. The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that this effect is mediated by glucocorticoid receptors. The 71-bp region between positions -169 and -98 contains a nucleotide stretch which is similar to the consensus sequence of the glucocorticoid responsive element (GRE). Partial alterations of this sequence resulted in decreased dexamethasone response. The GRE-containing region stimulated heterologous (SV40) promoter activity in response to dexamethasone treatment in an orientation- and position-independent manner. The GRE and the upstream AFP enhancer function independently from each other.     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。     

In vitro and in vivo characteristics of hepatic oval cells modified with human hepatocyte growth factor

Hepatocyte growth factor (HGF) is a multifunctional growth factor that controls cell scattering. It has been suggested that it regulates the proliferation of hepatic oval cells (HOCs). Using a HOC line that stably expresses the human HGF gene (hHGF), we investigated the in vitro proliferation and differentiation characteristics of hHGF-modified HOCs and explored their potential capacity for intrahepatic transplantation. A modified 2-acetylaminofluorene and partial hepatectomy (2-AAF/PH) model was established to activate the proliferation of oval cells in the rat liver. HOCs were transfected with the pBLAST2-hHGF plasmid and hHGF-carrying HOCs were selected based on blasticidin resistance. The level of hHGF secretion was determined via ELISA. Cell proliferation was determined using the MTT assay. Differentiation was induced by growth factor withdrawal. A two-cuff technique was used for orthotopic liver transplantation, and HOCs or hHGF-modified HOCs were transplanted into the recipients. The levels of biochemical indicators of liver function were measured after transplantation. An HOC line stably expressing hHGF was established. The transfected line showed greater hHGF secretion than normal HOCs. The hHGF gene promoted the proliferation capability of HOCs by reducing the peak time in vitro. The hHGF-modified HOCs differentiated into hepatocytes and bile duct epithelial cells upon growth factor withdrawal in vitro. In addition, hHGF-modified HOC transplantation significantly prolonged the median survival time (MST) and improved the liver function of recipients compared to HOC transplant recipients and nontransplanted controls. Our results indicate that hHGF-modified HOCs may have valuable properties for therapeutic liver regeneration after orthotopic liver transplantation.