The repressive function of AP2 transcription factor on the hepatocyte growth factor gene promoter.

Hepatocyte growth factor is an important multifunctional growth factor whose gene expression is tightly regulated at the transcriptional level. Previous studies from our laboratory have shown that several cis-acting elements are present in the promoter and proximal promoter region of the HGF gene. In this study, we have uncovered that AP2 transcription factor specifically binds to a regulatory site located at -230 to -260 in the upstream region of the HGF gene promoter. Gelshift and supershift assays confirmed that AP2 has high binding affinity to this region. Functional studies which introduced a mutation in the AP2 core binding region as well as cotransfection experiments using an AP2 expression vector revealed that AP2 exerts a repressive role on the HGF gene promoter activity. The AP2 binding site overlaps with those of NF1 and USF/E-box binding sites which we have recently shown to constitute a composite multifunctional docking site for the members of the NF1 and USF transcription factor families. An inverse correlation was noted between AP2 binding activity to this composite site and HGF gene expression in different cell lines. Therefore, AP2-mediated repression of the HGF gene promoter may be part of the molecular mechanism responsible for regulating HGF expression.

The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.

Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.

Liver regeneration.

Liver regeneration after the loss of hepatic tissue is a fundamental parameter of liver response to injury. Recognized as a phenomenon from mythological times, it is now defined as an orchestrated response induced by specific external stimuli and involving sequential changes in gene expression, growth factor production, and morphologic structure. Many growth factors and cytokines, most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-alpha, interleukin-6, tumor necrosis factor-alpha, insulin, and norepinephrine, appear to play important roles in this process. This review attempts to integrate the findings of the last three decades and looks toward clues as to the nature of the causes that trigger this fascinating organ and cellular response.

Induction of met proto-oncogene (hepatocyte growth factor receptor) expression during human monocyte-macrophage differentiation.

The met proto-oncogene encodes the cell surface receptor for hepatocyte growth factor (HGF) and transmits its multifunctional signals such as regulation of cell proliferation, motility, and morphogenesis. These pleiotropic actions attributable to HGF are mainly reported on cells of epithelial derivation which express the Met receptor. The HGF gene, on the other hand, is expressed in mesenchymally derived cells including peripheral blood leukocytes. Recently, we reported that Met receptor gene expression in epithelial cells is induced by inflammatory cytokines; currently, however, little is known concerning Met gene expression in mesenchymal cells. In the present study, we have explored the role of Met expression during monocyte-macrophage differentiation using THP-1 cells, a monocytic cell line, and monocytes freshly isolated from normal human peripheral blood. We have found that untreated monocytes do not express Met mRNA and protein. Upon incubation with differentiation inducers such as 12-O-tetradecanoylphorbol-13-acetate, lipopolysaccharide, a combination of interleukin (IL) 6 plus tumor necrosis factor (TNF) alpha, or IFN-gamma plus TNF-alpha, a pronounced increase in the amounts of Met mRNA and protein are seen in THP-1 cells. The expression of Met appears to correlate with the onset of differentiation of monocytes as noted by changes in cell morphology and adherence to culture plates, and the increased accumulation of Met protein was observed only in cells that differentiated and adhered to the culture dish. Moreover, Met was found to be phosphorylated on tyrosine residues, indicating that the receptor is potentially involved in signal transduction events. Addition of exogenous HGF to the activated cells resulted in the suppression of cell proliferation and an increase in cell motility. Reverse transcription-PCR and Western blot analyses revealed that untreated THP-1 cells contain HGF transcript and protein, and that HGF expression is inducible by addition of the differentiation agents such as 12-O-tetradecanoylphorbol-13 acetate or IL-6 plus TNF-alpha. Immune serum that is specific for neutralizing HGF activity markedly inhibited monocyte differentiation (50% reduction in cell attachment and process formation) induced by IL-6 and TNF-alpha. Moreover, we also found that the mRNA for Ron, which encodes a tyrosine kinase receptor for HGF-like protein (also known as macrophage-stimulating protein), is induced in THP-1 cells during the course of their differentiation to macrophages by IFN-gamma plus TNF-alpha. These findings indicate that the HGF and Met families may indeed be physiological regulators of monocyte-macrophage differentiation/maturation.

Hepatocyte growth factor is a mitogen for Schwann cells and is present in neurofibromas.

To characterize mitogens that might contribute to Schwann cell proliferation during development or in tumors, we tested the ability of hepatocyte growth factor (HGF) to stimulate Schwann cell division in vitro. HGF is a potent mitogen for purified rat Schwann cells; DNA synthesis in rat Schwann cells was stimulated 20-40-fold by 3-10 ng/ml HGF. Rat Schwann cells express c-met mRNA, encoding the HGF receptor, but not HGF mRNA, implying that HGF might act as a paracrine Schwann cell growth factor. HGF-stimulated Schwann cell proliferation differs from that of previously described Schwann cell mitogens in that its activity is abolished by forskolin and is not inhibited or potentiated by addition of transforming growth factor beta (TGF beta) or fibroblast growth factor (FGF). HGF is probably not a component of the axonal signal thought to cause Schwann cell division during development, as anti-HGF neutralizing antibodies failed to block neuron-stimulated Schwann cell proliferation. In contrast, mitogenic activity present in normal human adult nerves and in neurofibromas from patients with type 1 neurofibromatosis analyzed in the absence of forskolin is largely inhibitable by anti-HGF. Thus, HGF is a novel mitogen for Schwann cells in vitro and it is present in Schwann cell tumors, suggesting a potential role for HGF after wounding of peripheral nerves or in tumor growth.

Rapid induction of mRNAs for liver regeneration factor and insulin-like growth factor binding protein-1 in primary cultures of rat hepatocytes by hepatocyte growth factor and epidermal growth factor.

Liver regeneration factor belongs to the leucine-zipper family of transcription factors. It was originally cloned and characterized through differential screening of a regenerating rat liver cDNA library. The mRNA for liver regeneration factor-1 is barely detectable in normal rat liver but is dramatically induced after two-thirds hepatectomy, with a peak 1 to 3 hr after surgery. The nature of the signaling molecule(s) for this rapid induction is not known. It has been suggested that the liver regeneration factor-1 protein product, through complex interactions with other transcription factors such as c-Jun and Jun-B, controls expression of genes that are required during the G1 phase of hepatic growth. Hepatocyte growth factor has been shown to be the most potent mitogen for hepatocytes in vitro and in vivo. Plasma levels of hepatocyte growth factor rapidly (within 30 min) increase after loss of hepatic parenchyma induced by partial hepatectomy or carbon tetrachloride treatment. It has been postulated that hepatocyte growth factor plays a crucial role in stimulating the hepatocyte to enter the cell cycle. In this communication, we report that addition of pure hepatocyte growth factor to primary cultures of rat hepatocytes in the absence of serum and insulin results in rapid and transient induction of liver regeneration factor-1 mRNA (more than 20-fold) with a peak of expression 1 hr after treatment. The levels of jun-B and c-fos mRNAs, which are also known to be induced during the early hours of liver regeneration, were also increased after treatment of isolated hepatocytes with hepatocyte growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of c-MET proto-oncogene (HGF receptor) mRNA abundance by cytokines and hormones: evidence for rapid decay of the 8 kb c-MET transcript.

The c-MET proto-oncogene product is a transmembrane tyrosine kinase receptor which was recently shown to transmit an array of important cellular responses induced by Hepatocyte Growth Factor (HGF). These biological effects include induction of mitogenesis, motogenesis, morphogenesis, metastogenesis and anti-tumor activity on a variety of epithelial cells. All of these processes are known to be associated with normal and abnormal tissue growth and development. The 190 kDa c-MET protein is encoded by a major transcript of 8 kilobases (kb), which is reported to be expressed predominantly in epithelial tissues. The expression pattern of c-MET mRNA and protein are drastically modified in many tumor tissues and cell lines. Currently, no information is available on the molecular mechanisms that regulate c-MET mRNA level. In the present communication, we report for the first time that the inflammatory cytokines such as IL-1 alpha, IL-6 and TNF-alpha, as well as TGF-beta 1, EGF, HGF and the steroidal hormones (estrogen, progesterone, tamoxifen and dexamethasone) markedly influence the steady-state levels of the 8 kb c-MET mRNA in human carcinoma cell lines derived from human tissues such as ovary, breast and endometrium. We demonstrate that c-MET receptor protein is present at high levels in primary tumors of human ovaries (clear cell carcinomas). We present evidence that the 8 kb c-MET mRNA undergoes rapid degradation with a half-life of less than 30 min and that this decay can be quickly inhibited by cycloheximide. Our results suggest that the expression of the c-met proto-oncogene resembles that of an immediate early response gene.

Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by itself, did not have any detectable biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful production of bioactive recombinant HGF in large quantities, which will allow purification on the milligram scale of pro-HGF and will permit future studies to elucidate pathways involved in HGF activation by its target tissues.

Expression of HGF-SF in normal and malignant human tissues.

Hepatocyte Growth Factor (HGF) also known as Scatter Factor (SF) is a multifunctional cytokine that primarily acts on epithelial cells. Its effects range from enhancement of cell proliferation and cell growth, stimulation of cell migration and motility, promotion of angiogenesis and morphogenesis to inhibition of tumor cell growth. HGF-SF and its receptor proto-oncogene c-met are expressed in a variety of normal embryonic and adult tissues of mouse, rabbit, rat, human and probably other vertebrates. Though the precise physiological function of this cytokine is not understood presently, a large body of experimental data is being rapidly compiled which may help to elucidate the role of HGF-SF in normal and abnormal growth and development. The goal of this chapter is to discuss the current knowledge concerning HGF-SF expression in normal and malignant tissues.

The presence of hepatocyte growth factor in the developing rat.

Hepatocyte growth factor (HGF), a heparin-binding polypeptide mitogen, stimulates DNA synthesis in adult rat and human hepatocytes and in several other cells of epithelial origin. Recently, it was determined that scatter factor (SF), a protein that has been shown to cause the dispersion and migration of epithelial cells in culture, is identical to HGF. Moreover, the receptor for HGF was identified as the product of the proto-oncogene, c-MET, a tyrosine kinase-containing transmembrane protein. c-MET expression has been reported in a variety of adult and embryonic mouse tissues. Similarly, we and others have demonstrated that HGF is expressed in various adult rat and human tissues. In the present study, the tissue distribution of HGF during rat development was determined by immunohistochemistry using an HGF-specific polyclonal antiserum. Between day 12 and day 19, immunoreactivity for HGF was present in various locations such as hematopoietic cells, somites, squamous epithelium of the esophagus and skin, periventricular germinal matrix of the brain, bronchial epithelium, renal collecting tubules and chondrocytes. After day 19, HGF immunoreactivity was also present in the pancreas, submaxillary glands and neural tissues. In addition to immunolocalizing HGF in tissue sections, bioreactive and immunoreactive HGF was extracted and purified from rat fetuses. Other studies demonstrated the presence of HGF and c-MET mRNA in total fetal rat, and in fetal and neonatal rat liver. Addition of purified HGF to fetal and neonatal rat liver cultures enriched for hepatocytes stimulated DNA synthesis up to six-fold over controls. These findings strongly suggest a pivotal role for this potent regulator of growth and development.

Localization of hepatocyte growth factor (HGF) gene on human chromosome 7.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and a variety of epithelial cells in culture. The cDNAs for human and rat HGF have been cloned by different researchers, including ourselves; however, no information on the genomic structure and chromosome localization of the HGF gene is yet available. To investigate HGF's chromosomal localization, DNA from a battery of human-hamster somatic cell hybrids was digested with BglII and analyzed by Southern blot using a 2.3-kb human HGF cDNA as a hybridization probe. The gene encoding the human HGF was assigned to human chromosome 7. Restriction enzyme and Southern blot analyses using the HGF cDNA and HGF-specific oligonucleotides as probes suggest that the human HGF gene exists as a single-copy gene and is composed of several exons.

Patterns of alpha-1-adrenergic receptor expression in regenerating and neoplastic hepatic tissue.

As norepinephrine is a potent hepatocyte comitogen through binding to the alpha 1-adrenergic receptor, we have examined mRNA levels of the alpha 1a- and alpha 1b-adrenergic receptor subtypes in normal and regenerating rat hepatocytes as well as in several different rat hepatoma cell lines. All rat hepatomas examined lacked both alpha 1a- and alpha 1b-receptor message and receptor binding in radioligand binding experiments, suggesting that the growth of dedifferentiated neoplastic rat hepatocytes is not regulated by the alpha 1-adrenergic receptor. Interestingly, unlike the rat hepatomas analyzed, the human hepatocellular carcinoma cell line, HepG2, was positive for both alpha 1a and alpha 1b message at 4.5 kb, yet this cell line lacked receptor binding in radioligand binding assays. While normal and regenerating liver is negative for alpha 1a-receptor expression, it is positive for alpha 1b expression and is characterized by the presence of two bands at approximately 4.0 and 3.2 kb which peaked between 20 and 48 h after partial hepatectomy. A dramatic decrease in message level of the lower band and the continued presence of the upper band between 6 and 12 h after partial hepatectomy, and before the peak in DNA synthesis in regenerating rat liver, may correspond with observed differences in alpha 1-receptor function during liver regeneration.

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.

Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes.

Hepatocyte Growth Factor (HGF) (also known as Hepatopoietin A [HPTA] (1-9) is a heterodimeric heparin-binding polypeptide mitogen for hepatocytes distinct from other well-known growth factors. In this study, biologically active radioiodinated HGF was used to identify binding sites on intact hepatocytes in culture. The results show the presence of relatively low affinity binding sites due to the presence of heparin or heparin-like molecules and high affinity specific receptor binding sites on the cell surface of intact hepatocytes. Scatchard analysis of binding data indicates an apparent dissociation constant (Kd) of 3.5 nM with 120,000 sites per hepatocyte for the cell-surface receptor. Analysis of affinity cross-linked 125I-HGF-receptor complex by SDS-PAGE under non-reducing conditions reveals the presence of a distinct band with apparent Mr of 230,000. These data show that HGF exerts its biological effect on hepatocytes (stimulation of DNA synthesis) through a specific and unique cell-surface receptor.