The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis.
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Animals↗
Biomedical subjects
R Zarnegar
Publications and source records attributed to R Zarnegar.
Stimulatory effects of hepatocyte growth factor on normal and neoplastic human bronchial epithelial cells.
We examined the mitogenic, chemoinvasive, and chemotactic effects of hepatocyte growth factor (HGF) toward normal and neoplastic human epithelial cells derived from the bronchial mucosa. Primary cultures of human bronchial epithelial cells (HBE cells), immortalized bronchial epithelial cells (IB3-1 cells), and cells derived from a squamous cell carcinoma of the lung (128-88T cells) were used as targets. HGF was mitogenic for all three cell types as measured by bromodeoxyuridine (BrdU) labeling and colony-forming efficiency (CFE). With the use of BrdU labeling, 9.8-16.8% of nuclei were labeled in controls vs. 56.9-65.6% labeled nuclei in cells treated with HGF. HGF stimulated colony formation 3.6-6.2-fold over untreated control. Analysis by reverse transcription-polymerase chain reaction demonstrated the presence of the c-met gene, the receptor for HGF, in all three cell types. Cell lysates from all three cell types contained proteins that were recognized by a c-met antibody as determined by Western blotting. The gene for HGF was not expressed in any of the cell types, although it was expressed in control MRC5 fibroblasts. No HGF protein could be detected by Western blotting in the conditioned medium from epithelial cells, although it was readily detectable in medium conditioned by lung fibroblasts. HGF proved to be a powerful chemotactic agent for all three cell types and also stimulated invasion into Matrigel, an artificial basement membrane. The results indicate HGF acts mainly as a paracrine growth factor for cells derived from the human bronchus, and may play a role in the growth and progression of lung tumors.
Adenocarcinoma↗
Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma.
Hepatocyte growth factor (scatter factor) and its receptor, the c-met proto-oncogene product (c-MET), have been implicated in embryogenesis, tissue reorganization, and tumor progression. Little is known, however, of the expression and functional significance of these molecules in prostatic cells and tissue. In this investigation, we assessed the expression of hepatocyte growth factor (HGF) and c-MET in prostatic tissues and cell lines and also determined the effect of purified recombinant HGF on cell proliferation and scattering of prostatic carcinoma cell lines. HGF was expressed by human prostatic stromal myofibroblasts in primary culture but not by three human prostatic carcinoma cell lines (LNCaP, DU 145, and PC-3) as assessed by Northern blot analysis. HGF was also detected by reverse transcriptase-polymerase chain reaction in both benign and malignant tissues from radical prostatectomy specimens. c-MET transcripts were identified by Northern blot in two androgen-insensitive human prostatic carcinoma cell lines (DU 145 and PC-3) but not the androgen-sensitive LNCaP cell line. Additional evidence of linkage of androgen responsiveness and c-MET was provided by experiments in which androgen deprivation of normal rat prostates via castration produced a marked up-regulation of c-MET expression as determined by Northern blot and immunohistochemistry. c-MET protein was detected by immunohistochemical analysis in a substantial percentage (58 of 128 or 45%) of prostatic carcinomas and was found more often in metastatic growths of human prostatic carcinoma (15 of 20 patients) compared with primary tumors (43 of 108 patients; P < 0.005). Moreover, in Dunning R-3327 rat prostatic carcinoma cell lines, c-MET expression was highest in the androgen-insensitive subline with the highest metastatic capacity. Purified recombinant human HGF induced dose-dependent cellular proliferation and scattering in the DU 145 carcinoma cell line. These data indicate that HGF may function in the prostate gland as a paracrine growth factor, with synthesis by stromal cells and with biological target cells being the epithelial cells. Expression of the HGF receptor, c-MET, is up-regulated by androgen deprivation and c-MET appears to be preferentially expressed on androgen-insensitive, metastatic cells, suggesting a possible linkage of c-MET expression with prostatic carcinoma progression.
Animals↗
The mouse hepatocyte growth factor-encoding gene: structural organization and evolutionary conservation.
A mouse genomic phage library was screened by using a cDNA probe coding for mouse hepatocyte growth factor (HGF). Five overlapping genomic clones which contained the entire mouse HGF gene were isolated and characterized by restriction mapping, Southern hybridization and DNA sequencing. HGF spans about 65 kb and consists of 18 exons separated by 17 introns, similar to its human counterpart. The nucleotide (nt) sequences of the introns at the exon-intron junctions are GT-AG, analogous to those found in other eukaryotic genes. The exon-intron gene organization of HGF is highly homologous to that of several other genes encoding kringle-containing proteins, especially HGF-like protein and plasminogen. This result suggests that HGF probably evolved through gene duplication and/or exon shuffling events from an ancestral gene. Southern hybridization of genomic DNA from different species revealed that a high degree of homology exists among a variety of vertebrates, including chicken, when a mouse HGF cDNA was used as a probe. This evolutionary conservation of HGF strongly suggests that the protein may play an important role in normal cell physiology. Our current results on mouse HGF structure provide basic and detailed information to carry out further manipulation, such as gene targeting.
Animals↗
Structural and functional characterization of the mouse hepatocyte growth factor gene promoter.
To understand the molecular mechanisms underlying the regulation of hepatocyte growth factor (HGF) gene expression and to define the DNA sequences essential for its cell-type specific and inducible expression, we have isolated and characterized the 5'-flanking region of the HGF gene. A genomic clone containing 2.8 kilobases of the 5'-flanking region of the HGF gene has been isolated from a mouse liver genomic library. Sequence analysis showed that the promoter region of the mouse HGF gene contains a noncanonical TATA box (ATAAA). Further analysis of the 5'-flanking region revealed a number of putative regulatory elements, such as four interleukin-6 response elements (IL-6 RE), two potential binding sites for NF-IL6, a TGF-beta inhibitory element (TIE), a cAMP response element (CRE), two estrogen response elements (ERE) including one located in the first intron, a potential vitamin D response element (VDRE) which overlaps a chicken ovalbumin upstream promoter (COUP) transcription factor binding element, two liver-specific transcription factor (C/EBP) binding sites, and a B cell- and macrophage-specific transcriptional factor binding site (PU.1/ETS). To determine the location of sites that may be critical for the function of the HGF promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT). Transient transfection of chimeric plasmids demonstrated that the mouse HGF gene promoter containing 70 base pairs of the 5'-flanking sequences were active in mouse fibroblast NIH 3T3 cells and in human endometrial carcinoma RL95-2 cells. This basal transcription activity of the HGF promoter was modulated in NIH 3T3 and RL95-2 cells by multiple upstream elements. Three positive elements were identified at positions -2848 to -2674, -1386 to -1231, and -699 to -274, and three negative candidate elements were mapped to positions -1652 to -1386, -964 to -699, and -274 to -70, respectively. By the combination of a series of 5'-end deletion and internal deletion, a cell type-specific negative regulatory element in RL95-2 cells was localized to the nucleotide position -964 to -699. Moreover, the reporter plasmid containing interleukin 6 (IL-6) response element was responsive to IL-6 stimulation in stably transfected NIH 3T3 cells. Our findings revealed a complex pattern of transcriptional regulation of the mouse HGF gene expression.
Animals↗
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)
Activating Transcription Factor 3↗
Distinct morphological and mito-inhibitory effects induced by TGF-beta 1, HGF and EGF on mouse, rat and human hepatocytes.
TGF-beta 1 is known as a potent inhibitor of proliferation of rat and human hepatocytes. In this study we show that the effects of TGF-beta 1 are quite different on mouse hepatocytes. In rat and human hepatocytes, TGF-beta 1 inhibited DNA synthesis and also inhibited the morphological changes induced by growth factors in rat and human hepatocytes. In contrast, addition of TGF-beta 1 to mouse hepatocytes resulted in pronounced alterations in morphology of these cells. These changes were similar to those induced by HGF and EGF. The induction of structural changes by TGF-beta 1 was noted only in mouse hepatocytes. Mouse hepatocytes were also much more resistant to the mito-inhibitory effect of TGF-beta 1. These findings suggest profound differences in hepatocyte growth regulation between these species and may relate to observed differences in susceptibility to carcinogenesis.
Alkaloids↗
Modulation of hepatocyte growth factor gene expression by estrogen in mouse ovary.
Hepatocyte growth factor (HGF) is expressed in a variety of tissues and cell types under normal conditions and in response to various stimuli such as tissue injury. In the present study, we demonstrate that the transcription of the HGF gene is stimulated by estrogen in mouse ovary. A single injection of 17 beta-estradiol results in a dramatic and transient elevation of the levels of mouse HGF mRNA. Sequence analysis has found that two putative estrogen responsive elements (ERE) reside at -872 in the 5'-flanking region and at +511 in the first intron, respectively, of the mouse HGF gene. To test whether these ERE elements are responsible for estrogen induction of HGF gene expression, chimeric plasmids containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT) gene were transiently transfected into both human endometrial carcinoma RL 95-2 cells and mouse fibroblast NIH 3T3 cells to assess hormone responsiveness. Transfection results indicate that the ERE elements of the mouse HGF gene can confer estrogen action to either homologous or heterologous promoters. Nuclear protein extracts either from RL95-2 cells transfected with the estrogen receptor expression vector or from mouse liver bound in vitro to ERE elements specifically, as shown by band shift assay. Therefore, our results demonstrate that the HGF gene is transcriptionally regulated by estrogen in mouse ovary; and such regulation is mediated via a direct interaction of the estrogen receptor complex with cis-acting ERE elements identified in the mouse HGF gene.
3T3 Cells↗
Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene.
Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.
Animals↗
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.
Adolescent↗
Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver.
Hepatocyte growth factor and transforming growth factor-alpha are two well-known hepatomitogens for primary hepatocyte cultures. Here we report that these two growth factors also stimulate in vivo DNA syntheses in normal, unoperated, adult rat liver after 24-hr continuous intraportal infusion. As determined by an immunohistochemical staining technique, 5-bromo-2'-deoxyuridine incorporation was increased in a dose-dependent fashion after infusion of up to 10 micrograms of growth factor/100 gm body weight in the rat. Stimulation of DNA synthesis was seen in the periportal area. Pretreatment using intraportal infusion of collagenase (1 U/kg body weight) for 4 hr before administration of growth factor increased the labeling by 2- to 4-fold to a labeling index range of 48% to 52%. These results suggest that collagenases and possibly other proteases are involved in making hepatocytes competent to respond to growth factors at very early stages of liver regeneration.
Animals↗
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.
Cytokines↗
Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver.
We have previously shown that systemically injected hepatocyte growth factor (HGF) is primarily taken up by the liver. The present study shows that HGF injected systemically or through the portal circulation is retained primarily at periportal sites. The periportal retention of HGF seems to persist longer in regenerating liver. The percentage of the total HGF injected that was retained within the liver at 1 minute after injection varied with the dose. A maximal amount of 0.157 +/- 0.012 microgram of HGF per gram liver tissue is retained by normal liver. Analysis of the circulating form of HGF in the plasma showed a relative enrichment with time for the heterodimeric form of HGF. A portion of portally injected HGF, composed of both single chain and two chain (heterodimeric) form was excreted intact in the bile. This was found in both normal and regenerating liver. These studies show that the liver can sequester large amounts of HGF and that the sequestration occurs primarily at periportal sites. Our studies support the hypothesis that a nonlysosomal processing pathway for HGF is present in the liver.
Animals↗
Molecular cloning and characterization of cDNA encoding mouse hepatocyte growth factor.
A cDNA encoding mouse hepatocyte growth factor (HGF) has been cloned and completely sequenced by use of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent cloning. Sequence analysis reveals that mouse HGF, similar to its human and rat counterparts, consists of 728 amino acids, and both the alpha- and beta-chains are encoded in a single open reading frame. Strong homology exists in the primary structure of HGF among the three species of mouse, rat and human (more than 90%), especially in Kringle 1 of the alpha chain which is assumed to be an essential domain for binding of HGF to its receptor, c-MET, a proto-oncogene product. Our results suggest the existence of evolutionary pressure to conserve the distinct structure, and presumably the biological functions, of HGF.
Amino Acid Sequence↗
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.
Animals↗
On the presence of NK2 receptor subtypes in peripheral and central tissues.
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Animals↗
Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA.
Hepatocyte growth factor, also known as scatter factor, is a complete mitogen for hepatocytes that bears sequence and structural homology with plasminogen. Because it exists in both a mitogenically inactive single-chain form and an active two-chain form, we were interested in determining whether plasminogen activators could properly cleave single-chain hepatocyte growth factor to generate active two-chain hepatocyte growth factor. Herein we report that both urokinase-type plasminogen activator and tissue-type plasminogen activator can cleave single-chain hepatocyte growth factor, generating two-chain hepatocyte growth factor. When equal quantities of plasminogen activator-treated and activator-untreated hepatocyte growth factor are compared in serum-free in vitro bioassays, the treated hepatocyte growth factor is mitotically more active. Also, urokinase-type plasminogen activator was inactive against hepatocyte growth factor molecules with a mutated cleavage site. This suggests that urokinase-type and tissue-type plasminogen activator may be natural biological regulators of hepatocyte growth factor. Because the active form of hepatocyte growth factor is a powerful stimulator of DNA synthesis and cell motility, these findings may be relevant in understanding the role of plasminogen activators in the biology of cancer invasion and metastasis.
Animals↗
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.
Animals↗