PubMed Health⌕ Search

Biomedical subjects

G K Michalopoulos

Publications and source records attributed to G K Michalopoulos.

At least 55 records · Page 3Linked to original sources
PubMed1997

Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-alpha in rat liver epithelial cells.

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-alpha (TGF-alpha) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-alpha transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-alpha responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-alpha transfectants. We investigated the mechanisms by which TGF-alpha expression may influence the HGF/c-met pathway. We showed that most TGF-alpha transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-alpha, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or other exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-alpha and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-alpha and HGF.

Animals↗
PubMed1997

Bile ductular damage induced by methylene dianiline inhibits oval cell activation.

Administration of 2-acetylaminofluorene (2-AAF) given before a two-thirds partial hepatectomy (PHx), results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Our objective in this study was to examine the oval cell response and associated alpha-fetoprotein (AFP) gene expression by combining 2-AAF with selective hepatic damage caused by either carbon tetrachloride (CCl4) exposure or by PHx. We also examined oval cell response with the above two protocols (2-AAF/CCl4 and 2-AAF/PHx) as affected by previous bile ductular damage caused by 4,4'-methylene dianiline (4,4'-diaminodiphenylmethane, DAPM) exposure. DAPM is an aromatic diamine, known to cause bile ductular damage in both humans and animals. Using the protocols of 2-AAF/ CCl4 and 2-AAF/PHx, when DAPM was given 24 hours before the hepatic injury, no oval cell proliferation was seen (histological) and AFP expression was not detected by Northern blot analysis. These results provide direct evidence that oval cells are closely associated with the biliary epithelial cells and supports the theory that hepatic oval cells may originate from cells derived from either intraportal or periportal ductules.

2-Acetylaminofluorene↗
PubMed1996

Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.

Serum-free rat hepatocyte cultures can be stimulated to divide by the inactive, single-chain form of hepatocyte growth factor (scHGF), suggesting that hepatocytes contain a protein that can cleave scHGF to its biologically active, two-chain (tcHGF) form. We added radiolabeled scHGF to serum-free cultures and confirmed that tcHGF was being generated. Because scHGF can be cleaved to tcHGF by plasminogen activators (PAs), we next tested the cultures for active PA. Although little PA activity was initially present, the majority was of the urokinase type (u-PA) as determined by neutralization studies using either a polyclonal antibody against u-PA or, since u-PA functions in the context of its receptor (u-PAR), a monoclonal antibody against u-PAR. Considerable PA activity developed within 24 h, which was also neutralizable with antibody. To test whether the active, receptor-bound u-PA from the cell cultures was cleaving scHGF, iodinated scHGF was added to intact cells in the presence of the antibody against u-PAR. Comparison to control cultures determined that the antibody prevented scHGF cleavage. Analysis of cultures treated with HGF, epidermal growth factor, and transforming growth factor alpha (TGF-alpha) alpha showed these growth factors increased the hepatocyte PA activity in parallel with the mRNA for u-PA. TGF-beta had the opposite effect, and when TGF-beta was added to the culture system, conversion of scHGF to tcHGF was prevented in concert with the production of the type 1 PA inhibitor. When liver remnants from hepatectomized animals were assayed for active TGF-beta, elevated protein was found just prior to the appearance of PA inhibitor 1 message and protein. Collectively, our data show that in culture, active u-PA is present and cleaves scHGF to tcHGF in the context of its receptor. It also suggests that modulation of u-PA activity by various growth factors is relevant for regulating cleavage of scHGF to tcHGF both in vitro and in vivo.

Animals↗
PubMed1996

Synergistic effects of hepatocyte growth factor on human cord blood CD34+ progenitor cells are the result of c-met receptor expression.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor which, in addition to its mitogenic potency for primary hepatocytes, also has a role in the regulation of cell motility, cell growth and morphogenesis. In the present study, we show that c-met, the high-affinity receptor for HGF, is expressed on human cord blood (CB) CD34+ progenitor cells and CD34+Thy-1+ Lin-(lin-) cells. We have investigated the capacity of HGF to synergize with other growth factors to induce colony formation by CB CD34+ progenitor cells. CD34+ cells were cultured in semisolid medium containing serum with increasing concentrations of GM-CSF, G-CSF, macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), interleukin 3 (IL-3) and IL-11 alone or in combination with HGF. HGF acted as a potent synergist and enhanced, up to fourfold, colony formation induced by GM-CSF, G-CSF or M-CSF. HGF in combination with SCF, IL-3 or IL-11 did not induce proliferation of colony forming units-granulocyte macrophage (CFU-GM) above control levels. In serum-deprived cultures, HGF was only detectably synergistic with IL-11, and all other culture combinations showed no proliferation. To determine whether the stimulatory effect of IL-11 and the synergistic effect of HGF in the absence of serum could be attributed to the effect of these two cytokines on stem cells, IL-11-stimulated and unstimulated lin- cells were analyzed for expression of c-met. CD34+Thy-1+Lin- cells were positive for c-met, both in the presence and absence of IL-11 stimulation, and Northern analysis indicated that c-met RNA expression was upregulated in response to IL-11 compared to unstimulated controls. These results provide strong evidence for upregulation of the HGF receptor on primitive hematopoietic cells by IL-11, and for the synergistic role of HGF in colony formation by hematopoietic stem cells.

Antigens, CD34↗
PubMed1996

Expression of HGF mRNA in human rejecting kidney as evidenced by in situ hybridization.

In situ hybridization was performed to demonstrate hepatocyte growth factor (HGF) mRNA in two patients with normal kidney and in 23 patients with allograft nephrectomy. In situ hybridization was combined with immunohistochemistry to identify HGF-producing cells. In the two patients with normal kidney, no HGF mRNA was obtainable. In 15 of the 23 allograft patients, signals of HGF mRNA were detectable. In six of these 15 patients, the signals were present mainly at the medullocortex junction, and in the other nine patients at the cortex and/or medulla. Strong and frequent signals were present in gland-like structures in 15 cases. Some scattered signals were also present in the fibrosed glomeruli in five cases, in the thickened intimas of large arteries in three cases, and in the arterial muscle coats of two cases. Combined immunohistochemistry and in situ hybridization showed that HGF mRNA-positive cells in gland-like arrangements were also positive for cytokeratin and negative for factor VIII. Cells with HGF mRNA signal and located in the arterial media were also positive for actin. These findings suggest that HGF mRNA is transcribed both in the urinary tubular epithelium and in the mesenchymal cells (fibroblasts, and smooth muscle cells in chronic vascular rejection and endothelial cells and/or mesangial cells in transplant glomerulopathy) in human rejecting kidney.

Graft Rejection↗
PubMed1996

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.

Adult↗
PubMed1996

Hepatocyte growth factor mRNA in human liver cirrhosis as evidenced by in situ hybridization.

BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes, and HGF-producing cells have been reported to be Ito cells or endothelial cells in the sinusoid of the liver. No reports have been published about the localization of HGF mRNA in human liver cirrhosis. METHODS: In situ hybridization (ISH) for HGF mRNA was performed in 5 normal liver and 16 human liver cirrhosis specimens, using 1 RNA probe and 3 oligonucleotide probes labeled with 35S. RESULTS: A positive signal was obtained in 15 of these cases. In five normal liver specimens, signals of HGF mRNA were not obtainable. In 13 of the 15 cases of liver cirrhosis, HGF mRNA was present in the periphery of the regenerative nodules. This peripheral pattern was seen in regenerative nodules with irregular nodule to septal interfaces. Combined immunohistochemistry and ISH showed that vimentin and CD 68-positive cells consistent with macrophages expressed HGF mRNA in such cases. In three specimens with diffuse signal for HGF mRNA in the hepatic nodules, signals localized to the sinusoidal spaces. HGF mRNA-positive cells were spindled and polygonal in shape, suggesting endothelial, Kupffer, and/or Ito cells of origin. In the diffuse pattern the peripheral margins of the regenerative nodules appeared well-defined. In one case regenerative nodules with both diffuse and peripheral signal patterns were present in the same section. There was no relationship among HGF mRNA, etiology, and macroscopic appearance of liver cirrhosis. CONCLUSIONS: HGF gene transcription in human liver cirrhosis nodules may be heterogeneous, probably related to the degree of activity of the regenerative nodules. HGF appears to be produced by the mesenchymal cells, including Ito cells, macrophages (Kupffer cells), and endothelial cells in human liver cirrhosis.

Antigens, CD↗
PubMed1995

Hepatocyte growth factor inhibits cell proliferation in vivo of rat hepatocellular carcinomas induced by diethylnitrosamine.

In addition to its mitogenic, motogenic and morphogenic functions on various cell types, hepatocyte growth factor (HGF) also suppresses mitosis in several cancer cell lines, including carcinomas and sarcomas. Here we report that HGF is also mito-inhibitory in rat liver tumors induced by diethylnitrosamine. By using a double labeling technique employing [3H]thymidine and 5-bromo-2'-deoxyuridine to determine cell proliferation before and after HGF infusion, we determined that continuous infusion of 20 micrograms total HGF inhibited tumor cell proliferation by 50%. The labeling in non-tumor areas showed the reverse result in that the labeling was higher in HGF-treated rats than control rats. These results indicate that HGF has different effects on growth of normal and tumorous hepatocytes in vivo. These findings may be of relevance in understanding the role of HGF in hepatocarcinogenesis and provide added modalities for controlling growth of hepatocellular carcinomas.

Animals↗
PubMed1995

Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.

Hepatocyte growth factor (HGF), also known as scatter factor, is believed to play a primary role in liver regeneration. HGF is produced in an inactive single-chain form that can be cleaved in vitro to the active two-chain form by tissue-type and urokinase-type plasminogen (PLG) activators (tPA and uPA). We have now documented the de novo appearance of active uPA in livers from male Fischer F344 rats that underwent 70% partial hepatectomy (PHx) as early as 1 minute after surgery. Western blot analyses of protein extracts from liver remnants that were obtained immediately after surgery and periodically until 24 hours after PHx indicate that the quantity of uPA remains fairly constant in PHx samples. In contrast, the uPA receptor (uPAR) dramatically increases, beginning within 1 minute after PHx. This results in enhanced activity of uPA, as seen by direct zymography on cryostat sections. The uPA present in remnant liver homogenates from rats that underwent PHx is the primary agent that cleaves single-chain HGF to its two-chain form, because cleavage can be prevented when antibody against uPA is included in the liver homogenates. Furthermore, heterodimeric HGF, which is not present in normal liver, increases in the liver remnants from rats that underwent PHx, correlative to uPAR. The presence of active uPA is one of the earliest responses yet documented after PHx. These findings imply that both uPA and uPAR are involved in activating endogenous HGF in the regenerating livers of animals that underwent PHx.

Animals↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

Comparative effects of hepatocyte growth factor and epidermal growth factor on motility, morphology, mitogenesis, and signal transduction of primary rat hepatocytes.

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatocyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal-associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of 32P-loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal-associated F-actin. Two cytoskeletal-associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF-stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20-fold at 1 min and remained phosphorylated over 50-fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20-fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF- or EGF-specific motility, morphology, and mitogenicity in hepatocytes.

Animals↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗