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The effects of drugs which change the bile-salt-independent fraction of bile flow on NaK and Mg activated ATPases were studied in membrane fractions rich in bile canaliculi. The administration of phenobarbital caused no induction of these enzymes which could explain the increased bile flow observed in the rat. Rose bengal, in addition to its strong photoxidative inhibition of both ATPases, inhibits the NaK ATPase of rat and rabbit bile canaliculi in the absence of light. A closely related substance, uranine, inhibits neither bile flow nor NaKATPase. Inhibition of this enzyme by rose bengal may therefore be responsible for the observed effects of this dye on bile flow independent of bile salts. [ABSTRACT FROM PUBLISHER]

Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl(4)-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.

The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in β-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1β, TNF-α, lymphotoxin-β, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-β and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.

Background/aims: Resident macrophages and myofibroblasts derived from hepatic stellate cells play a key role in liver wound healing. We previously reported that these sinusoidal cells secrete the growth arrest-specific protein 6 (Gas6) and express Axl, one of its receptors. Here we address the role of Gas6 in the healing process during acute liver injury., Methods: Toxic hepatitis was induced by a single carbon tetrachloride injection in Gas6 deficient (Gas6(-/-)) mice and liver recovery was compared with wild-type animals., Results: Gas6 deficiency did not cause any change in CCl(4)-induced liver damage. At 72 h, an efficient tissue repair was observed in wild-type animals whereas in Gas6(-/-) mice, we noticed a defective wound healing accounted by reduced Kupffer cell activation revealed by a decrease in the induction of CD14, TNF-alpha, IL6 and MCP-1. Gas6-deficiency, by limiting cytokine/chemokine release, prevents hepatocyte proliferation, recruitment of circulating monocytes and accumulation of myofibroblasts in healing areas. We also report a direct chemotactic effect of Gas6 on circulating monocytes which might explain defective macrophage infiltration in liver necrotic areas of Gas6(-/-) mice. Interestingly in Gas6(-/-) mice, we observed a high and constitutive expression of Axl and an induction of the suppressor of cytokine signaling SOCS1 after CCl(4) treatment., Conclusions: The lower level of cytokines/chemokines in Gas6(-/-) mice after CCl(4) injury, is the consequence of an inhibitory signal arising from Axl receptor overexpression, leading to delayed liver repair in deficient mice.

The protein product of the growth arrest-specific gene 6 (Gas6) is a secreted ligand for tyrosine kinase receptors, among which Axl is the most widely distributed and displays the highest affinity for Gas6. The Gas6/Axl signaling pathway has been increasingly implicated in growth and survival processes occurring during development and tissue repair. In liver, after an acute or chronic injury, repair involves macrophages and hepatic stellate cells (HSC) activated into myofibroblastic cells (HSC/MFB), which produce cytokines and matrix proteins. We investigated the expression and the role of Gas6 and its receptor Axl in liver repair. Three days after CCl4-induced liver injury in the rat, we detected the expression of Gas6 in ED1-positive macrophages as well as in desmin-positive HSC, which accumulated in injured areas. Axl, the high-affinity receptor for Gas6, was detected in macrophages, HSC, and HSC/MFB. In vitro, expression of gamma-carboxylated Gas6 was strongly induced in HSC along with their transformation into myofibroblasts, and it exerted an anti-apoptotic effect on both HSC and HSC/MFB mediated by the Axl/PI3-kinase/Akt pathway. In conclusion, Gas6 is a survival factor for these cells and we suggest that Gas6, secreted by macrophages and HSC/MFB in vivo after liver injury, promotes HSC and HSC/MFB survival and might support transient HSC/MFB accumulation during liver healing.

gamma-Glutamyl transpeptidase (GGT) plays key roles in glutathione homeostasis and metabolism of glutathione S-conjugates. Rat GGT is transcribed via five tandemly arranged promoters into seven transcripts. The transcription of mRNA V is controlled by promoter 5. Previously we found that GGT mRNA V-2 was responsible for the induction of GGT in rat alveolar epithelial cells by 4-hydroxynonenal (HNE). In the current study, the underlying mechanism was investigated. Reporter deletion and mutation analysis demonstrated that an electrophile-response element (EpRE) in the proximal region of GGT promoter 5 (GP5) was responsible for the basal- and HNE-induced promoter activity. Gel-shift assays showed an increased binding activity of GP5 EpRE after HNE exposure. The nuclear content of NF-E2-related factor 2 (Nrf2) was significantly increased by HNE. The recruitment of Nrf2 to GP5 EpRE after HNE treatment was demonstrated by supershift and chromatin immunoprecipitation assays. The tissue expression pattern of GGT mRNA V was previously unknown. Using polymerase chain reaction, we found that GGT mRNA V-2 was expressed in many tissues in rat. Taken together, GGT mRNA V-2 is widely expressed in rat tissues and its basal and HNE-induced expression is mediated through EpRE/Nrf2 signaling.

Gamma-glutamyl transpeptidase (GGT) plays critical roles in glutathione homeostasis and metabolism. Rat GGT is a single-copy gene from which seven types of GGT mRNA with a common protein encoding sequence, but different 5'-untranslated regions, may be transcribed. We previously showed that type V-2 was the predominant form of GGT mRNA in rat L2 epithelial cells, and that it could be induced by 4-hydroxynonenal (HNE) through the electrophile response element (EpRE) located in GGT promoter 5 (GP5). Here, we report transcription factors binding to GP5 EpRE and the involved signaling pathways. Immunodepletion gel shift assays demonstrated that GP5 EpRE bound JunB, c-Jun, FosB, and Fra2 from unstimulated cells, and that after exposure to HNE, EpRE binding complexes contained nuclear factor erythroid 2-related factor (Nrf) 1, Nrf2, JunB, c-Jun, FosB, c-Fos, Fra1, and Fra2. HNE-induced binding of Nrf2 and c-Jun in GP5 EpRE was confirmed by chromatin immunoprecipitation assays. Using reporter assays and specific inhibitors, we found that HNE induction of rat GGT mRNA V-2 was dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), but not protein kinase C or phosphatidylinositol 3-kinase. Pretreatment with ERK and p38MAPK inhibitors also blocked HNE-increased EpRE binding. HNE-increased nuclear content of Nrf1, Nrf2, and c-Jun in L2 cells was partially blocked by inhibition of either ERK1/2 or p38MAPK and completely blocked by simultaneous inhibition of both MAPKs. In conclusion, HNE induces GGT mRNA V-2 through altered EpRE transcription factor binding mediated by both ERK and p38MAPK.

Background & Aims: The growth arrest-specific gene 6 (Gas6) protein is a vitamin K-dependent protein that binds to the Axl subfamily of tyrosine kinase receptors and exerts antiapoptotic and proliferative effects. Because Gas6 plays a role in development and tissue remodelling, we studied its expression as well as that of its high-affinity receptor Axl in a well-characterized model of hepatic regeneration from precursor oval cells., Methods: Hepatic regeneration was induced by treating rats with acetylaminofluorene followed by partial hepatectomy., Results: Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 and 14 after hepatectomy and declined thereafter. Oval cells expressed Gas6 protein and messenger RNA (mRNA). Axl mRNA hepatic levels paralleled the number of oval cells, and immunohistochemistry showed Axl expression in these cells. WB-F344 cells, a hepatocytic precursor cell line, also expressed Gas6 and Axl. Addition of Gas6 significantly increased the number of WB-F344 cells cultured with or without serum. Gas6 did not increase cell entry in the S phase of the cell cycle but inhibited 15-d-prostaglandin J2-induced WB-F344 cell apoptosis., Conclusions: Our data demonstrate an expression of Gas6 and of its receptor Axl by oval cells during hepatic regeneration. Because the Gas6/Axl couple protects from apoptosis a hepatocytic precursor cell line, these results strongly suggest that the Gas6/Axl couple favors oval cell accumulation in regenerating liver by an autocrine/paracrine mechanism.

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.

Liver fibrosis is potentially reversible after removal of the injurious agent. Fibrosis resolution is characterized by apoptosis of hepatic myofibroblasts and degradation of extracellular matrix components. Matrix metalloproteinase-2 (MMP-2) is involved in matrix remodeling. In the liver, it is synthesized by myofibroblasts, secreted as a proenzyme, and activated by membrane type-MMPs (MT-MMP) such as MT1-MMP. The goal of this work was to determine whether apoptosis induction in human hepatic myofibroblasts modulates the gene expression of MMP-2 and/or its activation by MT1-MMP. Induction of apoptosis by cytochalasin D or C(2)-ceramide did not modulate MMP-2 mRNA expression. In contrast, apoptosis was associated with marked activation of pro-MMP-2, as shown by gelatin zymography, which revealed the presence of the 59-kd active form, whereas untreated cells only expressed the 66-kd proform. SB-203580, a specific inhibitor of p38 (MAPK), selectively abrogated both C(2)-ceramide-induced apoptosis and pro-MMP-2 activation. Apoptosis-induced pro-MMP-2 activation was inhibited by the tissue inhibitors of metalloproteinases (TIMP)-2 but not by TIMP-1, implying involvement of an MT-MMP-mediated process. Induction of apoptosis by cytochalasin D and C(2)-ceramide upregulated MT1-MMP protein expression and MT1-MMP mRNA expression. In conclusion, apoptosis of hepatic myofibroblasts induces pro-MMP-2 activation through increased MT1-MMP expression. HEPATOLOGY 2002;36:615-622.)

Gamma glutamyltransferase (GGT) is a plasma membrane bound enzyme that initiates the degradation of glutathione. The presence of several promoters in the rat GGT gene indicates strict control and regulation of its expression. The aim of this study was to investigate whether the GGT gene was regulated differently after butyrate-induced differentiation and oxidative stress exposure of rat colon carcinoma cells and whether the regulation was related to the glutathione level. The activity of GGT was upregulated in a time-and-dose dependent manner after both butyrate and menadione incubations. The presence of antioxidants blocked the menadione but not the butyrate mediated induction of the enzyme. The level of intracellular glutathione was reduced after menadione, but not after butyrate incubations. Depletion of glutathione alone did not alter GGT activity. Reactive oxygen species (ROS) were not produced after incubations with butyrate, while menadione incubations produced ROS. The multiple GGT mRNA transcripts (mRNA I-V) that originate from the five distinct promoters were all present in the cell line. Incubations with butyrate enhanced mRNA II and IV transcripts whereas a reduction in mRNA IV-1 was noted during menadione incubations. The level of total GGT mRNA (I-V) was not altered when related to the amount of total beta-actin mRNA. We conclude that GGT activity can be upregulated by at least two distinct mechanisms during differentiation and oxidative stress. Apparently, the regulation of the enzyme is not directly linked to the intracellular level of glutathione.

gamma-Glutamyl transpeptidase (GGT), a major enzyme of glutathione (GSH) homeostasis, is often used as a biliary marker to follow the differentiation of hepatic precursor cells. The expression of the GGT gene is driven by different promoters and yields multiple mRNAs, depending on the cell type or the stage of differentiation. In the present study, we analyzed the GGT mRNA expression pattern by quantitative reverse transcriptase-polymerase chain reaction or by in situ hybridization i) in the liver, in vivo, at early stages of development; ii) in oval cells, which proliferate and differentiate into hepatocytes in response to galactosamine injury in vivo; and finally, iii) during hepatoblast differentiation, in vitro. We show that GGT gene transcription originates from promoters P3, P4, and P5 in rat hepatic precursor cells. Differentiation of these cells induces profound alterations in GGT gene expression, leading to extinction of promoters P4 and P5, when they differentiate into the hepatocytic pathway, and to extinction of promoters P3 and P5 when they differentiate into the biliary pathway. This diversity in GGT mRNA expression provides unique molecular probes to follow hepatic precursor cell differentiation. Furthermore, the identification of factors governing GGT P5 and P4 promoter expression should provide further insight into the molecular events that occur as the liver precursor cell differentiates into the hepatic lineages.

Gamma-glutamyl transpeptidase (GGT) is an enzyme located at the external surface of epithelial cells. It initiates extracellular glutathione (GSH) breakdown, provides cells with a local cysteine supply and contributes to maintain intracellular GSH level. GGT expression, highly sensitive to oxidative stress, is a part of the cell antioxidant defense mechanisms. We describe recent advances in GGT gene structure and expression knowledge and put emphasis on the complex transcriptional organization of that gene and its conservation among different species. GGT gene structure has been elucidated in rat and mouse where a single gene is transcribed from multiple promoters into several transcripts which finally yield a unique polypeptidic chain. Analysis of rat, mouse, human and pig cDNA and gene sequences reveals a large conservation of the transcriptional organization of that gene. This complex structure provides flexibility in GGT expression controlled at the promoter level, through multiple regulatory sites, and at RNA level by alternate 5' untranslated sequences which may create a diversity in the stability and translational efficiency of the different transcripts. In conclusion, transcription of the GGT gene from several promoters offers multiple DNA and RNA targets for various oxidative stimuli and contributes to a broad antioxidant cell defense through GGT induction and subsequent cysteine supply from extracellular glutathione.

Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adult rat liver by Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization analysis. We show that the GGT activity in the 18-day-old fetus results from the transcription of the gene from the promoter P3 in the hepatocytes. In contrast, the GGT promoter P4 activity appears to be specific of biliary cells in normal as well in cholestatic liver. Thus, sequences unique to the GGT transcripts initiated on these two alternate promoters provide unique molecular probes to discriminate between the biliary and the hepatocytic phenotypes in liver differentiation and cell lineage studies.

In rat undifferentiated hepatoma cells, the gamma-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.

The expression of gamma-glutamyl transpeptidase (GGT) has been associated with the emergence of a functional blood-brain barrier. We have undertaken a precise localisation of this enzyme in the cerebral cortical vessels of different species, by immunocytochemistry using a polyclonal antibody and light and electron microscopy. GGT immunoreaction product was present on the luminal surface of endothelial cells in 1-day-old to 3-month-old rats, whereas in the mouse, monkey and human cortex, this protein was present in astrocytic endfeet around the vessels. No labelling was observed in the other cellular components of the vessel walls, such as pericytes, fibroblasts, smooth muscle cells and perivascular cells. The localisation of GGT in astrocytes in mice, monkeys and humans suggests that these cells could play a role in the detoxication of lipophilic xenobiotic substances that cross the endothelial barrier. In these species, astrocytes can be viewed as a second line of defense against xenobiotics, beyond the capillary endothelium.

In the small intestine and in HTC hepatoma cells, the gamma-glutamyl transpeptidase (GGT) single-copy gene is transcribed into a 2.5 kb and a 2.2 kb mRNA. Cloning of the GGT cDNA sequences from HTC cells demonstrates that the 2.5 kb mRNA (mRNA(IV-1)) differs from the other rat GGT transcripts by a 371-base unique leader sequence which maps in the gene as 2 separate exons upstream of the 3 promoters which have been previously characterized. We established that the transcription of these two mRNAs is initiated on a new promoter (promoter IV) and occurs in the small intestine, in the epididymis, and in some hepatoma cells. The primary transcript initiated on GGT promoter IV is then alternatively spliced into the 2.5 kb mRNA(IV-1) or the 2.2 kb mRNA(IV-2) which is shorter in its 5'-untranslated sequence. The rat GGT gene exhibits a complex transcriptional organization leading to the transcription of five mRNAs from four independent promoters in a tissue-specific manner. The expression of the GGT promoter IV in the HTC hepatoma cells as well as in the small intestine could reveal that the HTC-transformed cells originate from liver precursor cells which still have the capacity to evolve toward different lineages. Thus, the GGT promoter IV will be valuable to isolate factors involved in the differentiation and carcinogenic processes.

The expression of gamma-glutamyl transpeptidase (GGT) mRNA in tissues of the adult rat male reproductive tract was examined. Northern blot analysis of total RNA revealed that GGT mRNA expression occurs primarily in the initial segment and caput epididymidis. Multiple GGT mRNAs of varying sizes were detected in the testis and in different regions of the epididymis: testis, 2.4 and 2.8 kb; efferent ducts, 2.2 and 2.5 kb; initial segment, 2.2, 2.4, and 2.5 kb; caput, 2.2, 2.4, and 2.5 kb; corpus, 2.2 and 2.5 kb; cauda, 2.2 and 2.5 kb; ductus deferens, 2.2 and 2.4 kb. Ribonuclease (RNase) H removal of the poly(A) tail from testicular and epididymal GGT mRNA revealed that multiple GGT mRNAs were not generated by differences in the length of the poly(A) tail. Northern blot analysis of poly(A)+ mRNA with four GGT mRNA 5' untranslated region (UTR)-specific cRNA probes showed that the multiple GGT mRNAs expressed in the testis and epididymis were due to differences in the lengths and nucleotide compositions of the 5' UTR. We hypothesize that transcription of multiple GGT mRNAs from different promoters on the single-copy GGT gene is the molecular basis that underlies the region-specific expression of GGT mRNAs along the rat male reproductive tract.

In rat, gamma-glutamyl transpeptidase (GGT) is encoded by multiple mRNAs (mRNAI, mRNAII, mRNAIII, and mRNAIV) that differ only in their 5' untranslated regions and are transcribed from a single-copy gene. Using oligonucleotides designed from the 5' untranslated sequences of the GGT mRNAII and mRNAIII, we amplified a 3.4-kb genomic sequence which contains the promoter region for mRNAII. The sequence flanking the two initiation start sites for mRNAII contains consensus motifs for several potential regulatory proteins and a TATA-like element at the expected position 26 bp upstream from the predominant start site. The sequence from positions -528 to +72 associated with the chloramphenicol acetyltransferase (CAT) reporter gene drives a promoter activity in LLC-PK1, a pig kidney cell line. Deletion analysis revealed that the region from nucleotides -528 to -322 mediates an activation of the promoter activity, whereas the sequence from -322 to -114 has a negative effect. Furthermore, the structural organization of the 5' end of the GGT gene reveals that the GGT mRNAIII is transcribed from a third promoter located upstream from the promoter II on the GGT gene. By Northern blot analysis, the promoter II was found to be expressed only in the kidney and in the epididymis. We also identified two new mRNA species which are expressed in the H5 hepatoma cells. Therefore, the GGT gene expression reveals a strong tissue- or cell-specific pattern which is based on the transcription of several mRNA species from multiple promoters.