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Autophagy is a lysosomal degradation pathway of cellular components that regulates macrophage properties. Macrophages are critically involved in tumor growth, metastasis, angiogenesis and immune suppression. Here, we investigated whether macrophage autophagy may protect against hepatocellular carcinoma (HCC). Experiments were performed in mice with deletion of the autophagy gene Atg5 in the myeloid lineage (ATG5 Mye-/- mice) and their wild-type (WT) littermates. As compared to WT, ATG5 Mye-/- mice were more susceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis, as shown by enhanced tumor number and volume. Moreover, DEN-treated ATG5 Mye-/- mice exhibited compromised immune cell recruitment and activation in the liver, suggesting that macrophage autophagy invalidation altered the antitumoral immune response. RNA sequencing showed that autophagy-deficient macrophages sorted from DEN mice are characterized by an enhanced expression of immunosuppressive markers. In vitro studies demonstrated that hepatoma cells impair the autophagy flux of macrophages and stimulate their expression of programmed cell death-ligand 1 (PD-L1), a major regulator of the immune checkpoint. Moreover, pharmacological activation of autophagy reduces hepatoma cell-induced PD-L1 expression in cultured macrophages while inhibition of autophagy further increases PD-L1 expression suggesting that autophagy invalidation in macrophages induces an immunosuppressive phenotype. These results uncover macrophage autophagy as a novel protective pathway regulating liver carcinogenesis., (© 2021. The Author(s).)

Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of alcoholic liver disease. We have previously demonstrated that CB2 receptor protects against alcoholic liver disease by inhibiting alcohol-induced inflammation and steatosis via the regulation of Kupffer cell activation. Here, we explored the mechanism underlying these effects and hypothesized that the anti-inflammatory properties of CB2 receptor in Kupffer cells rely on activation of autophagy. For this purpose, mice invalidated for CB2 receptor (CB2(Mye-/-) mice) or for the autophagy gene ATG5 (ATG5(Mye-/-) mice) in the myeloid lineage, and their littermate wild-type mice were subjected to chronic-plus-binge ethanol feeding. CB2(Mye-/-) mice showed exacerbated alcohol-induced pro-inflammatory gene expression and steatosis. Studies in cultured macrophages demonstrated that CB2 receptor activation by JWH-133 stimulated autophagy via a heme oxygenase-1 dependent pathway. Moreover, JWH-133 reduced the induction of inflammatory genes by lipopolysaccharide in wild-type macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in wild-type mice, but not in ATG5(Mye-/-) mice demonstrating that macrophage autophagy mediates the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Altogether these results demonstrate that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagy-dependent pathway.

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5(fl/fl) LysM-Cre mice, referred to as atg5(-/-)) and their wild-type (Atg5(fl/fl), referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5(-/-) mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5(-/-) mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5(-/-) mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5(-/-) mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5(-/-) macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5(-/-) mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.

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.

Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti-inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis. Wild-type (WT) mice fed an alcohol diet showed an induction of hepatic classical (M1) and alternative (M2) markers. Cotreatment of alcohol-fed mice with the CB2 agonist, JWH-133, decreased hepatic M1 gene expression without affecting the M2 profile. In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol-induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH-133-treated mice and its enhancement in CB2-/- animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned-medium experiments showed that preventing M1 polarization in CB2-activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase-1 (HO-1) mediated the anti-inflammatory effects of CB2 receptors. Indeed, alcohol-fed mice treated with JWH-133 showed increased hepatic expression of macrophage HO-1, as compared to vehicle-treated counterparts. In keeping with this, JWH-133 induced HO-1 expression in cultured macrophages, and the HO-1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH-133 on lipopolysaccharide-induced nuclear factor-kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol-induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2 agonists as potential therapeutic agents for the management of alcoholic liver disease., (Copyright © 2011 American Association for the Study of Liver Diseases.)

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.

Unlabelled: The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions., Conclusion: CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

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.

Differentiation of hepatic precursor cells in the biliary lineage has rarely been investigated, owing to the lack of convenient in vitro models. In this study, we used sodium butyrate and culture on Matrigel to promote differentiation of WB-F344 rat liver epithelial cells along the biliary phenotype. This differentiation was assessed by following the expression of phenotypic markers at the protein or mRNA level. Sodium butyrate induced cytokeratin 19 expression and gamma-glutamyltranspeptidase activity, together with a large increase in gamma-glutamyltranspeptidase mRNA IV, a transcript expressed at high levels in biliary cells. We also observed an increase in aquaporin-1 and beta4 integrin mRNAs, encoding two proteins expressed in adult biliary cells. Culture on Matrigel increased cytokeratin 19, gamma-glutamyltranspeptidase, and BDS7 expression in WB-F344 cells which still expressed aquaporin-1 and beta4 integrin. These results show that WB-F344 cells are able to differentiate in vitro along the biliary pathway, making them a candidate model for analyzing the molecular events associated with the hepatoblast-biliary cell transition.

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) 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.

Glucocorticoids are known to promote the formation of zymogen granules in acinar cells of the exocrine pancreas in vivo as well as in vitro. To gain insight into the mechanism of this regulation, we studied the effects of glucocorticoids on the synthesis of two components of the secretory granule membrane, the glycoprotein 2 (GP-2) and the gamma-glutamyl transpeptidase (GGT). It was demonstrated that following adrenalectomy, degranulation of pancreatic acinar cells is accompanied by a sharp decrease in GGT and GP-2 synthesis as measured by mRNA and protein accumulation. The decline of GGT synthesis was prevented by glucocorticoid replacement therapy, whereas GP-2 synthesis could be maintained with either glucocorticoid or estradiol treatment. These in vivo observations were corroborated and extended in an in vitro study using AR42J pancreatic cells. With this cell line, it was demonstrated that dexamethasone induces the formation of zymogen granules and the accumulation of a specific GGT transcript (mRNA III) by decreasing its degradation rate. At the same time, the GP-2 mRNA level was not modified by the hormonal treatment. These data demonstrate that glucocorticoids exert a positive control on the GGT expression in pancreatic cells at a post-transcriptional level. GGT, an enzyme of the glutathione metabolism, could play a significant role in protein packaging in secretory cells.

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.

gamma-Glutamyl transpeptidase (GGT) is an enzyme encoded by multiple mRNAs (mRNAI to mRNAIV) that, in the rat, are transcribed from a single copy gene in a tissue-specific manner. In the liver, GGT expression is up-regulated in transformed cells, and this induction is the most widely used marker of liver cell transformation. We characterized the GGT mRNA species expressed in the liver (mRNAIII), and we report that this mRNA differs from the other GGT mRNA species by a 275-base alternate 5'-end sequence. Its transcription occurs on a specific promoter (promoter III) that maps on the GGT gene upstream of the two promoters coding for the GGT mRNAI and mRNAII. In hepatoma cells, mRNAIII expression is related to the differentiation state of the cells. We have shown that, in Reuber H-35-derived cell lines, the GGT mRNAIII is transcribed in cells that express a differentiated phenotype (Fao), but not in the dedifferentiated C2 and H5 variants. Moreover, we observed a reexpression of the GGT mRNAIII species in the C2 Rev7 variant, which has reverted from C2 toward a differentiated hepatocyte phenotype. In the proximal promoter III region, we identified a sequence that strongly enhances transcriptional activity in Fao and C2 Rev7 cells, but not in the dedifferentiated C2 variant. This motif interacts with nuclear proteins belonging to the NF-1 and NF-Y families that govern GGT promoter III expression in differentiated hepatoma cells.

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.

gamma-Glutamyl transpeptidase (GGT) is an enzyme that plays a key role in interorgan glutathione transport. Three mRNAs (mRNAI, mRNAII, and mRNAIII) are known to encode the GGT precursor; they are initiated on three separate promoters on the single GGT gene. In this work, we identified by Northern blot and RNase H analysis a new GGT mRNA (mRNAIV). This mRNA differs from the others in its 5'-noncoding sequence. This mRNA species is the predominant GGT mRNA expressed in HTC hepatoma cells and in the small intestine in which its level increases from the base to the apex of the microvillus. The analysis of the GGT gene expression pattern in kidney, mammary gland, small intestine, liver, preneoplastic liver, and HTC hepatoma cells reveals a strong tissue or cell specificity. The mRNAIII was found in all the tissues and cells; in contrast, the expression of mRNAI, mRNAII, and mRNAIV is limited in normal tissues to the kidney and to the small intestine, the two tissues that display the highest enzyme activity. The synthesis of these three mRNAs is linked to the development of the kidney proximal tubule and to the differentiation of the enterocyte. The tissue and cell specificity of the GGT gene expression is based upon the use of multiple promoters that are controlled independently by specific cell factors.

Two gamma-glutamyl transpeptidase mRNAs (mRNAI and mRNAII), with alternate 5'-untranslated regions, are expressed in the rat kidney. Oligonucleotides were designed based upon these two alternate 5' sequences and used as primers to amplify GGT genomic DNA sequences. The genomic organization of the mRNAI and mRNAII 5'-untranslated sequences reveals that the mRNAs are encoded from two separate promoters which are 2.1 kbp apart on the single GGT gene. A 2775 base pair genomic sequence, which contains the proximal GGT promoter, was cloned from two overlapping amplified fragments. S1 mapping analysis shows that the kidney GGT mRNAI is transcribed from several start sites on this promoter which displays neither a classical TATA box nor Sp1 binding sites. Chimeric plasmids, including the GGT promoter region for mRNAI, associated with the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently expressed in a kidney (LLCPK) and in a hepatoma (HTC) cell line. A sequence extending 308 bases upstream from the major GGT mRNAI start site drives a promoter activity which is 5-fold higher in LLCPK than in HTC cells and is sufficient to confer cell specificity to the GGT proximal promoter.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.

Two different cDNAs have been isolated and characterized from a rat kidney cDNA library. The two cDNA sequences are identical in the coding region and in the 144 bases upstream from the initiation codon but have alternate sequences (154 and 138 bases) at their 5' ends. Primer extension analysis on kidney mRNA reveals that both cDNAs are full-length and correspond to two mRNAs of nearly the same size (2142 and 2127 bases). Synthesis of two mRNAs with alternative 5' ends can be explained only by initiation at two separate promoters on the single rat gamma-glutamyl transpeptidase (GGT) gene. The alternate 5' end nucleotide sequences were used as probes to detect the corresponding mRNAs in several rat tissues. In the kidney, the expression of both RNAs was detected by in situ hybridization in the distal part of the proximal convolutions of the renal tubule. Northern blot analysis of kidney mRNAs reveals that the expression of both mRNAs increases from birth to the adult stage. Neither of these two transcripts is expressed in the liver or in seminal vesicles in which a larger mRNA (2.4 kilobase pairs) is transcribed from the same gene. Thus, two GGT mRNAs, initiated on two separate promoters on the single GGT gene, are expressed in the rat in a tissue-specific manner and coordinately regulated.

Gamma glutamyltransferase (GGT) is a membrane-bound enzyme that is involved in glutathione metabolism and aminoacids uptake. GGT activity is stimulated by a number of hormones and pharmacological agents in certain animal tissues. In rat liver, adrenalectomy causes a 2-fold decrease in GGT activity and hydrocortisone treatment of adrenalectomized animals specifically stimulates this enzyme activity. In a highly differentiated hepatoma cell line, Fao, GGT activity is similar to rat liver and is under glucocorticoids control. These hormones specifically stimulate GGT activity (2- to 3-fold). Translation and transcription inhibitors prevent the hormonal effect. The stimulation of GGT activity is therefore probably due to an increase in GGT mRNA synthesis. The results reported suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of both the glucocorticoid effects on differentiated cells as well as the modulation of membrane-bound enzymes biosynthesis.

The effect of ethanol on protein synthesis in the C2 rat hepatoma cell line was analyzed by two-dimensional gel electrophoresis after the labeling with [35S]methionine of cells that were untreated or had been treated with 180 mM ethanol. In this cell line, this concentration of ethanol is known to induce gamma-glutamyl transpeptidase, a marker of alcoholism in man (Barouki et al., Hepatology 1983; 3: 323-329). In the present work we demonstrate that ethanol, besides causing a slight decrease in overall protein synthesis (less than 25%), primarily regulates the expression of two unique proteins among 1500 labeled products that were analyzed: one of these was induced and did not correspond to gamma-glutamyl transpeptidase, and one was repressed after 20 h of ethanol treatment. We conclude that the set of hepatic proteins altered by ethanol is likely to be very limited in number, which reflects the specificity of alcohol action on protein synthesis in the C2 cell line.

We have determined the chromosomal location of the human gene for gamma-glutamyltransferase (GGT). This study was done by in situ hybridization of human metaphase spreads with a rat cDNA probe specific for this enzyme and constructed from two clones previously characterized in our laboratory. The final construct had a 1.6-kb-long insert covering 92% of the coding sequence for GGT. The new insert was also freed of any GC tails introduced for the cDNA cloning, because we observed that these sequences were responsible for a high background. Using this probe for the analysis of 136 human metaphase spreads, we observed a strong specific signal on chromosome 22 at the interface of q111-112 and a minor peak in q131. Thus GGT might represent a new marker for the study of certain diseases which have chromosomal abnormalities at these loci.

The clone C2 derived from a rat hepatoma cell line was used to investigate the mechanism of the induction of gamma-glutamyltransferase by ethanol. gamma-glutamyltransferase activity was detected in the C2 cell (1.4 mU per mg protein), and its kinetic properties were similar to normal rat liver gamma-glutamyltransferase. Ethanol provoked a dose- and time-dependent increase in gamma-glutamyltransferase activity, the maximum (2- to 3-fold) occurring 48 hr after the addition of ethanol (180 mM). In contrast, the activity of five other enzymes tested were not markedly modified by ethanol. Propanol was more potent than ethanol in inducing gamma-glutamyltransferase (5-fold stimulation), whereas methanol had no effect. The release of the enzyme in the medium was increased by ethanol and propanol. Several observations argue in favor of an increase in the biosynthesis of gamma-glutamyltransferase after ethanol addition: (i) ethanol increased the maximal velocity of the enzyme and did not modify the affinity for its substrates. It did not alter gamma-glutamyltransferase subcellular distribution; (ii) ethanol had no immediate effect when added directly to the assay mixture; (iii) the lag period and the time course of the increase in gamma-glutamyltransferase activity were those expected for an induction process; (iv) the increase in gamma-glutamyltransferase activity was prevented by cycloheximide and actinomycin D suggesting that ethanol acted at the transcriptional level. The effect of ethanol was not mimicked by acetaldehyde. In conclusion, we have demonstrated that ethanol increases the biosynthesis of gamma-glutamyltransferase in a rat hepatoma cell line which provides a new in vitro system.

Gamma-Glutamyltransferase activity was detected in the plasma membrane of the highly differentiated hepatoma cell line Fao, (0.93 mU/mg cell protein). Dexamethasone (1 microM) provoked a 2-3-fold increase in the activity of the enzyme in the presence of fetal calf serum. Maximal induction occurred 48-72 h after addition of the glucocorticoid to the cell culture medium. The hormonal specificity was demonstrated by the relative potencies of several glucocorticoids and sex steroids: hydrocortisone and corticosterone increased gamma-glutamyltransferase activity while tetrahydrocorticosterone and all sex steroids tested were ineffective. The effect of dexamethasone on gamma-glutamyltransferase activity wa specific since the activities of several other plasma membrane enzymes were not modified. The mechanism of the dexamethasone-induced increase in gamma-glutamyltransferase activity was neither by modification of the affinity of the enzyme for its substrates nor by alteration of the subcellular distribution of the enzyme. This increase was prevented by cycloheximide and actinomycin D. The data presented are consistent with a specific glucocorticoid receptor-mediated induction of gamma-glutamyltransferase activity in Fao cells. The kinetic parameters of the induction process by glucocorticoids are very similar to those found in adult rat liver. These results suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of glucocorticoid effects on differentiated cells.

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.

gamma-Glutamyl transferase (gamma-GT) was assayed in the serum and liver biopsies of children affected with either progressive idiopathic cholestasis (PIC, Byler's disease), or other types of cholestatic (biliary atresia, cholestasis of various origins) and non-cholestatic diseases. The mean liver gamma-GT activity was increased significantly only in PIC and biliary atresia. In contrast, the serum gamma-GT activity, raised in children with evident damage to the main bile ducts or to the interlobular bile ducts, was normal in children with PIC. Although the mechanism for such a discrepancy between high liver and normal serum gamma-GT activities in PIC is still speculative, this peculiarity could prove to be of use in leading to a better understanding of the disease.

The increase in serum gamma-glutamyl transpeptidase (GGT) is a well known marker of chronic alcoholism in man. We have previously shown that ethanol (180 mM) induces GGT activity 2-3-fold in the C2 rat hepatoma cell line. In this study, we have analyzed the interaction of ethanol with steroid hormones and drugs in this well defined cell culture system. Dexamethasone (100 nM), a synthetic glucocorticoid agonist, completely prevented the induction of GGT by ethanol, but had no effect when added alone. This inhibitory effect was also observed with other corticosteroids, but not with sex steroids; it was prevented by RU 486, a glucocorticoid antagonist. These observations suggest that dexamethasone acts through a high affinity glucocorticoid receptor. Conversely, ethanol did not interfere with the glucocorticoid induction of alanine aminotransferase in the same cell. We have analyzed the metabolism of ethanol in the C2 cells. These cells lack significant alcohol dehydrogenase activity as well as any cytochrome P-450 Alc immunoreactivity. Dexamethasone did not modify the disappearance of ethanol in the culture medium of those cells. We conclude that glucocorticoid hormones interact with ethanol at the cellular level, and that this interaction does not involve a modification of alcohol metabolism.

The biosynthesis of gamma-glutamyl transpeptidase was investigated in hepatoma tissue culture cells. Pulse-chase experiments using [35S]methionine labeling have shown that the two glycosylated subunits of the enzyme (Mr = 58,000 and 29,000) derive from a single glycosylated precursor (Mr = 79,000 at early times). Only one polypeptide chain was immunoprecipitated from cell-free translation products and was shown to correspond to the nonglycosylated precursor (Mr = 64,000). Treatment with endoglycosidase H was used to probe for the transfer of the proteins from the endoplasmic reticulum to the Golgi and demonstrated: (i) that the precursor is at least partially cleaved in the endoplasmic reticulum; (ii) that part of the precursor is transferred to the Golgi where the processing of the oligosaccharide chains takes place. None of the precursor forms were detected at the surface of the cell where the mature enzyme was found. Tunicamycin, an inhibitor of protein glycosylation, did not prevent the proteolytic processing of the enzyme, but delayed the appearance of the mature enzyme at the cell surface. Monensin, which is known to alter Golgi functions, significantly delayed the acquisition of complex type oligosaccharides and the appearance of the enzyme at the cell surface. It did not, however, alter the proteolytic processing of the precursor of gamma-glutamyl transpeptidase. Taken together, these results show that gamma-glutamyl transpeptidase is synthetized as a single precursor which is at least partially cleaved in the endoplasmic reticulum. Part of the precursor is transferred to the Golgi where its oligosaccharide chains are processed.

We have screened a cDNA library (20,000 clones) made from rat kidney poly(A)+ RNA, using an oligonucleotide probe that was a mixture of 14-base DNA oligomers containing all 32 possible sequences coding for residues 32-36 of the gamma-glutamyl transpeptidase (EC 2.3.2.2.) heavy chain. We isolated and sequenced two cDNAs corresponding to the mRNA coding for the entire length of the enzyme precursor. The nucleotide sequence that we obtained (2072 bases) reveals an open reading frame of 1707 nucleotides coding for the common precursor of both enzyme subunits. The amino acid sequence begins with the 21 residues located at the NH2-terminal hydrophobic region of the heavy subunit. We show that this sequence, which is not processed, is the only possible signal peptide in the sequence. Five potential N-glycosylation sites are present in the gamma-glutamyl transpeptidase sequence. Using one of the two cDNA clones as probe, a 2.2-kilobase sequence was detected by blot analysis in rat kidney and human fetal liver RNA.

gamma-Glutamyl transpeptidase (GGT) genomic sequences were isolated from rat and human libraries using a rat GGT cDNA as a cross-species hybridization probe. Characterization of the human GGT clones by restriction mapping clearly establishes that at least four different GGT genes or pseudogenes are present in the human genome. All the rat genomic clones cover a 12.5-kilobase sequence and exhibit a unique restriction pattern. A precise quantitation of the rat GGT gene copy number by Southern blot analysis demonstrates that this sequence is present as a single copy/rat haploid genome. Therefore, the GGT gene organization is different between rat and human species; this raises the possibility of different regulatory mechanisms in the two species.