gamma-GT mRNA Research Articles

Type I gamma-GT mRNA is expressed in B16 melanoma and levels correlate with pigmentation.

Gamma-glutamyl transpeptidase (gamma-GT), an ectoenzyme involved mainly in glutathione metabolism, is expressed in B16 melanoma cells. B16 melanoma cells under continuous culture conditions show a phenotypic drift from melanotic to amelanotic and re-melanotic stages. We have investigated the regulation of gamma-GT in B16 melanoma cells under such different pigmentary conditions. High levels of gamma-GT messenger RNA (mRNA) and activity were detected in pigmented B16 melanoma cells, whereas in amelanotic B16 melanoma cells the levels were very low. Treatment with lactic acid, a known inhibitor of tyrosinase gene expression, also led to the down-regulation of gamma-GT mRNA and activity. Thus our results indicate that gamma-GT regulation depends on the pigmentation status in pigment cells. We have also assessed the levels of gamma-GT in normal murine melanocytes (melan-a cells). It was seen that melan-a cells express very low levels of gamma-GT. As gamma-GT is known to be regulated in a tissue-specific manner, and is expressed from as many as six promoters giving rise to six different types of mRNAs each having unique 5' ends, we have further investigated the type of gamma-GT mRNA expressed in B16 melanoma and melan-a cells. In this study, we have conclusively demonstrated that type I mRNA transcript of gamma-GT is expressed in B16 melanoma and melan-a cells.

N-acetylcysteine does not protect against type II cell injury after prolonged exposure to hyperoxia in rats.

Although the antioxidant properties of N-acetylcysteine (NAC) in vitro are widely accepted, the efficacy of NAC in the prevention of O2 toxicity in vivo is poorly documented. The aim of our study was to investigate the presumed protective effect of NAC on hyperoxic lung injury, focusing on gamma-glutamyltransferase (gamma-GT) activity and glutathione (GSH) levels in lung tissue, epithelial lining fluid (ELF), and isolated rat type II cells immediately after their isolation and 48 h later when kept in culture in normoxia. Thirty-four male Wistar rats were divided in three groups (n = 10-14) and were exposed to air or to 60 or 85% O2 for 7 days. One-half of the rats in each group received 200 mg/kg NAC intraperitoneally one time per day from 3 days before exposure until the end of the experiment, and the other one-half received the vehicle. In the 85% O2-exposed animals, NAC led to more respiratory distress and weight loss. NAC did not prevent the rise in bronchoalveolar lavage lactate dehydrogenase and alkaline phosphatase, but it did prevent the rise in calculated ELF volume. NAC decreased GSH levels (1.4-fold) and gamma-GT activity (1.8-fold) in the air-exposed type II cells. In the 60% O2-exposed group, no effects of NAC were seen (except for a decrease in gamma-GT mRNA expression), but, in the 85% O2-exposed group, NAC gave rise to higher GSH (2.6-fold) and higher gamma-GT activity (2.9-fold) in the ELF and lower GSH (6.9-fold) and higher gamma-GT activity (3.6-fold) in the type II cells. Even in culture, GSH levels remained 1.5-fold lower than in the cells from the air-exposed animals and 2-fold lower than in the cells from the 85% O2-exposed animals. There was increased DNA damage (as assessed by thymidine incorporation) and apoptosis after hyperoxia, especially after 60% O2, and this effect was amplified after NAC treatment. Although protective at the endothelial side, NAC treatment led to adverse effects at the epithelial side, despite, or probably because of, restoration of the ELF GSH levels in the presence of high O2 levels. Because NAC is rapidly metabolized to cysteine, it is plausible that the effects of NAC are manifested through the toxic effects of cysteine.

Changes in gamma-glutamyltransferase activity in rat lung tissue, BAL, and type II cells after hyperoxia.

The effect of hyperoxia on gamma-glutamyltransferase (gamma-GT), an important enzyme for the uptake of precursor molecules for intracellular synthesis of glutathione (GSH), has not been established. Our aim was to investigate the effects of prolonged subtoxic levels of hyperoxia on gamma-GT activity and GSH levels in lung tissue, epithelial lining fluid (ELF), and isolated rat type II cells immediately after their isolation and 48 h later when kept in culture in normoxia. Seventeen male Wistar rats were divided in three groups (n = 5-7) and were exposed to air or to 60 or 85% O2 for 7 days. Pulmonary gamma-GT activity increased in the 60 and 85% O2-exposed animals (1.6- and 3.2-fold, respectively), and tissue GSH levels increased only in the 60% O2 group (1.3-fold). In isolated type II cells from 60 and 85% O2-exposed animals, gamma-GT activity decreased by -70 and -88%, respectively, which was supported by cytochemical staining. Type II cell gamma-GT mRNA expression tended only to decrease after 85% O2. Type II cell gamma-GT activity strongly correlated with ELF gamma-GT (r = 0.60, P < 0.001), and ELF gamma-GT strongly correlated with ELF GSH (r = 0.75, P < 0.0001). When in culture, type II cell gamma-GT activity and GSH levels remained, respectively, 2.5- and 1.9-fold lower in the 60% O2-exposed group, but, in the 85% O2-exposed group, gamma-GT activity increased 2.1-fold, and GSH levels dropped to the levels of the control cells. Hyperoxia led to a concentration-dependent decrease in gamma-GT activity in rat type II cells, possibly by direct inactivation, but led to an increase in whole lung tissue gamma-GT. There seemed to be a negative feedback between intracellular GSH levels and type II cell gamma-GT activity. gamma-GT levels in the ELF were correlated with type II cell gamma-GT activity, but ELF gamma-GT did not seem to play an active role in the regulation of the ELF GSH pool. Hyperoxia decreased ELF GSH levels, possibly by increased degradation of GSH in the parenchymal lung tissue as a result of the increased gamma-GT activity.

Ontogeny of gamma-glutamyltransferase in the rat lung.

gamma-Glutamyltransferase (gamma-GT) is a key enzyme in the metabolism of glutathione and glutathione-substituted molecules. The gamma-GT gene is expressed in two epithelial cells of the adult lung, the bronchiolar Clara cell and the alveolar type II cell. Because pulmonary glutathione metabolism may be important in the perinatal period, we studied gamma-GT ontogeny in the developing rat lung. In the late fetal and early postnatal lung, gamma-GT mRNA was below detectable limits on Northern blots. Pulmonary gamma-GT protein and enzyme activity were present at low levels after fetal day 18. gamma-GT protein appeared as a high-molecular-mass band (>95 kDa), with small amounts of enzymatically active gamma-GT heterodimer. Between the 2nd and 3rd postnatal wk, pulmonary gamma-GT mRNA expression increased in association with an increase in gamma-GT protein and enzyme activity that reached adult lung levels. At this time, gamma-GT protein appeared predominantly in the heterodimeric form with small amounts of the >95-kDa protein. Immunocytochemistry revealed that, in the fetal and early postnatal lung, gamma-GT was expressed only in the alveolar type II cell, whereas the Clara cell became the major site of gamma-GT mRNA and protein expression by 2-3 wk and in the adult. Type II cells isolated from the fetal lung express gamma-GT mRNA and synthesize the >95-kDa form of gamma-GT in excess of the heterodimer. These studies demonstrate that the alveolar type II cell is the only cell producing gamma-GT in the newborn lung and that it synthesizes a form of gamma-GT that appears to differ from that produced at a later time point by the Clara cell.

Three alternative promoters of the rat gamma-glutamyl transferase gene are active in developing lung and are differentially regulated by oxygen after birth.

The rat gamma-glutamyl transferase mRNA transcripts I, II, and III are derived from three alternative promoters, P(I), P(II), and P(III). In the adult only mRNA III is expressed in the lung. We show that mRNA III gene expression is developmentally regulated in the fetal lung; it is first expressed in gestation. In contrast to the adult lung, the fetal lung expresses mRNA I, II, and III. The switch from the fetal to the adult pattern of gammaGT mRNA expression begins within the first 24 h of birth and is complete by 10 d of age. gammaGT mRNA II disappears within 24 h, mRNA I disappears by 10 d leaving mRNA III as the sole transcript. Alveolar epithelial type 2 cells (AT2) isolated from the adult lung express only mRNA III. When cultured in 21% O2 mRNA III is maintained, but when cultured in 3% O2 the fetal pattern of mRNA I, II and III expression is induced. When AT2 cells in hypoxia are exposed to carbon monoxide, mRNA II is suppressed suggesting that a heme-binding protein (responsive to oxygen) may suppress mRNA II expression and may be responsible for the decrease in lung mRNA II seen after birth. A reporter gene under the control of DNA sequences from the gammaGT P(III) promoter is activated in transient transfection studies in response to hyperoxia, while a deletion construct retaining an antioxidant responsive element is not. Oxygen appears to regulate each of the alternative promoters of the gammaGT gene, such that P(II) is rapidly repressed by a heme-dependent mechanism, P(I), is more gradually repressed by a nonheme mechanism and P(III) is activated by a putative oxygen response element. We hypothesize that similar oxygen-dependent mechanisms regulate other genes in the developing lung at birth.

Identification of a Sixth Promoter That Directs the Transcription of γ-Glutamyl Transpeptidase Type III RNA in Mouse

We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.

Gamma-Glutamyl transpeptidase is increased by oxidative stress in rat alveolar L2 epithelial cells.

The tripeptide glutathione (GSH) is used by cells to detoxify hydroperoxides, produced during oxidative stress, and is consumed in the process. Previous studies have indicated that cells can be protected against oxidative stress by extracellular GSH through its degradation catalyzed by the exoenzyme gamma-glutamyl transpeptidase (gamma GT) and its de novo synthesis within the cytosol. We hypothesized that gamma GT would be increased as part of the adaptation of cells to oxidative stress. We examined whether oxidative stress could increase gamma GT activity, protein, and mRNA in a lung epithelial cell line (L2). Cultures were subjected to H2O2-mediated toxicity by 15 min of exposure to the redox cycling quinone, menadione. Menadione (50 microM) caused an initial decrease (27 +/- 9% of baseline after 15 min) in intracellular GSH, followed by resynthesis to levels significantly higher than baseline (335 +/- 40% after 24 h, P < 0.001). This elevation was prevented by acivicin, a gamma GT inhibitor. Menadione also caused a dose-dependent increase in gamma GT enzymatic activity (715 +/- 125% of control at 24 h after 15 min of exposure to 100 microM menadione, P < 0.001) that was prevented by actinomycin D. Western blot analysis indicated increased levels of gamma GT protein with increasing menadione. A concentration-dependent increase in gamma GT-mRNA was also observed. Previous investigation has demonstrated that an increase in gamma GT activity enhances the capacity of cells to utilize extracellular GSH. The findings presented here are consistent with a role for gamma GT in cellular adaptation to oxidative stress.

Type VI RNA is the major gamma-glutamyl transpeptidase RNA in the mouse small intestine.

Mouse gamma-glutamyl transpeptidase (gamma GT) is encoded by a single copy gene with at least five and probably six different promoters directing the transcription of six types of gamma GT RNAs. In mouse small intestine, only Type I, V, and VI gamma GT RNAs are detected, and ribonuclease protection assays reveal that Type VI represents more than 90% of gamma GT RNA. To investigate the structure of intestinal gamma GT RNA in greater detail, we cloned and sequenced mouse intestinal gamma GT cDNAs. Seven of eight informative clones were Type VI and consisted of Type VI unique exons, VIa and VIb (as described previously by us) (Rajagopalan, S., Wan, D.-F., Habib, G. M., Sepulveda, A. R., McLeod, M. R., Lebovitz, R. M., and Lieberman, M. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6179-6183) as well as common 3' sequences. Exon VIb contains two alternative splice acceptors, one previously identified by us and the other 17 bases 5' of this site. Another clone contained a previously unidentified gamma GT mRNA designated as Type VII. Type VII consists of a unique 5' exon which is 315 base pairs upstream of the exon VIa splice donor site and is spliced to exon VIb. Regulation of gamma GT expression in the small intestine is complex and involves at least three previously described promoters, alternative splicing, and a previously undescribed exonic sequence (Type VII RNA) 5' of promoter VI.

The mouse gamma-glutamyl transpeptidase gene is transcribed from at least five separate promoters.

The mouse gamma-glutamyl transpeptidase (gamma GT) gene encodes six distinct mRNAs that differ in their 5'-untranslated regions but appear to code for the same protein. To elucidate the mechanisms that generate these different mRNAs we determined the transcription start sites of gamma GT kidney mRNAs and investigated the ability of the 5'-flanking regions of mRNAs I, II, IV, V, and VI to direct transcription of chloramphenicol acetyltransferase (CAT) reporter gene constructs in a mouse kidney cell line. Types I, II, and VI mRNAs show heterogeneous start sites, whereas types IV and V have more precise initiation sites. Only the type V 5'-flanking region contains a TATA-like element. The highest CAT activities were observed with 416 base pairs of type II and 240 base pairs of type IV flanking regions. We have also shown that types II and IV represent the predominant gamma GT mRNAs in kidney; therefore, these CAT activities correlate well with the relative amount of each gamma GT mRNA. This study shows that the mouse gamma GT gene is transcribed from at least five and possibly six different promoters. In addition, the gamma GT promoters show cell specificity because no significant CAT activity was detected when these constructs were introduced into NIH-3T3 fibroblasts.

Up-regulation of gamma-glutamylcysteine synthetase activity in melphalan-resistant human multiple myeloma cells expressing increased glutathione levels.

Levels of intracellular glutathione (GSH) and the GSH-related enzymes gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-glutamyltranspeptidase (gamma-GT) were measured in the melphalan-resistant human multiple myeloma cell line 8226/LR-5 and were compared to those measured in the drug-sensitive 8226/S and doxorubicin-resistant 8226/Dox40 cell lines. Both GSH and gamma-GCS activity, the rate-limiting step in the de novo synthesis of GSH, were elevated by a factor of approximately 2 in the melphalan-resistant 8226/LR-5 cells relative to the other two lines. gamma-GT activity was not elevated significantly in the /LR-5 cells. Northern analysis with a probe specific for the large subunit of human liver gamma-GCS identified two bands (3.2 and 4.0 kb), both of which were increased by a factor of 2-3 in the 8226/LR-5 line. Levels of gamma-GCS mRNA expression were comparable in the /S and /Dox40 cell lines. Levels of gamma-GT mRNA were similar in the /S and /LR-5 lines but were reduced in the /Dox40 cells. These data suggest that the increased GSH levels associated with resistance to melphalan in the 8226/LR-5 myeloma cells is attributable to up-regulation of gamma-GCS. This observation is consistent with recent demonstrations of up-regulation of gamma-GCS in melphalan-resistant prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells, suggesting that increased expression of gamma-GCS may be an important mediator of GSH-associated resistance mechanisms.

Human lung expresses unique gamma-glutamyl transpeptidase transcripts.

gamma-Glutamyl transpeptidase (EC 2.3.2.2, gamma GT) is a membrane-bound ectoenzyme that plays an important role in the metabolism of glutathione. It is composed of two subunits, both of which are encoded by a common mRNA. We examined the expression of gamma GT in human lung tissue by Northern blot analysis and screening a cDNA library made from human lung poly(A)+ RNA. Our results show that there are two gamma GT mRNA populations in human lung tissue. We define these as group I (2.4 kb) and group II (approximately 1.2 kb) transcripts. In the present communication, we characterize the unique lung transcript. Sequence analysis of representative clones shows that group I transcripts are virtually identical to those previously isolated from liver and placenta but possess a unique 5' untranslated region. In marked contrast, group II transcripts appear to be human-lung-specific. Group II transcripts appear on Northern blots probed with full-length or 3'-biased gamma GT cDNA. Sequence analysis of group II clones shows them to be homologous with group I clones in the region that encodes the reading frame for the light chain; however, they possess a series of unique 5' untranslated regions, which suggests that they arise from lung-specific message processing. Additionally, approximately 50% of the isolated group II clones contain 34 nt substitutions compared with the "wild-type" gamma GT transcripts. These data indicate that human lung expresses unique gamma GT transcripts of unknown function as well as the classical form. The abundant group II transcripts may encode part of a heterodimer related to gamma GT or represent processed lung-specific pseudogenes.