Expression and regulation of gamma -glutamyl transpeptidase-related enzyme in tracheal cells

Pravin D. Potdar, Kaya L. Andrews, Paul Nettesheim, and Lawrence E. Ostrowski

Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Glutathione plays an essential role in protecting the pulmonary system from toxic insults. gamma -Glutamyl transpeptidase-related enzyme (GGT-rel) is a novel protein capable of cleaving the gamma -glutamyl peptide bond of glutathione and of converting leukotriene C4 to leukotriene D4. A rat homologue of GGT-rel was identified and was found to be highly expressed in cultures of differentiating rat tracheal epithelial (RTE) cells. The 2.6-kb cDNA predicts a 572-amino acid protein with 79% identity to human GGT-rel. GGT-rel was weakly expressed in normal trachea but was strongly induced by epidermal growth factor in cultures of RTE cells. GGT-rel was also highly expressed in lung tumors induced by inhalation of isobutyl nitrite. These results demonstrate that GGT-rel 1) is expressed in normal tracheal cells, 2) can be induced by epidermal growth factor, and 3) is elevated after chemical exposure. The induction of high levels of GGT-rel may play an important role in protecting the lung from oxidative stress or other toxic insults.

glutathione; oxidative stress; pulmonary epithelium

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GLUTATHIONE (gamma -glutamylcysteinylglycine; GSH), the most abundant intracellular nonprotein thiol, is the major reductant of cellular thiol groups and participates in many important biological processes, including the gamma -glutamyl cycle and the conjugation of carcinogens and toxins. The membrane-bound enzyme gamma -glutamyl transpeptidase (GGT) catalyzes the first step in the extracellular breakdown of GSH into its constituent amino acids, which can then be transported into the cell and used to maintain the level of intracellular GSH (24). GGT is abundant in tissues with a secretory or absorptive function, such as kidney and pancreas (6). Mice deficient in GGT appear normal at birth but grow slowly and suffer premature death, demonstrating the importance of GGT to normal homeostasis (17). Increased GGT activity is also widely used as a marker of preneoplastic lesions in the liver during chemical carcinogenesis (12).

In addition to GGT, a second enzyme capable of cleaving GSH has been described by Heisterkamp et al. (13) and Morris et al. (19). This enzyme, referred to as GGT-related enzyme (GGT-rel), was cloned from a human placental cDNA library. GGT-rel, when transfected into NIH/3T3 cells, stimulated the degradation of GSH and the conversion of leukotriene C4 into leukotriene D4. However, GGT-rel expression was not demonstrated in any mouse tissue examined, including embryo and placenta, and its biological role is unknown.

The role of GSH and the enzymes involved in GSH metabolism have been the focus of several recent studies of pulmonary cells. For example, exogenous GSH has been shown to protect cultured bovine pulmonary artery endothelial cells from menadione toxicity (3), and oxidative stress increases gamma -glutamylcysteine synthetase and GSH (23). In alveolar epithelial cells, oxidative stress increases GGT transcription and activity (15), and increased levels of GGT and gamma -glutamylcysteine synthetase have been shown to provide increased resistance to oxidative stress (18). In addition, increased GSH levels may be part of an adaptive response to long-term ozone exposure (8). Glutathione S-transferases (GSTs), which catalyze the addition of the thiol group of GSH to many xenobiotics, may also protect the airway from toxic compounds. A lack of the GST-M1 isoenzyme has been associated with a higher risk of lung cancer (7). Thus GSH and the enzymes responsible for its synthesis and metabolism are of significant importance to the protective mechanisms of the airways in response to a variety of chemical insults.

In our laboratory, we have been investigating the regulation of mucociliary differentiation of rat tracheal epithelial (RTE) cells using a model system in which primary RTE cells undergo many of the processes that occur during injury and repair (14). We have previously shown that the pathway of differentiation can be altered by changing the concentrations of growth factors and hormones used to culture RTE cells. For example, epidermal growth factor (EGF) stimulates mucous cell differentiation and mucus production (11), whereas the removal of EGF increases ciliated cell differentiation (5).

In this work, differential display was used to identify genes for which expression was altered during differentiation of RTE cells under specific conditions. We have identified and cloned the rat homologue of GGT-rel and have demonstrated that its expression can be strongly upregulated by EGF. Additionally, GGT-rel expression was observed to be elevated in lung adenomas and carcinomas from rats chronically exposed to isobutyl nitrite. The increased levels of GGT-rel expression suggest that GGT-rel plays an important role in the metabolism of gamma -glutamyl compounds in the airways after injury. Induction of GGT-rel expression may be a protective mechanism in the airways after exposure to toxic insults.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Oligonucleotides were obtained from Genosys (The Woodlands, TX) or Research Genetics (Huntsville, AL). All radioactive materials were obtained from Amersham (Arlington Heights, IL) except 35Sequetide, which was obtained from NEN (Boston, MA). All amplifications were performed in a GeneAmp polymerase chain reaction (PCR) system 2400 or 9600 (Perkin-Elmer, Branchburg, NJ). All tissue culture media and reagents were obtained from Sigma Chemical (St. Louis, MO) with the exception of EGF and rat tail collagen, which were purchased from Collaborative Biomedical Products (Bedford, MA), and bovine pituitaries, which were purchased from Pel-Freeze (Rogers, AR).

Cell culture. Primary RTE cells were cultured using the method originally described by Kaartinen et al. (14) with modifications described in detail previously (22). Briefly, tracheas were obtained from 10- to 14-wk-old male Fischer 344 rats, filled with 1% Pronase, and incubated overnight at 4°C. Primary RTE cells were plated at a density of 2.4 × 104 cells/cm2 on collagen-coated Transwell clear membranes (Costar, Cambridge, MA; see Ref. 2). For all experiments reported here, the cells were submerged in growth factor-supplemented Dulbecco's modified Eagle's medium + F-12 medium [complete medium (CM)] until they reached confluency (day 8). On day 8, parallel cultures were divided into the different treatment groups as described for the individual experiments. Cultures were refed daily beginning on day 5. Previous studies have demonstrated that, under these conditions, RTE cells grow predominantly as a pseudostratified mucociliary epithelium with no obvious fibroblast contamination (5, 11, 14). Recent studies from our laboratory have shown that submersion of RTE cells in CM (SUB cultures) inhibits ciliogenesis (21), whereas withdrawal of EGF and cholera toxin (CT) from the media (-EGF/-CT) promotes ciliated cell differentiation (5). Cells were harvested for RNA at different time points, according to the experimental design.

RNA isolation. Cultured cells were collected by scraping with a rubber policeman directly into guanidinium thiocyanate solution and processed for RNA isolation by the method of Chomczynski and Sacchi (4). The same procedure was used to isolate RNA from rat tissues, except the frozen tissues were ground to a powder in liquid N2, then added to the guanidinium thiocyanate solution. RNA from tracheal epithelium was obtained by simply flushing excised tracheas with the guanidinium thiocyanate solution as described by Ostrowski et al. (20). To remove DNA contamination, total cellular RNA (15-30 µg) was incubated for 30 min at 37°C with 10 units of RNasin ribonuclease inhibitor (BRL, Gaithersburg, MD) and 10 units of ribonuclease free deoxyribonuclease I (Stratagene, La Jolla, CA). After extraction with phenol-chloroform, the supernatant was ethanol precipitated in the presence of 0.3 M sodium acetate. The precipitated RNA was dissolved in diethyl pyrocarbonate-treated water and was used for differential display and reverse transcriptase (RT)-PCR analysis.

Lung tumors. Frozen adenomas, carcinomas, and nonneoplastic lung tissue from Fischer 344 rats treated with isobutyl nitrite were obtained from the National Toxicology Program study, courtesy of Dr. R. Maronpot (National Institute of Environmental Health Sciences). Animals used for this study were exposed by inhalation to 75 or 150 parts/million of isobutyl nitrite for 6 h/day, 5 days/wk for 2 yr. Samples were homogenized in guanidinium thiocyanate solution and were processed as above.

Differential display. Differential display (16) was performed using a Delta RNA Fingerprinting Kit (Clontech, Palo Alto, CA). Two micrograms of purified RNA (days 14-16) from SUB cultures, air-liquid interface (ALI) cultures grown in CM, and ALI cultures grown in -EGF/-CT were compared. These conditions were shown previously to result in differential expression of ciliated and mucous cell-specific genes (1, 11). Aliquots of cDNA were amplified with an arbitrary primer, 5'-ATTAACCCTCACTAAATGCTGGTGG-3' (P3), and an oligo(dT) primer, 5'-CATTATGCTGAGTGATATCTTTTTTTTAG-3' (T3), using Advantage KlenTaq Polymerase (Clontech). The PCR cycling conditions were as described by the manufacturer. The amplified cDNAs were then separated on a denaturing 6% polyacrylamide gel. The gel was dried and exposed to film. Differentially expressed bands of interest were cut from the gel and were reamplified using the same set of primers, according to the manufacturer's instructions. The products of the PCR reaction were separated on a 3% NuSieve/Seakem GTC agarose gel (FMC Bioproducts, Rockland, ME). The isolated band was used as a probe for initial confirmation of the differential expression of these genes by Northern analysis and for cloning into the pCRII vector using the TA Cloning Kit (Invitrogen, San Diego, CA).

Cloning of rat GGT-rel. Rapid amplification of cDNA ends (RACE) was performed using a rat brain Marathon-Ready cDNA library (Clontech) essentially as described by the manufacturer. GGT-rel specific primers were 5'-GCTTCATTTCAACGTGCTGAAAGG-3' and 5'-AAGTTCGCAGTAGGGCAGAGGTGG-3' for 3'-RACE and 5'-GGCAAACTTGAGCGTCTCTACAAG-3' and 5'-TCAATCTGCTGGCGGATGTG CT-3' for 5'-RACE. Products were cloned into the pCRII vector as above.

Sequence analysis. Plasmid DNA was isolated using the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI) and was sequenced using a Sequenase Kit (Amersham) or using an ABI PRISM 377 DNA sequencer and Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). The final sequence was analyzed using the Genetic Computer Group (GCG) sequence-analysis package (version 8; GCG, Madison, WI). Sequences were also analyzed using the MacVector and AssemblyLIGN programs (International Biotechnologies, New Haven, CT).

Northern analysis. Northern analysis was performed using standard procedures essentially as previously described (20). Ten to twenty micrograms of RNA were electrophoresed through a 1.2% agarose-1.0 M formaldehyde gel. Ethidium bromide staining demonstrated equal loading before transfer to the membrane. Gels were immersed in 50 mM NaOH for 15 min and were washed two times in 10× sodium chloride-sodium phosphate-EDTA (SSPE) buffer for 15 min, and RNA was transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) by capillary blotting overnight with 10× SSPE buffer. RNA was cross-linked to the membrane with a Stratalinker (Stratagene) and then was prehybridized and hybridized in QuikHyb hybridization solution (Stratagene), according to the manufacturer's instructions. Nonspecifically bound radioactivity was removed by washing the membrane in 2× standard sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) two times for 15 min at room temperature followed by a 30-min wash in 0.1× SSC and 0.1% SDS at 58°C. The blots were exposed to Kodak film at -70°C with intensifying screens. For estimation of changes in the expression level of GGT and GGT-rel, Northern blots were analyzed using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). To avoid any cross-hybridization between GGT and GGT-rel, duplicate Northern blots were prepared and probed separately for each gene. Blots were also probed with a beta -actin cDNA. Results for GGT and GGT-rel were then normalized to the expression level of beta -actin as a control for loading and transfer. For the results shown, two completely independent RTE cultures were analyzed with reproducible results.

Labeling of probes. A rat GGT probe was prepared by RT-PCR of RNA isolated from RTE cell cultures with primers specific for rat GGT. The upstream primer was 5'-AGCCAGGTAAGCAACCGCTTTC-3' and the downstream primer was 5'-CCGCCTTTTCTGGAATCTGAG-3' (bases 1319-1679 of rat GGT). An annealing temperature of 58°C was used. The 361-bp product was cloned and was verified by sequencing. Probes for GGT-rel and GGT were generated by digestion of plasmid DNA to remove the insert. The cDNA insert was isolated from a low melting point agarose gel, and 25 ng of insert DNA were labeled with the Rediprime DNA Labeling System (Amersham). Probes were purified on NucTrap columns (Stratagene).

RT-PCR analysis. One microgram of RNA from rat brain, heart, kidney, liver, lung, spleen, testis, trachea, or cultured RTE cells was reverse transcribed and amplified with an RNA PCR kit (Perkin-Elmer). After reverse transcription, the cDNA samples were amplified using specific primers for rat GGT (as above) or GGT-rel. Primers used for rat GGT-rel were 5'-TGAAGGGAGGGTGAACGTGTAC-3' (upstream) and 5'-TCAATCTGCTGGCGGATGTGCT-3' (downstream). Typical PCR cycling conditions were 95°C for 1 min followed by 35-45 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s, followed by 72°C for 7 min. An aliquot of each reaction was electrophoresed through a 3% NuSieve-Seakem GTC agarose gel and was visualized by ethidium bromide staining.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of a differentially expressed cDNA during RTE cell differentiation. To isolate genes for which the expression changes during mucous or ciliated cell differentiation, differential display was performed using RNA isolated from RTE cells cultured under three different conditions (described in MATERIALS AND METHODS) that have previously been shown to influence the extent of mucous and ciliated cell differentiation (5, 11, 21). Samples from two independent cultures (days 14-16) were analyzed in parallel. Overall, the differential display banding pattern was highly reproducible between the cultures and between these three conditions (Fig. 1A). With the use of various primer combinations, several differentially expressed bands were identified. One cDNA, which was abundantly expressed in RTE cells cultured in CM at an ALI and in SUB cultures but was not expressed in RTE cells cultured at an ALI in -EGF/-CT (Fig. 1A), was chosen for further characterization in this study.


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Fig. 1.   Differential display analysis of RNA from tracheal cells. RNA isolated from cultures of rat tracheal epithelial (RTE) cells grown under three different conditions [complete medium (CM), submerged in media (SUB), and withdrawal of epidermal growth factor and cholera toxin (-EGF/-CT)] were used for differential display as described in MATERIALS AND METHODS. A: RNA isolated from 2 independent experiments was processed in parallel, and amplified cDNAs were analyzed on a denaturing 6% polyacrylamide gel. Differentially expressed cDNA (arrow) was isolated for further study. B: isolated cDNA from A was reamplified, gel purified, and used as a probe on a Northern blot containing 20 µg of RNA from RTE cells cultured under the same conditions used for differential display. Probe hybridized strongly to an mRNA of ~2.8 kb in CM and SUB cultures, but its expression was markedly reduced by day 14 in -EGF/-CT cultures, verifying the differential expression of this cDNA.

The cDNA in this band was isolated, reamplified, and used as a probe on Northern blots. The probe detected an abundant transcript of ~2.8 kb in CM and SUB cultures fed with complete growth medium throughout the culture period (Fig. 1B). However, in -EGF/-CT cultures, this message was weakly expressed on day 14 and was not detectable by day 16 (Fig. 1B). This result confirms the differentially expressed pattern observed by differential display.

Cloning and sequencing of rat GGT-rel cDNA. The reamplified cDNA was cloned, and several independent clones were sequenced. The sequence data indicated that during the differential display procedure the cDNA was primed with the arbitrary primer (P3) at both ends. A search of GenBank revealed a high degree of homology between the cloned 373-bp cDNA fragment and human GGT-rel (amino acids 247-270). No significant homology with any other gene was found. To determine the full-length sequence for rat GGT-rel, primers were designed based on the partial sequence, and 5'- and 3'-RACE was performed using a rat brain cDNA library. Several clones for both the 5'- and 3'-ends were obtained and sequenced. The composite 2.6-kb cDNA contained a 1,716-nucleotide open reading frame and codes for a predicted protein of 61,600 kDa. An alignment of the predicted rat and human GGT-rel protein sequences (Fig. 2) demonstrated 79% identity and 82% similarity. For comparison, an alignment of rat and human GGT also showed 79% identity (83% similarity). Rat GGT-rel contains eight potential N-linked glycosylation sites (Fig. 2), four of which are conserved between the rat and human protein. Rat GGT-rel also contains a two-amino acid insertion (positions 533 and 534) and a 16-amino acid deletion (after position 443) compared with the human protein. Overall, these results indicate that the differentially expressed cDNA is the rat homologue of GGT-rel.


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Fig. 2.   Sequence analysis of rat gamma -glutamyl transpeptidase-related enzyme (GGT-rel). Clones covering the full-length cDNA for rat GGT-rel were isolated by rapid amplification of cDNA ends (RACE) and were sequenced. An alignment of the predicted translation of rat GGT-rel (top line) with human GGT-rel (bottom line) shows 79% identity and 82% similarity between the rat and human proteins. Possible N-linked glycosylation sites are marked with an asterisk. Rat GGT-rel contains a 16-amino acid deletion (443) and a 2-amino acid insertion (533-534) compared with human GGT-rel. The sequence of rat GGT-rel has been deposited in GenBank (accession number U76252).

Expression of GGT-rel in rat tissues. To examine the expression of GGT-rel in different tissues, cDNA was used to probe a Northern blot of RNA isolated from rat tissues and RTE cell cultures. As expected, RNA from RTE cells cultured in CM demonstrated a strong hybridization signal (Fig. 3A). A faint signal was also observed in RNA isolated from tracheal epithelium by flushing the lumen with lysis solution. However, no message was detected by Northern analysis in all other tissues (brain, heart, kidney, liver, lung, spleen, and testis) tested. To examine the expression of GGT-rel at a more sensitive level, the same tissues were analyzed by RT-PCR using primers specific for GGT-rel. After 45 cycles of amplification using the GGT-rel specific primers, a clear product was visible in all tissues tested (brain, heart, kidney, liver, lung, spleen, testis, and trachea; Fig. 3B). Amplifications performed without RT or without RNA template were routinely negative, indicating the product obtained was dependent on the presence of GGT-rel mRNA. These results indicate that GGT-rel is expressed in many tissues of the adult rat, although at low levels.


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Fig. 3.   Expression of GGT-rel in rat tissues. RNA was isolated from the indicated tissues, and ~20 µg were size fractionated on a formaldehyde-agarose gel, transferred to a nylon membrane, and probed with a rat GGT-rel cDNA. A: RTE cells cultured in CM demonstrated a strong hybridization signal, whereas RNA isolated from tracheal lumen showed only a faint hybridization signal. All other tissues were negative by this method. B: reverse transcriptase (RT)-polymerase chain reaction was used to analyze rat tissues for low levels of GGT-rel expression. One microgram of RNA was reverse transcribed and then amplified for 45 cycles using GGT-rel-specific primers. A specific product of the expected size was observed in all tissues analyzed. No product was observed in control reactions without RT (lanes marked -). STD, standard.

Effect of EGF and CT depletion on GGT-rel expression. As shown above, GGT-rel is strongly expressed in cultures grown in CM but was not detected in cells cultured 7 days in the absence of EGF and CT. To examine the regulation of GGT-rel by EGF and CT, RTE cells were cultured for 7 days in CM, media without EGF, media without CT, or media without EGF and CT. Northern analysis showed that removal of CT from the media had no effect on the expression of GGT-rel (Fig. 4A), whereas the removal of EGF alone reduced GGT-rel expression substantially (72% reduction when normalized to the expression of beta -actin). The removal of both EGF and CT caused a further decrease in the expression of GGT-rel. In contrast, the removal of EGF or EGF and CT only slightly reduced GGT expression (-EGF, 36% reduction, -EGF/-CT, 30% reduction; Fig. 4B). These results indicate that EGF has a major effect on the expression of GGT-rel in RTE cells.


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Fig. 4.   Effect of epidermal growth factor (EGF) and cholera toxin (CT) withdrawal on GGT-rel and GGT expression. RNA was isolated from RTE cells cultured in CM, media without EGF (-EGF), media without CT (-CT), and media without EGF and CT for 7 days (days 8-15). Twenty (GGT-rel) or fifteen (GGT) micrograms of RNA were used for Northern analysis. Filters were reprobed with a beta -actin cDNA for normalization, and phosphorimage analysis was used to estimate relative expression levels. A: GGT-rel expression was reduced by the removal of EGF from the media (30% of CM) but was unaffected by the removal of CT alone. B: in contrast to GGT-rel, GGT expression was reduced only slightly by the removal of EGF.

Expression of GGT-rel during differentiation in the presence and absence of EGF. To examine the level of expression of GGT-rel during mucociliary differentiation and its regulation by EGF, RTE cells were cultured in media with or without EGF for 7 days after the formation of the ALI. RNA was isolated from parallel cultures at daily intervals and was analyzed for the level of GGT-rel, GGT, and beta -actin transcripts by Northern blotting (Fig. 5, A and B). Removal of EGF from the media resulted in a substantial decrease in the level of GGT-rel expression. When normalized to beta -actin expression and compared with RTE cells cultured in CM, RTE cells cultured in the absence of EGF showed a 57% reduction in GGT-rel expression by day 13 (Fig. 5C). By day 15, the level of GGT-rel expression in cultures grown in the absence of EGF was 3.9-fold less than in the CM cultures. The level of GGT expression also decreased after the removal of EGF, although the magnitude of the reduction was <50% on day 15 (Fig. 5C). These results show that GGT-rel is expressed throughout differentiation of RTE cells in vitro and that the removal of EGF causes a substantial reduction in the expression of GGT-rel.


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Fig. 5.   Expression of GGT-rel during differentiation of RTE cells in the presence or absence of EGF. Parallel RTE cultures were grown in media with or without EGF for 7 days (days 8-15). RNA was isolated at daily intervals throughout the culture period and was analyzed by Northern blotting for the expression of GGT-rel (A) and GGT (B). -E, without EGF. C: relative expression levels were determined by phosphorimage analysis after normalization to the signal for beta -actin and are expressed as a percentage of control. Results are the average of 2 independent experiments. Error bars indicate the range of values; in some cases the error bars are covered by the symbol. Withdrawal of EGF resulted in decreased expression of GGT-rel.

Induction of GGT-rel by EGF. To determine if EGF could induce GGT-rel expression directly, RTE cells were grown in media without EGF from days 8 to 13. On days 13 and 14, some of the EGF-depleted cultures were refed with media containing 25 ng/ml EGF. Cells were harvested from the different groups 8, 24, and 48 h after the addition of EGF to the EGF-depleted cultures. Northern analysis demonstrated a 2.4-fold increase (average of 2 experiments) in GGT-rel expression 24 h after addition of EGF (Fig. 6A). This induction was maintained for at least 48 h (2.2-fold, average of 2 experiments), although the level of GGT-rel expression in cultures treated with EGF did not reach the level in parallel cultures grown continuously in CM. GGT expression was also increased after addition of EGF, although not as strongly (1.7-fold at 24 h and 1.4-fold at 48 h, average of 2 experiments; Fig. 6B). EGF is therefore capable of rapidly inducing the expression of GGT-rel in RTE cells.


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Fig. 6.   Induction of GGT-rel expression by EGF. RTE cells were cultured in the absence of EGF for 5 days (days 8-13) and then were refed with media with (+E) or without (-E) 25 ng/ml of EGF. RNA was isolated at different time points and was analyzed by Northern blotting for the expression of GGT-rel (A) and GGT (B). Data from 2 independent experiments were collected by phosphorimage analysis and were normalized to the expression of beta -actin. GGT-rel was induced rapidly in cultures refed with media containing EGF, increasing >2-fold within the first 24 h after EGF addition. GGT expression also increased in response to EGF, although to a lesser degree (1.4-fold at 48 h).

Expression of GGT-rel in lung tumors. As shown above, EGF induced GGT-rel expression in RTE cells. Transforming growth factor (TGF)-alpha , which activates the EGF receptor, is overexpressed by transformed RTE cells (9) and also by many other transformed cell lines and tumors. Therefore, GGT-rel expression was examined in several lung tumor samples. RNA was isolated from normal lung, adenomas, and carcinomas from rats treated with isobutyl nitrite and also from normal untreated lung. Northern analysis demonstrated a strong signal for GGT-rel in both the carcinomas and adenomas compared with normal untreated lung (Fig. 7). The normal lung tissue obtained from an animal treated with isobutyl nitrite also showed a high level of GGT-rel expression. GGT-rel expression was particularly high in one of the carcinomas (Fig. 7, lane 5). These results show that GGT-rel is expressed at high levels in lung tissue and tumors from animals chronically exposed to isobutyl nitrite.


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Fig. 7.   Expression of GGT-rel in lung tumors. RNA was isolated from normal lung tissue and adenomas, carcinomas, and nonneoplastic lung tissue from rats chronically exposed to isobutyl nitrite. Twenty micrograms of RNA were separated by electrophoresis and were examined for the expression of GGT-rel by Northern analysis (A). All of the treated animals showed elevated levels of GGT-rel expression compared with normal lung. One carcinoma demonstrated a substantially increased level of GGT-rel expression. B: hybridization with a beta -actin cDNA demonstrates approximately equal loading of all samples.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using differential display to analyze changes in gene expression during mucociliary differentiation of RTE cells, we have identified a rat homologue of human GGT-rel. This is the first evidence that GGT-rel is expressed in airway epithelial cells. Heisterkamp et al. (13) were unable to demonstrate GGT-rel expression in any of the mouse tissues they analyzed and only observed a very low level of expression in two human cancer cell lines. In contrast, our studies have demonstrated a high level of GGT-rel expression by normal RTE cells in primary culture. In agreement with the results of Heisterkamp et al. (13), GGT-rel expression was not detected by Northern analysis using whole lung or other rat tissues as a source of RNA. However, GGT-rel expression was detectable by RT-PCR in all rat tissues examined, including brain, heart, kidney, liver, lung, spleen, testis, and trachea. This suggests that, similarly to GGT, GGT-rel is widely distributed, although constitutively expressed at much lower levels. Alternatively, it is possible that only a small number of specialized cells express GGT-rel in any particular tissue or that GGT-rel is only highly expressed under certain conditions. In support of the latter hypothesis, our results show that GGT-rel expression can be induced by EGF. Treatment of EGF-deprived RTE cultures with 25 ng/ml of EGF caused a rapid and sustained increase in GGT-rel expression. This demonstrates that GGT-rel expression can be regulated by growth factors and may link the expression of GGT-rel to increased levels of proliferation. In addition, high levels of GGT-rel expression were observed in lung carcinomas, adenomas, and normal lung tissue from rats exposed by inhalation to isobutyl nitrite, a chemical that causes hyperplasia of the respiratory airway epithelium. Increased levels of GGT expression have been observed in many tumor types, and its expression is frequently used as an early marker for hepatocarcinogenesis (12). Whether GGT-rel expression is induced in tumors as a result of chemical exposure or as a direct result of the neoplastic process is unclear. Additional experiments will be needed to elucidate the possible roles of GGT-rel in these conditions.

GGT-rel has many potential functions in airway epithelial cells. For example, GGT-rel could function in the defense mechanisms of the airways by supplying reducing equivalents to the cell via GSH. Forman and co-workers (3, 15, 18, 23) have demonstrated that GSH can protect pulmonary cells from oxidative stress and that oxidative stress can induce GGT and gamma -glutamylcysteine synthetase. Because GGT-rel has been shown to catalyze some of the same reactions as GGT (13), high levels of GGT-rel could also provide protection from oxidative stress. GGT-rel may also play a role in the secretory functions of RTE cells. RTE cell cultures grown in media containing EGF show a high percentage of mucous cells and actively produce and secrete mucus and other products. The withdrawal of EGF decreases mucus production and also reduces the level of GGT-rel expression. Thus GGT-rel may play a role in the secretory pathway, perhaps by providing essential precursors for the synthesis of secreted molecules. In addition, GGT-rel could function in the metabolism of important biological mediators. Human GGT-rel was shown to catalyze the conversion of leukotriene C4 into leukotriene D4, and leukotrienes can cause bronchoconstriction and changes in vascular permeability in the lung (10). At present, the physiological function(s) of GGT-rel in the airways is unknown, but the present work indicates that further studies are warranted.

In summary, the experiments presented here have identified a rat homologue of GGT-rel and demonstrated that GGT-rel is expressed in a wide variety of rat tissues. GGT-rel was shown to be inducible to high levels in cultures of primary RTE cells by EGF. High levels of GGT-rel expression were also observed in several lung tumors and in lung tissue chronically exposed to a carcinogen. Under these conditions, GGT-rel, in addition to GGT, may be important to the metabolism of gamma -glutamyl compounds, and future studies will need to distinguish between these two enzymes.

    ACKNOWLEDGEMENTS

We thank Dr. Bob Maronpot for supplying the rat lung tumors used in this study, Daniel Hart for assistance with automated sequencing, and Drs. Doug Bell and Tom Eling for reviewing the manuscript. We also acknowledge Dr. John Groffen for helpful discussions of this work.

    FOOTNOTES

P. D. Potdar was funded by Rameshwardas Birla Smarak Kosh, Mumbai India; the National Cancer Institute short-term scientist exchange program; and National Institute of Environmental Health Sciences.

Present address of P. D. Potdar: Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 400012, India

Address for reprint requests: L. E. Ostrowski, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.

Received 9 June 1997; accepted in final form 14 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Andrews, K. L., P. Nettesheim, D. J. Asai, and L. E. Ostrowski. Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation. Mol. Biol. Cell 7: 71-79, 1996[Abstract].

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AJP Lung Cell Mol Physiol 273(5):L1082-L1089