ARTICLE |
Correspondence to: Wilma M. Frederiks, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: w.m.frederiks@amc.uva.nl
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Summary |
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Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.
Key Words: glucose-6-phosphate dehydrogenase, enzyme histochemistry, immunohistochemistry, in situ hybridization, image analysis, tongue epithelium
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Introduction |
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GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) is a housekeeping enzyme that catalyzes the first step in the pentose phosphate pathway that produces riboses, which are incorporated into nucleic acids, and NADPH, the major cytoplasmic reducing compound (
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Materials and Methods |
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Tissue
Six male Wistar rats, weighing 200250 g, and six mature male New Zealand White rabbits, with a mean weight of 5 kg, were used. Animals were sacrificed with an overdose of barbiturates. The tongue was immediately removed and divided into three parts, the anterior, central, and posterior parts. Small fragments of all parts with a thickness of up to 5 mm were frozen in liquid nitrogen and stored at -80C. Cryostat sections 8 µm thick were cut at -25C on a motor-driven Bright cryostat fitted with a rotary retracting microtome, picked up on clean glass slides, and stored at -30C.
Histochemical Localization of G6PD Activity
Cryostat sections were allowed to dry at room temperature (RT) for 5 min and were then incubated for the demonstration of G6PD activity according to
Immunohistochemistry of G6PD Protein
Rat G6PD protein was demonstrated in rat tongue using a polyclonal rabbit anti-yeast G6PD antibody (Sigma; dilution 1:200) according to
Rabbit G6PD protein was demonstrated using a polyclonal goat anti-rabbit G6PD antibody (dilution 1:50) (
In Situ Hybridization of G6PD mRNA
The procedure to localize G6PD mRNA was performed as described by
Immunohistochemistry of Proliferating Cell Nuclear Antigen (PCNA)
PCNA was detected immunohistochemically in cryostat sections of rat tongue. They were air-dried (1 hr or overnight) and fixed for 2 min in 4% formaldehyde in phosphate buffer, pH 7.4. After dehydration and rehydration in a series of ethanol (50%70%90%96%90%70%50%), sections were rinsed in PBS and then incubated with PBS containing 10% normal goat serum. Between all further incubation steps, sections were thoroughly rinsed in PBS. Sections were incubated with the mouse monoclonal antibody PC10 against PCNA (Dako; dilution 1:100) for 60 min and then incubated with biotinylated rabbit anti-mouse Ig (Dako; dilution 1:200) in the presence of 10% normal rat serum. Sections were incubated with a streptavidinbiotinhorseradish peroxidase complex (Dako; dilution 1:100) prepared 30 min in advance and mixed shortly before use with an equal volume of PBS and 20% normal rat serum. Peroxidase activity was detected by incubating the sections for 10 min in a medium containing 0.5 mg/ml diaminobenzidine, 3 mM H2O2, and 50 mM Tris-HCl buffer (pH 7.6).
Image Analysis and Processing
Serial sections of anterior, central and posterior parts of tongue from six rats and six rabbits were used to quantify G6PD activity, protein, and mRNA with image analysis. In each section (one section per part of the tongue per animal), three areas were selected in the basal cell layers and three in the upper cell layers of the epithelium. In each selected area, three measurements were made and mean gray values were calculated. Gray values were converted into absorbance values by using a set of neutral density filters (Kodak, Rochester, NY) (
Statistics
Results were tested statistically for differences using the Student's t-test, and p=0.05 was taken as level of significance.
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Results |
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Localization of G6PD Activity
G6PD activity was not significantly different in the anterior, central, and posterior parts of tongue epithelium of rat and rabbit (not shown). Fig 1A shows the localization pattern of G6PD activity in the median sulcus of rat tongue. The epithelium in this region is mainly organized as filiform papillae, which consist of stratified squamous epithelium including a basal germinative layer and cornified projections. Among the filiform papillae, scattered fungiform papillae were present, with one taste pore on top covered by a slightly keratinized layer. G6PD activity in epithelium was very high in comparison with that in other tissue components of the tongue such as muscle, connective tissue, and nerves (Fig 1A). Activity was hardly present in the connective core of papillae.
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The following distribution patterns of G6PD activity were observed in epithelium. Basal cell layers contained low activity, whereas activity became higher towards upper cell layers (Fig 1A and Fig 2A). Activity was not detectable in nuclei of epithelial cells. Control incubations did not result in any significant precipitate in these layers (Fig 1B). Highest amounts of final reaction product were found in keratinized superficial layers (Fig 1A), but high amounts were also found after the control reaction and can therefore be considered to represent nonspecific staining (Fig 1B). Formation of final reaction product in the keratinized layer was prevented by incubating sections in the presence of 10 mM N-ethylmaleimide. This finding suggests that SH groups in keratin were responsible for nonspecific staining. Addition of 10 mM epiandrosterone, a specific inhibitor of G6PD, to the incubation medium containing substrate and co-enzyme resulted in similar staining patterns and intensities as those in the control reactions, which confirms that SH groups in keratin caused nonspecific staining in superficial layers but that staining was specific in basal and upper cell layers.
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Similar G6PD activity distribution patterns were observed in tongue of rabbit. The overall activity was lower than that found in rat but the distribution pattern was similar, with the basal cell layers containing lower activity than the upper cell layers.
Image analysis revealed that rat tongue epithelium contained twofold higher activity than that of rabbit, whereas activity in the upper epithelial cell layers was almost threefold higher than that in basal cell layers in both rat and rabbit (Table 1).
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Immunohistochemical Localization of G6PD Protein
G6PD protein distribution patterns in epithelium of rat tongue are shown in Fig 2B. Highest amounts were observed in basal cell layers and lower levels in the upper cell layers. Protein was not present in keratinized layers. Nuclei of epithelial cells were negative (Fig 2B). Control incubations resulted in the absence of staining except for the external (luminal) surface of tongue epithelium, which must be due to food residues because G6PD activity was not present here (Fig 1A). Table 1 shows that the amounts of protein in basal cell layers of both species were at least twofold higher than those in the upper cell layers.
In Situ Hybridization of G6PD mRNA
Fig 2C shows the localization of G6PD mRNA in the anterior part of rat tongue. Similar results were obtained with both mRNA probes, G6PD1 and G6PD3. The distribution patterns of G6PD mRNA showed that mRNA levels were highest in basal cell layers of the epithelium and decreased towards upper cell layers. In general, mRNA levels were about 40% higher in basal cell layers than in upper cell layers (Table 1). mRNA was absent in the keratinized superficial layers. RNase treatment completely abolished staining.
Immunohistochemical Localization of PCNA
Proliferating cells were localized immunohistochemically by detection of PCNA in rat tongue. Fig 2D shows that nuclei positive for PCNA were present only in basal epithelial cell layers. Control incubations resulted in the absence of staining.
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Discussion |
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In this study, we have shown that G6PD activity, protein, and mRNA have distinct distribution patterns in tongue epithelium of rat and rabbit. The activity of the enzyme is extremely high in tongue epithelium. For example, G6PD activity is here 50-fold higher than in rat liver (
The histochemical reaction to demonstrate G6PD activity is specific. The specific reaction is defined as the test minus control reaction (
Image analysis showed that G6PD activity in tongue epithelium is twofold higher in rat than in rabbit. Both animals showed a non-homogeneous distribution of G6PD activity over the epithelial layers. Highest activity was found in the upper cell layers of the epithelium, whereas the basal cell layers showed lower activity. The ratios of the activities in the upper and basal cell layers were similar for both animals. The different activities in the two species may be explained by their diets: rats are omnivorous, whereas rabbits are herbivorous. Previous studies have shown that differences in feeding habits, mastication, and mechanical stress can modify characteristics of epithelial cells of the tongue (
The study of distribution patterns of G6PD protein in serial sections was performed to correlate amounts of protein and activity. Protein content was highest in cells of the basal layers in both rat and rabbit. This means that areas containing relatively high amounts of protein express low activity, whereas the areas with relatively low amounts of protein show high activity. Because G6PD mRNA showed similar distribution patterns as the protein, we conclude that the enzyme is mainly synthesized in cells of the basal layers, and becomes activated posttranslationally in upper cell layers. There are indications that the enzyme is synthetized in a glycosylated form, which is less active, and that the enzyme becomes activated by removal of oligosaccharide groups (
PCNA localization revealed that the basal cell layers of tongue epithelium are the only cell layers that contain proliferating cells. This means that, in these cells, low levels of G6PD activity sustain proliferation, as was shown in previous studies (
In summary, our findings suggest that in tongue epithelium of rat and rabbit, G6PD is mainly synthesized in the basal cell layers, where it is not very active. It is activated in upper cell layers, indicating that its main function in tongue epithelium is NADPH production for protection against oxidative stress. Our quantitative localization study of G6PD activity, protein, and mRNA clearly shows the functional importance of posttranslational regulation of activity of G6PD.
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Acknowledgments |
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We wish to thank Dr Frank van den Berg and Dr Eleonora Aronica for their help in performing the in situ hybridization of G6PD, Mr Jan Peeterse for photographic contributions, and Ms Trees Pierik for preparing the manuscript.
Received for publication February 15, 2000; accepted February 28, 2000.
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