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Expression of 5-Reductase in the Human Temporal Lobe of Children and Adults¹

Departments of Clinical Biochemistry (B.S., M.W., S.S., L.W., D.K.), Neurosurgery (J.S.), and Internal Medicine (G.R., H.-U.S.), University of Bonn, Bonn, Germany

Address all correspondence and requests for reprints to: Prof. D. Klingmüller, Institut für Klinische Biochemie, Universität Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Androgens exert important biological effects on the brain, and 5-reductase plays a crucial role in androgen metabolism. Therefore, we investigated the expression of the two isozymes of 5-reductase in the human temporal lobe to determine the predominant isoform and to elucidate the existence of possible sex differences and differences between children and adults. We studied biopsy materials from the temporal lobe of 34 women, 32 men, and 12 children. Quantification of 5-reductase 1 and 2 messenger ribonucleic acid (mRNA) was achieved by competitive RT-PCR. 5-Reductase activity was determined in tissue homogenates using [1,2-³H]androstenedione as the substrate. Only 5-reductase 1 mRNA was expressed in human temporal lobe tissue; 5-reductase 2 mRNA was not expressed. 5-Reductase 1 mRNA concentrations did not differ significantly in the cerebral cortex of women [25.9 ± 7.9 arbitrary units (aU); mean ± SEM] and men (20.4 ± 2.8 aU) or in the cerebral cortex (23.3 ± 4.4 aU) and the subcortical white matter of adults (32.6 ± 5.6 aU), but they were significantly higher in the cerebral cortex of adults than in that of children (6.4 ± 2.3 aU; P < 0.005). The apparent K_m of 5-reduction did not show significant differences between the two sexes. In conclusion, 5-reductase 1 mRNA is expressed in the temporal lobe of children and adults, but 5-reductase 2 mRNA is not. 5-Reductase 1 mRNA concentrations did not differ significantly in the sexes, but they were significantly higher in specimens of adults than in those of children.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
ANDROGENS exert important biological effects on the brain either directly or after 5-reduction or aromatization (1, 2, 3, 4). Specific receptors for androgens have been identified in several regions of the brain, through which androgens could effect a genomic response (5).

5-Reduction represents a major route of ⁴-androgen metabolism. 5-Reductase (EC 1.3.99.5) uses NADPH to reduce the double bound of a variety of steroid substrates with generalized 3-oxo--4,5 structures (6). Recent cloning and expression studies reported the isolation of complementary DNAs (cDNAs) for two different isozymes (types 1 and 2) of 5-reductase in rat as well as human tissues (7, 8). In addition to biochemical and pharmacological differences, the type 1 and type 2 messenger ribonucleic acids (mRNAs) are differentially expressed in human tissues. 5-Reductase 2 is the predominant isoform found in male accessory sex organs, whereas 5-reductase 1 is present in tissues such as liver and nongenital skin (9).

5-Reductase activity has been demonstrated in neural tissue from various animal species and human fetuses (1, 3, 10, 11, 12). To date, there is little information on the androgen metabolism in the human brain at different ages. Systematic studies in human brain tissue are lacking. Although 5-reductase enzymatic activity has been studied in only a few frontal lobe and temporal lobe specimens of adults (13, 14), 5-reductase has not yet been studied at the molecular level in cortical tissue from children and adults. Only one study reported 5-reductase 1 expression in a few human cerebellum, hypothalamus, and pons tissue specimens that were collected postmortem (9).

The cloning of 5-reductase 1 and 2 cDNAs has enabled this investigation of the isozyme expression of 5-reductase. It was designed to investigate the expression of 5-reductase isozymes in the human temporal lobe in a large number of specimens from children and adults to determine the predominant isoform and to elucidate the existence of possible sex differences and differences between children and adults. To extend and confirm the results obtained in the mRNA quantification experiments, 5-reductase enzyme activity was also determined.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Biopsy materials removed at neurosurgery from 34 women (32.5 ± 1.3 yr; mean ± SEM), 32 men (34.8 ± 1.6 yr), and 12 children (8 ± 1.4 yr) with temporal lobe epilepsy undergoing partial temporal lobe resection were used.

Steroids and reagents

[1,2-³H]Androstenedione (42 Ci/mmol) was purchased from New England Nuclear Corp. (Dreieich, Germany). It was purified by thin layer chromatography to assure a purity greater than 98%. Nonradioactive steroids were purchased from Steraloids, Inc. (Wilton, NH) or Sigma Chemical Co. (Deisenhofen, Germany). NADPH, Taq polymerase, and ribonuclease (RNase)-free deoxyribonuclease I (DNase I) were purchased from Boehringer Mannheim (Mannheim, Germany). Trizol reagent and the Superscript II preamplification system were obtained from Life Technologies (Paisley, UK). The pCR-script cloning kit and the RNA in vitro transcription kit were purchased from Stratagene (La Jolla, CA). The QIAquick PCR purification kit and the RNeasy total RNA kit were obtained from Qiagen (Hilden, Germany). Primers were obtained from Genosys (Cambridge, UK) or PE Applied Biosystems (Weiterstadt, Germany; Table 1).

Temporal lobe biopsy materials were separated into cortex and subcortical white matter by inspection, transferred into liquid nitrogen immediately after removal, and stored at -80 C. Cortex tissue specimens were obtained from 19 women, 16 men, and 9 children; white matter tissue specimens were obtained from 6 women, 7 men, and 1 child; and both cerebral cortex and white matter tissue specimens were obtained from 9 women, 9 men, and 1 child, respectively.

Liver tissues were obtained in a transplantation program from biopsies to exclude liver diseases from the Department of Surgery, University of Bonn (Bonn, Germany), and prostate tissues were obtained from the Department of Urology, Waldkrankenhaus Bonn (Bonn, Germany). Tissues were transferred to liquid nitrogen immediately after removal and stored at -80 C.

The study was approved by the local ethics committee, and informed consent was obtained from all tissue donors.

mRNA quantification

mRNAs of 5-reductase 1 and 2 were quantified with only a few modifications according to a nested competitive RT-PCR protocol previously described (15).

Total RNA was extracted from 25–50 mg tissue using the Trizol reagent. Traces of DNA were removed by treatment with RNase-free DNase I, followed by a second RNA extraction. RNA was taken up in RNase-free H₂O and quantified by its spectrophotometric absorption at 260 nm.

Competitive RNA standards were prepared by overlap extension mutagenesis of 5-reductase 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or by single step mutagenesis of 5-reductase 2, resulting in the loss of 7, 10, and 11 bp for 5-reductase 1, GAPDH, and 5-reductase 2, respectively, as previously described [5-reductase 1/GAPDH (15, 16), 5-reductase 2 (17)]. The mutant cDNAs (5-reductase 1/GAPDH) were cloned with the pCR-script cloning kit. From these plasmids, cDNA templates were amplified using primer pairs spanning the mutagenized cDNA fragment and the T7 promoter region (T7 primer and GAPDH reverse primer/T7 primer and 5-reductase 1 reverse primer; Table 1). Mutant 5-reductase 2 PCR products were produced by a PCR with a 5'-primer containing the T7 promotor and 5-reductase 2 5'-sequence and a 3'-primer containing the 5-reductase 2 3'-sequence with a deletion of 11 bp. These templates were cleaned using the QIAquick PCR purification kit and used to generate standard RNA by in vitro transcription. Successful mutagenesis was confirmed by sequencing on a semiautomated sequencer (373A, PE Applied Biosystems, Foster City, CA). RNA in vitro transcription was performed using an RNA in vitro transcription kit with T7 polymerase; cDNA templates were removed by treatment with RNase-free DNase I (1 U/µg template). Standard RNA was extracted with the RNeasy total RNA kit, and its concentration was measured spectrophotometrically.

To estimate the amount of standard RNA required for quantification of individual RNA samples, 4–10 RNA samples of the respective tissue groups were pooled. To aliquots of these mixtures containing 250 ng RNA each, defined amounts of standard RNAs were added. Serial dilutions ranged from 500 pg to 5 attograms (ag) for GAPDH and from 100 pg to 1 ag for 5-reductase 1 and 2. Each mixture containing the respective amount of RNA standard, and patient RNA was reverse transcribed followed by PCR amplification. The optimal titration point was defined as the concentration of standard RNA at which PCR products yielded signals of comparable intensity for standard and native RNA (Fig. 1). A stock solution was prepared containing standard RNAs for 5-reductase 1, 5-reductase 2, and GAPDH at the optimal titration point. The concentration of this stock solution was selected in a way that 1 µL stock was sufficient for the RT of 250 ng total RNA. RT was performed at 42 C for 60 min using 100 U Superscript II (Superscript preamplification system). The resulting cDNA was diluted 20-fold with water, and PCR was performed in a final volume of 20 µL containing 2 µL diluted cDNA, 10 mmol/L Tris-HCl (pH 8.3), 40 mmol/L KCl, 1.5 mmol/L MgCl₂, 200 µmol/L of each deoxy-NTP, 0.5 U Taq polymerase, and 4 pmol of each primer (Table 1). One primer of the primer pairs used for GAPDH PCR or nested PCR (5-reductase 1 and 2) was labeled with fluorescent dyes. PCR amplification was carried out in microtiter plates in a Unoblock (Biometra, Gottingen, Germany). Initial denaturation at 94 C for 4 min was followed by 32 (GAPDH) or 35 (5-reductase 1 and 2) PCR cycles. Cycling conditions were 94 C for 35 s, 55 C for 50 s, and 72 C for 90 s. A final extension step of 5 min at 72 C was used. Nested PCR of 5-reductase 1 and 2 was performed under the same conditions.

View larger version (64K): [in this window] [in a new window]   Figure 1. Titration of RNA standards for 5-reductase 1 and 2. Nested PCR was performed from total RNA of liver tissue (5-reductase 1; a), prostate tissue (5-reductase 2; b), and temporal lobe tissue [5-reductase 1 (c) and 5-reductase 2 (d)]. Each lane corresponds to cDNA reversely cotranscribed from 250 ng total RNA with decreasing standard RNA concentrations of either 5-reductase 1 or 5-reductase 2. The amounts of standard RNAs were, from left to right, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 ag, 10 ag, and 1 ag. The optimal titration point for 5-reductase 1 standard RNA is 10 pg in liver tissue and 100 fg in temporal lobe tissue for 250 ng total RNA each. For 5-reductase 2 standard RNA, the optimal titration point is 1 pg in prostate tissue and below 1 ag in temporal lobe tissue for 250 ng total RNA each.

Determination of 5-reductase activity

5-Reductase activity in the biopsy materials was determined with the following modifications according to methods previously described (18, 19). In brief, samples were homogenized in ice-cold 10 mmol/L Tris-chloride buffer (pH 7.4), and 1 mmol/L ethylenediamine tetraacetate with a Douncer homogenizer (Kontes Co., Vineland, NJ). Either these homogenates were used, or a crude nuclear fraction and a crude supernatant containing the microsomes were prepared by centrifuging the homogenates at 1000 x g for 15 min. The precipitate was then suspended in the same buffer and rehomogenized to obtain the presumable nuclear fraction. The supernatant containing the microsomes was not diluted further. 5-Reductase activity was determined by incubation with [1,2-³H]androstenedione as substrate. Standard assays for kinetic studies were carried out in triplicate. Standard assays contained the 1,2-³H-labeled androstenedione at concentrations varying from 0.05–3.5 µmol/L, 3 mmol/L NADPH, 0.08 mol/L Tris citrate (pH 7.5), and 5 mmol/L MgCl₂ and the brain homogenates in a final volume of 200 µL. Incubations, separation of the 5-androstanes by thin layer chromatography, and calculations of 5-reductase activity rates were performed as previously described (18, 19, 20).

The pH optimum of 5-reduction was determined in cortex homogenates using a procedure similar to that described for 5-reductase activity, except that a series of buffers (0.08 mol/L Tris citrate) with different pH values (4.0–9.5) were used instead of a single buffer. These experiments were carried out in duplicate.

Statistical analysis

Results were calculated as the mean ± SEM. The statistical difference between groups was calculated using the Mann-Whitney U test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Determination of titration points for 5-reductase 1 and 2 mRNA in reference tissues and temporal lobe tissue

To validate our mRNA quantification protocol, we determined the expression of 5-reductase 1 mRNA in liver tissue and the expression of 5-reductase 2 in prostate tissue. The use of competitive RT-PCR requires the amount of standard RNA that yields a signal of approximately equal density when coamplified with total RNA. The optimal titration point for liver tissue was 10 pg standard RNA for 5-reductase 1 and 250 pg for GAPDH based on 250 ng total RNA (Fig. 1a). For total RNA from prostate tissue, the equivalent titration point was 1 pg standard RNA for 5-reductase 2 and 250 pg for GAPDH (Fig. 1b).

In the same way, the titration points of 5-reductase 1 and 2 in human temporal lobe tissue were determined. The optimal titration point was 100 fg standard RNA for 5-reductase 1 and 250 pg for GAPDH based on 250 ng total RNA (Fig. 1c). For 5-reductase 2, even a RNA standard amount of 1 ag did not yield a detectable fluorescence signal of native RNA (Fig. 1d). Conclusively, 5-reductase 2 mRNA is not expressed in the human temporal lobe; only illegitimate transcription was detectable in each sample when the standard RNA was omitted in the RT step.

Expression of 5-reductase 1 mRNA in temporal lobe tissue from children and adults

5-Reductase 1 mRNA concentrations in the cerebral cortex did not differ significantly between women [25.9 ± 7.9 arbitrary units (aU); n = 28] and men (20.4 ± 2.8 aU; n = 25), but they were significantly higher in the cerebral cortex of adults (23.3 ± 4.4 aU; n = 53) than in that of children (6.4 ± 2.3 aU; n = 10; P < 0.005; Fig. 2a). No significant differences in 5-reductase 1 mRNA expression were observed between the cerebral cortex (23.3 ± 4.4 aU; n = 53) and the subcortical white matter of adults (32.6 ± 5.6 aU; n = 31; Fig. 2). Also, 5-reductase 1 mRNA concentrations did not differ significantly in the subcortical white matter of women (26.4 ± 6.8 aU; n = 15) and men (38.5 ± 8.7 aU; n = 16; Fig. 2b). As only two white matter specimens from children were available, a statistical analysis of the mRNA expression in the white matter of adults and children is impossible. However, in the 2-yr-old boy studied, the 5-reductase 1 mRNA concentration was 2.8 aU, and in the 13-yr-old girl, it was 3 aU, which means a low level of expression compared to the expression levels in adults.

View larger version (14K): [in this window] [in a new window]   Figure 2. Expression of 5-reductase 1 mRNA in human cortex tissue (a) and subcortical white matter tissue (b) of children and adults.

5-Reductase activity in the temporal lobe of children and adults

Studies were then performed to characterize 5-reductase activity in temporal lobe tissue. Due to the limited amount of tissue available, studies of 5-reductase activity had to be performed on a smaller number of specimens than studies of 5-reductase 1 mRNA expression. However, using androstenedione as the substrate, 5-reductase activity was present in all studied temporal lobe specimens of children and adults. As summarized in Table 2, in cortex tissue the apparent K_m of 5-reduction did not show significant differences between crude nuclear fractions and crude supernatants containing the microsomes or between the two sexes. Although only two children’s specimen could be studied, no obvious difference concerning the K_m value of 5-reduction between children and adults was present; the maximal veloity of 5-reduction in the two children was higher than that in almost all adults.

View this table: [in this window] [in a new window]   Table 2. Kinetic parameters of 5-reduction in 1000 x g precipitates presumably containing the nuclear fractions (nucl.) and in the respective supernatants containing the microsomes (micros.) of cerebral cortex tissue

View this table: [in this window] [in a new window]   Table 3. Comparison between kinetic parameters of 5-reduction in homogenates of cortex and white matter tissue (white m.)

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of pH on 5-reductase activity in cortex homogenates of a 34-yr-old man and a 34-yr-old woman. Homogenates were incubated with [3H]androstenedione (1 µmol/L) and 3 mmol/L NADPH at 37 C for 1 h at a range of pH values. Representative data are shown from single experiments performed in duplicate.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Two isozymes of 5-reductase (types 1 and 2) with differential tissue distribution and biochemical and pharmacological differences have been identified in humans (7, 8). Our study is the first to determine the expression of 5-reductase isoforms in a large number of fresh human temporal lobe tissue specimens. The highly sensitive nested competitive RT-PCR approach used permitted us to demonstrate that the almost exclusive 5-reductase gene expressed in the human temporal lobe is the type 1 isoform. This is in accordance with a study of Thigpen and co-workers, who found exclusively 5-reductase 1 mRNA expression in tissue samples collected postmortem from cerebellum, hypothalamus, pons, and medulla oblongata (9), and with studies on rat brain tissue reporting a predominant expression of 5-reductase 1 (23, 24).

Our data show that 5-reductase 1 mRNA is present in cortex tissue as well as in the subcortical white matter of children and adults. To confirm and extend these experiments, 5-reductase activity was measured in tissue homogenates. The enzyme activity was present in all tissue specimens under investigation. The apparent K_m values and the pH profile of 5-reduction substantiated the predominant expression of the type 1 isoform. The apparent K_m values determined in tissue homogenates (either cortex tissue or white matter tissue) or in crude nuclear fractions or crude supernatants containing the microsomes varied between 0.99 µmol/L (mean) and 2.47 µmol/L (mean). When COS cells were transfected with human 5-reductase 1 cDNA, the apparent K_m value obtained for androstenedione as the substrate was 1.7 µmol/L (7), so our results are in accordance with the presence of 5-reductase 1 in the human brain.

The broad pH optimum (6.0–8.5) centered at pH 8 of 5-reduction indicates the predominant presence of 5-reductase 1 in human brain, because in cell extracts prepared from 293 cells transfected with the 5-reductase 2 cDNA, 5-reduction had a sharp pH optimum at pH 5.0, and in 293 cells transfected with the 5-reductase 1 cDNA, it showed a broad pH optimum between pH 6–8.5 (8).

One report on the subcellular distribution of 5-reductase demonstrated that 5-reductase enzyme activity was highest in the nuclear fraction in human fetal brain when androstenedione or progesterone was used as the substrate (10), whereas in another study, using testosterone as the substrate, the microsomal fraction displayed high activity rates in rat brain tissue preparations (25). However, using androstenedione as the substrate, we could not find obvious differences between the kinetic studies of 5-reductase in 1000 x g precipitates and the respective supernatants of cerebral cortex homogenates.

In our study the expression levels of 5-reductase 1 mRNA and 5-reductase activity did not differ significantly in cerebral cortex and subcortical white matter tissue. In the rat and mouse brain, however, 5-reductase activity appears to be highly concentrated in the subcortical white matter, whereas the cerebral cortex possesses a much lower activity (14). In other animal species (hamster, bull, pig, and monkey) and in brain tissue from a 61-yr-old woman, these researchers found the 5-reductase activity to be more concentrated in the cerebral cortex than in the white matter. The reasons for these discrepancies may refer to differences between the species.

The expression levels of 5-reductase 1 mRNA did not differ significantly between the sexes, nor could obvious sex differences concerning the kinetics of 5-reduction be detected. As studies on the mRNA expression in human brain are still lacking, only a comparison of our results with data obtained for the enzymatic activity of 5-reductase is possible. Our findings are consistent with previous studies in which no significant sex differences concerning 5-reductase activity were found in neural tissue of nonhuman primates during fetal development (26) or in rodents during postnatal development (27, 28).

An important finding of this study is the fact that 5-reductase 1 mRNA expression was significantly higher in cortex specimens from adults than in those from children as well as in tissue specimens from two postmenopausal women (aged 50 and 53 yr) not receiving sex hormone replacement therapy.

Similar results were reported on the expression of 5-reductase 1 in human skin tissue (9). At this point it is of interest that the researchers found a steep increase in 5-reductase 1 during puberty by immunoblotting. The data presented suggest that there is a low 5-reductase 1 mRNA expression in the brain during childhood, which is further induced during puberty, when serum sex steroid hormones increase.

The physiological significance of 5-reduction in the brain remains unclear. The brain is an important target for the effects of androgens; specific receptors have been identified in several regions of the brain, so androgens could effect a genomic response (5). Based on differences in substrate affinities and tissue distribution of the steroid 5-reductase isozymes observed in the rat, it has been concluded that type 2 may play an anabolic and type 1 a catabolic role in the metabolism of androgens and other steroid hormones (23). However, the physiological role of steroid 5-reductase isozymes in most tissues to date awaits elucidation.

The metabolism of androgens occurring in the human brain may subserve different physiological purposes at different times of life, and this may account for the differences in the expression levels of 5-reductase 1 in children and adults. On the other hand, the ubiquitous distribution of 5-reductase in animal brain suggests that the 5-reduced metabolites may be concerned with more general effects rather than exclusively with the regulation of specific brain mechanisms, such as controlling reproductive function (1, 2). In contrast to reproductive and neuroendocrine actions of steroids via intracellular receptors that regulate transcriptionally directed changes in protein synthesis, certain pregnanes and androstanes rapidly alter central nervous system excitability and produce behavioral effects (29). 5-Reduced metabolites of progesterone alter -aminobutyric acid_A receptor function, behavior, drug metabolism, and neural development (3, 29). Therefore, the effects of those metabolites may involve both genomic and nongenomic actions.

In conclusion, the present study is the first to determine 5-reductase isozyme expression in the human temporal lobe of children and adults. We found mRNA expression of 5-reductase 1 and 5-reductase activity in the temporal lobe of children and adults. In contrast, 5-reductase 2 mRNA was not detectable. The expression levels of 5-reductase 1 did not differ significantly between the sexes or between cerebral cortex and subcortical white matter tissue, but they were significantly lower in children than in adults (P < 0.005). Many questions regarding the biological role of 5-reductase in the human brain are still unanswered, and further efforts are required to delineate and understand the physiological role of 5-reductase activity in the brain.

	Acknowledgments

 
We thank Dr. D. W. Russell, University of Texas Southwest Medical Center (Dallas, TX), for the human 5-reductase cDNAs; Prof. M. Nuri, Department of Urology, Waldkrankenhaus Bonn (Bonn, Germany), for the supply of prostate tissue; and Dr. M. Wolff, Department of Surgery, University of Bonn (Bonn, Germany), for the supply of liver tissue.

	Footnotes

 
¹ This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Kl 524/4–1). Presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, 1996 (Abstract P3-474).

Received February 4, 1998.

Revised June 12, 1998.

Accepted June 18, 1998.

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