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Department of Reproductive Medicine, University of California-San Diego School of Medicine, La Jolla, California 92093-0633
Address all correspondence and requests for reprints to: Dr. Ismail H. Zwain, Department of Reproductive Medicine, University of California-San Diego School of Medicine, La Jolla, California 92093-0633. E-mail: izwain{at}ucsd.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (P450c17),
the key steroidogenic enzyme for androgen synthesis, and evaluate
DHEA production by highly purified astrocytes,
oligodendrocytes, and neurons. Using RT-PCR, we have demonstrated for
the first time that astrocytes and neurons in the cerebral cortex of
neonatal rat brain express P450c17. The presence of P450c17 in
astrocytes and neurons was supported by the ability of these cells to
metabolize pregnenolone to DHEA in a dose-dependent manner
as determined by RIA. These data were further confirmed by production
of androstenedione by astrocytes using progesterone as a
substrate. However, cortical neurons express a low transcript of
P450c17 messenger RNA and produce low levels of DHEA and
androstenedione compared with astrocytes. Oligodendrocytes neither
express the messenger RNA nor produce DHEA. The production
of DHEA by astrocytes is not limited to cerebral cortex,
as hypothalamic astrocytes produce DHEA at a level 3 times
higher than that produced by cortical astrocytes. Cortical and
hypothalamic astrocytes also have the capacity to metabolize
DHEA to testosterone and estradiol in a dose-dependent
manner. However, hypothalamic astrocytes were 3 times more active than
cortical astrocytes in the metabolism of DHEA to
estradiol. In conclusion, our data presented evidence that astrocytes
and neurons express P450c17 and synthesize DHEA from
pregnenolone. Astrocytes also have the capacity to metabolize
DHEA into sex steroid hormones. These data suggest that as
in gonads and adrenal, DHEA is biosynthesized in the brain
by a P450c17-dependent mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In gonads and adrenal gland, P450c17 enzyme is responsible for the conversion of pregnenolone (P5) into DHEA (5, 6). However, the presence of this enzyme in brain is a matter of controversy. P450c17 messenger RNA (mRNA) was detected in rat and mouse embryonic brain tissues (7) and in whole brain and cerebral cortical tissues of young adult rat brains (8, 9) using RT-PCR. In contrast to these studies, neither P450c17 protein (10), mRNA (11), nor activity (12) was detected in adult rat brain tissues. Moreover, mixed glial cells and astrocytes from rat and mouse embryo brains in cultures did not express P450c17 mRNA, as determined by RT-PCR (11), and were unable to metabolize radioactive P5 to DHEA, as determined by TLC/HPLC (12). Thus, the cellular origin of DHEA and the mechanism of its biosynthesis in brain remain an open question.
Evidence accumulated during the last years documented that macrophages
are involved in the regulation of steroidogenesis in gonads (for
review, see Ref. 13). These cells produce abundant quantities of nitric
oxide (NO), tumor necrosis factor- (TNF), and interleukin-1ß
(IL-1ß), which at low concentrations are able to inhibit androgen
production (14, 15, 16, 17, 18, 19) in ovary and testis via inhibition of P450c17 gene
expression (14, 15, 16, 17, 18). Residual macrophages in thecal-interstitial cell
cultures have been reported to decrease androgen production by these
cells (14, 15, 16, 17, 18, 19, 20).
Microglia cells (macrophages of brain) are also known as the main
sources for NO, TNF, and IL-1ß in the brain (21, 22, 23). These cells
were found to be the major contaminant cell type (5%) in primary glial
cell cultures (24) and could persist more than 4 months in culture
(25). It, therefore, is possible that the presence of contaminant
microglia cells in glial cell cultures could result in inhibition of
gene expression and biosynthesis of P450c17 enzyme and make its
detection difficult. This may explain the unsuccessful attempts (11, 12) to identify P450c17 mRNA and its activity in vitro. We,
therefore, attempted to determine the gene expression of P450c17 and
biosynthesis of DHEA in the brain using highly purified
microglia-free astrocytes, oligodendrocytes, and neurons. We
demonstrated that astrocytes express P450c17, produce
DHEA, and are able to metabolize this hormone to
testosterone (T) and estradiol (E2). Neurons
express low levels of P450c17 mRNA and produce low concentrations of
DHEA, whereas oligodendrocytes neither express P450c17 nor
produce DHEA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The microglia-free astrocytes and oligodendrocytes were extensively washed with serum-free medium (SFM) and cultured for 48 h in SFM with various treatments.
Isolation and culture of neurons
Neuronal culture was performed as previously described by Hertz
et al. (31) with modification. Briefly, cerebral cortical
and hypothalamic tissues from neonatal rat brains was mechanically
dispersed, and cells were cultured in medium containing 5% horse serum
for 3 days at 37 C in culture plates precoated with
poly-L-lysine substrates. Cell cultures were then exposed
for 24 h to cytosine arabinoside (40 µM) to
eliminate the nonneuronal cells, including microglia and macroglia
cells (astrocytes and oligodendrocytes). To remove any possible
remaining contaminant microglia, neurons were successively plated three
times with systematic changing of culture plates as described above.
Neurons were then cultured in precoated plates in SFM. After 48 h
in culture, neurons were washed and cultured for an additional 48
h in SFM with various treatments. The purity of the neurons was 99%,
as judged by immunocytochemical analysis of neurofilment protein
(Sigma Chemical Co., St. Louis, MO), a specific protein
marker for neurons. No microglia cells were detected in neuronal cell
cultures, as determined by immunocytochemical analysis of Leu M5.
The purity of isolated neurons, glial cells, astrocytes, and oligodendrocytes was analyzed using cells from three different experiments. Before treatment of cells with steroids, six-well culture plates containing astrocytes, oligodendrocytes, and neurons (one plate of each cell type) from each experiment were randomly selected for assessment of cell purity by immunocytochemical analysis. The purity of the isolated cells in the three experiments was 99%.
Preparation of samples for quantitative analysis of steroids and
P450c17 mRNA
At the conclusion of culture times, astrocytes,
oligodendrocytes, and neurons were removed by mild trypsinization, and
cell viability was determined by trypan blue staining. Media from
astrocyte, oligodendrocyte, and neuron cultures were collected,
centrifuged, and stored at -20 C until analysis of steroid
concentrations using a RIA kit (Diagnostic System Laboratories, Inc.,
Webster, TX). The minimum detection limit of the DHEA
assay was 9 pg/ml. The intra- and interassay variations of the assay
were 3.2% and 6.4%, respectively. The cross-reactivity of the
DHEA antibody with other steroids was determined by the
manufacturer of the RIA kit. This antibody was mainly shown to
cross-react with isoandrosterone (0.733%), androstenedione
(A4; 0.460%), 5-androstane-3,17-dione (0.240%),
11-deoxycortisol (0.061%), progesterone (P4;
0.045%), androsterone (0.035%), and T (0.028%).
Total RNA from cultured cells was isolated using the guanidine phenol-chloroform extraction method, treated with deoxyribonuclease to remove contaminant DNA, and stored at -70 C until analysis of steroidogenic enzyme gene expression by RT-PCR. The integrity of the RNA was demonstrated by analysis of ribosomal RNAs using ethidium bromide staining.
RT-PCR
RT-PCR analysis was performed as previously described by Zwain
et al. (27). Briefly, total RNA was reverse transcribed
using Moloney murine leukemia virus reverse transcriptase
(Perkin-Elmer, Branchburg, NJ) in the presence of the
oligo(deoxythymidine) primer. One tenth of the RT reaction was used as
a template for amplification by PCR using AmpliTaq Gold DNA polymerase
(Perkin-Elmer). The specific sense and antisense oligonucleotide
primers used in amplification of P450c17 complementary DNA (cDNA) were
prepared as previously described by Stromstedt and Waterman (14). PCR
products were resolved on 2% agarose gel, stained with ethidium
bromide, and visualized under UV light. The identities of the PCR
products were further confirmed by restriction digestion and Southern
blot analyses. RNA from rat ovary and testis was also extracted and
used as experimental positive controls for RT-PCR analysis. A negative
control was included where reverse transcriptase was omitted in the RT
reaction.
Southern analysis
Southern blot analysis of P450c17 was performed as previously
described by Zwain et al. (27). Briefly, PCR products were
fractionated on 2% agarose gel, denatured, transferred onto Nytran
nylon membranes (Schleicher & Schuell, Inc., Keene, NH),
and immobilized by UV cross-linking. Membranes (blots) were
prehybridized for 4 h at 37 C in the prehybridization buffer,
which consisted of 6 x SSC (standard saline citrate) containing
50% deionized formamide, 10% dextran sulfate, 1% SDS, and 100
µg/ml sheared and denatured salmon sperm DNA. Blots were then
hybridized for 18 h at 37 C in the prehybridization buffer
containing 2 x 106 cpm/ml of
-32P-labeled specific P450c17 internal oligonucleotide
probe, prepared as previously described by Stromstedt and Waterman (9).
Membranes were washed and exposed to x-ray film at -70 C for 3–10
h.
Treatment of astrocytes with antisense oligonucleotide
To determine the specificity of metabolism of DHEA
by astrocytes, the biosynthesis of cytochrome P450 aromatase enzymes
(P450arom) was inhibited by treatment of astrocytes with antisense
oligonucleotide to P450arom cDNA using a transfection kit from
Life Technologies (Gaithersburg, MD). A 20-mer
phosphorthioate antisense and a sense oligonucleotide overlapping the
initiation codon of P450arom cDNA were designed and HPLC purified as
previously described by Ackermann et al. (32). The sense
oligonucleotide was used as a negative experimental control. Astrocytes
were seeded in 12-well plates and cultured for 48 h at 37 C in the
presence and absence of the antisense (1 and 10 µM) or
sense (10 µM) oligonucleotides. At the conclusion of
culture time, media were harvested, centrifuged, and stored at -20 C
until analysis of the E2 concentration by RIA. To
determine whether the antisense oligonucleotide has real impact on
P450arom, RNA from treated and untreated astrocytes was extracted and
analyzed for P450arom gene expression by RT-PCR as described above. The
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
cytotoxcity assay was used to determine the viability of astrocytes
treated with the antisense and sense oligonucleotides as previously
described by Schlingeniepen and Klinger (33). The
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay
kit was purchased from Sigma Chemical Co. (St. Louis, MO).
Oligonucleotides had no effect on the viability of astrocytes compared
with the controls where cells were cultured alone.
Statistical analysis
Student’s unpaired t test was used when only two
individual groups were compared. For multiple comparison, data were
analyzed by one-way ANOVA with post-hoc Bonferroni test
using the Statistical Analysis System (SAS Institute, Cary, NC;
StatView software). Results are expressed as the mean ±
SD of three replicate cultures. The statistical
analysis was performed for three independent experiments and gave the
same results. P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lane 1) and neurons (Fig. 1A, lane 3), but not oligodendrocytes (Fig. 1A, lane 2), isolated from
neonatal rat brains express P450c17 mRNA. The mRNA level of P450c17 in
the cortical neurons was extremely low compared with that in astrocytes
(Fig. 1A, lane 3 vs. lane 1). These data were further
confirmed by RT-PCR/Southern blot analysis using a specific P450c17
oligonucleotide probe (Fig. 1B, lanes 1–3). The restriction digestion
enzyme analysis of PCR products has been used to confirm the identity
of P450c17 in astrocytes and neurons. The PCR products from astrocytes
and neurons were reamplified and digested with the restriction enzyme,
NCO1. Two fragments of the expected sizes were generated in samples
from astrocytes and neurons (data not shown). These DNA fragments were
identical in size to fragments from adult rat ovary and testis.
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(A and C, left panel),
culture of cerebral cortical astrocytes and neurons for 48 h in
the presence of increasing concentrations of P5
resulted in a dose-dependent increase in DHEA production.
At a concentration of 10-6 M
P5, DHEA production by cortical
astrocytes was 10 times higher than that by cortical neurons.
Oligodendrocytes were not able to convert P5 into
DHEA (Fig. 2B, left panel). These data were
further confirmed by the ability of astrocytes and neurons, but not
oligodendrocytes, to convert P4 into
A4 in a dose-dependent fashion (Fig. 2, A–C,
right panel).
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). At a
concentration of 10 µM ketoconazole, DHEA
production by cortical astrocytes was inhibited by 84% compared with
that in the controls where cells were cultured with only
P5.
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). At a concentration of
10-6 M P5,
DHEA production by hypothalamic astrocytes was 3 times
higher than that by cortical astrocytes (Fig. 4).
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, purified and unpurified astrocytes
converted P5 into DHEA in a
dose-dependent manner. However, DHEA production by
astrocytes containing contaminant microglia cells was extremely low
compared with that by microglia-free astrocytes.
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) and E2 (Fig. 6B) levels in the cultured medium. However, the production of
E2 by hypothalamic astrocytes was higher than
that by cortical astrocytes, indicating that hypothalamic astrocytes
are more active than cortical astrocytes in aromatization of androgen
to estrogen.
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). The
effect of trilostane on 3ßHSD activity in astrocytes was further
confirmed by the ability of trilostane to inhibit the conversion of
P5 to P4 (Fig. 7B).
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). At a dose of 10 µM, the
antisense oligonucleotide inhibited the conversion of T
(10-7 M) to E2 by
50-fold compared with that in controls where astrocytes cultured alone
or with 10 µM sense oligonucleotide (Fig. 8). To confirm
the specificity of the antisense oligonucleotide action,
semiquantitative RT-PCR analysis using ß-actin as an internal
standard housekeeping gene was employed to assess the effects of the
oligonucleotide on the mRNA level of P450arom in astrocytes. Treatment
of cells with the oligonucleotide resulted in a dose-dependent
inhibition of P450arom mRNA, whereas the sense oligonucleotide was
without effect (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Identification of P450c17 mRNA in astrocytes and neurons coupled with the ability of these cells to convert P5 to DHEA reveals de novo biosynthesis of DHEA in brain via a P450c17-dependent mechanism and strongly supports the idea that DHEA is a neurosteroid. However, a hypothetical biochemical pathway for DHEA formation in brain was also suggested depending on the findings that treatment of brain tissues (43) and brain glial tumor cell lines (C6 rat glioma cells) (44) with various chemicals, especially FeSO4, liberated P5 and DHEA. The P450c17 inhibitor, SU-10603, did not reverse FeSO4-induced DHEA formation in C6 cells, suggesting that FeSO4 effect is mediated via a P450c17-independent mechanism (44). However, our data did not exclude the possibility that biosynthesis of DHEA in brain is also induced by another unknown enzyme(s).
The discrepancy between our data and those reporting the inability of
mixed glial cells and astrocytes in cultures to metabolize
P5 to DHEA (12) and express P450c17
(11) may be attributed to the purity of cells, particularly to the
presence of contaminant microglia cells in the cultures. In this study
we attempted to eradicate microglia cells from glial cell cultures by
treating cells with L-leucine methyl-ester, followed by
successive plating of cells with systematic changing of culture flasks.
By combining these successive purification methods, no cell expressing
microglia/macrophage-specific protein marker could be detected.
However, no effort was made by other studies (11, 12) to remove
microglia from cell cultures. Residual microglia cells was reported in
the primary astrocyte cultures (12). Our data presented evidence that
the presence of microglia cells in astrocyte cell cultures resulted in
a dramatic inhibition of the conversion of P5 to
DHEA. However, the mechanism of microglia action is not
known. In gonads, NO, TNF, and IL-1ß are mediated gonadal
macrophages-induced inhibition of androgen production by Leydig and
thecal-interstitial cells (14, 15, 16, 17, 18, 19) via inhibition of P450c17 gene
expression (14, 15, 16, 17, 18). As microglia cells secrete high concentrations of
NO, TNF, and IL-1ß in brain, it is possible that these factors may
also mediate the microglia effect in inhibition of DHEA
biosynthesis in astrocytes. However, further studies are required to
determine the role of these macrophage/microglia cell-secretory factors
in the regulation of steroidogenesis in astrocytes.
Brain tissues are able to metabolize DHEA to its
hydroxylated metabolites, 7-hydroxy-DHEA and
7ß-hydroxy-DHEA, leading to the formation of
androstenediol and androstenetriol. Astrocytes were reported to be
responsible for this metabolic activity in the brain (45). In the
present study we have demonstrated that astrocytes from hypothalamus
and cerebral cortex are also capable of metabolizing DHEA
to T and subsequently to E2. This process
requires the enzymatic activities of 3ßHSD, 17ßHSD, and P450arom
enzymes that are responsible for the conversion of DHEA to
A4, A4 to T, and T to
E2, respectively. The gene expressions and
activities of 3ßHSD (46), 17ßHSD (Zwain, I., and S. S. C.
Yen, unpublished data), and P450arom (27) have been demonstrated in
astrocytes of rat brains, supporting the ability of these cells to
metabolize DHEA to sex steroid hormones. The specificity
of the metabolism of DHEA by astrocytes was confirmed by
the ability of trilostane and antisense oligonucleotide to P450arom
cDNA to inhibit the metabolism of DHEA to androgen and
androgen to estrogen, respectively.
Hypothalamic astrocytes appear to be more active than cortical astrocytes in the metabolism of DHEA to estrogen, as reflected by the accumulation of E2 in the cultured medium. These data are consistent with studies reporting high P450arom activity in hypothalamic tissues compared with other regions of rat brain (47). The high rates of production and metabolism of DHEA by hypothalamic astrocytes suggest that this hormone may be involved in the regulation of hypothalamic neuronal function, particularly GnRH neurons, whether directly or indirectly through its metabolite, E2. However, further studies are required to address this issue.
Although DHEA sulfate (DHEAS) was abundantly present in brain tissues, DHEA sulfotransferase (D-Stase), the enzyme responsible for sulfonation of DHEA, was reported to be absent in whole human and rat brain tissues (48). The absence of D-STase protein (49) and its mRNA (50) was also reported in human brain tissues using immunohistochemical and Northern blot analyses, respectively. However, a low activity of D-STase was recently detected in several regions of brain, with the highest level in the hypothalamus and pons (51). The question remains to be answered whether this low activity of D-STase is responsible for the formation of DHEAS at high concentrations in the brain (51). Thus, biosynthesis of DHEAS in the brain remains an possibility. However, DHEAS was reported to be produced by human adrenal tumor tissues from P5 sulfate (P5-S) via a P450c17-dependent mechanism (52). As P5-S is produced in high concentrations in brain, it is therefore possible that DHEAS is synthesized in the brain via the same mechanism. Further investigations are required to validate this suggestion using purified microglia-free astrocytes and neurons.
The exact role and significance of the biosynthesis of
DHEA in brain are not yet known. However, several
functional activities have been reported for DHEA in the
central nervous system, being an inhibitor of the 
-aminobutyric
acidA receptor (53) and an activator of
N-methyl-D-aspartate (54) and 
-receptor (54).
DHEA was also shown to increase memory and learning of
adult rats (55). This effect may be explained by the ability of
DHEA to increase neuronal communication in brain by
increasing the extension of neuronal processes, enhancing the
connection between isolated neurons, and increasing neuronal and glial
survival and differentiation (55, 56). An antiaggression effect for
DHEA was also reported; administration of
DHEA to castrated male mice resulted in a decrease in the
aggressive behavior of these animals toward lactating intruders (57).
In a recent study, DHEA and DHEAS have also been shown to
protect embryonic rat hippocampal neurons against excitatory amino
acid-induced neurotoxicity, suggesting that these neurosteroids may be
used as neuroprotective agents to reduce risk of age-related
neurodegenerative disorders (58). A beneficial effect was reported for
DHEA in reducing the risk of Alzheimer’s disease (59). It
has not yet been determined whether this effect is mediated by
DHEA itself or by its metabolite,
E2, which has been demonstrated to decrease the
risk of senile dementia associated with Alzheimer’s disease in
premenopausal women and to improve their cognitive performance (60). A
remarkable correlation between blood DHEA/DHEAS and a
feeling of well-being has also been reported by clinical studies of
people 65 yr of age and older (61). The de novo biosynthesis
of DHEA in brain, as demonstrated by the present study,
may reveal a crucial role for this steroid hormone in the regulation of
neuronal function in the central nervous system.
In conclusion, these results demonstrated for the first time that hypothalamic and cortical astrocytes in vitro express P450c17 steroidogenic enzyme and are able to synthesize and secrete DHEA and metabolize this hormone to testosterone and estradiol. Cortical neurons in vitro are also able to express a low level of P450c17 mRNA and produce a small amount of DHEA. However, it is not known whether astrocytes and neurons in vivo have the same capacity to produce DHEA as they do in vitro or whether these cells possess adequate enzymatic activity to metabolize DHEA to testosterone and estradiol in vivo. Further studies are required to address these issues. The data of the present study suggest that, as in gonads and adrenal, the P450c17 enzyme is responsible for the biosynthesis of DHEA in the brain.
Received July 13, 1998.
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References |
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