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Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism
Neurochemistry International 58 (2011) 620–624
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Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism A˚sa Fex Svenningsen a,1, Grzegorz Wicher a,b, Johan Lundqvist b, Hanna Pettersson b, Mikael Corell a, Maria Norlin b,* a b
Departments of Neuroscience, University of Uppsala, Sweden Department of Pharmaceutical Biosciences, University of Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 November 2010 Received in revised form 17 December 2010 Accepted 20 January 2011 Available online 12 February 2011
The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor b, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Steroid Hydroxylation Neuroprotection Glia Primary culture
1. Introduction The steroid-hydroxylating enzyme CYP7B1 is involved in metabolism of several physiological steroids in the CNS and elsewhere (Rose et al., 1997; Martin et al., 2001; Pettersson et al., 2008). This enzyme has been associated with a number of processes, including neuronal viability, endocrine signaling, cholesterol homeostasis and effects on memory and cognition (Rose et al., 1997; Jellinck et al., 2001; Yau et al., 2003; Pettersson et al., 2008; Schu¨le et al., 2009). Altered CYP7B1 levels and/or function have been linked to inflammation, malignancy and neurodegenerative conditions. Although expressed in many tissues in humans and animals,
Abbreviations: CYP, cytochrome P450; DHEA, dehydroepiandrosterone; DMEM, Dulbeccos’s modified Eagle’s medium; DPN, diarylpropionitrile; ER, estrogen receptor; PPT, propylpyrazoletriol; TLC, thin layer chromatography. * Corresponding author at: Department of Pharmaceutical Biosciences, Division of Biochemistry, University of Uppsala Box 578, S-751 23 Uppsala, Sweden. Tel.: +46 18 471 4331; fax: +46 18 558778. E-mail address: [email protected] (M. Norlin). 1 Present address: Institute of Molecular Medicine, Department of Neurobiology Research, University of Southern Denmark, Odense, Denmark. 0197-0186/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.01.024
CYP7B1 was first isolated from rodent brain (Rose et al., 1997). Wellknown substrates for CYP7B1 include the neurosteroids dehydroepiandrosterone (DHEA) and pregnenolone, which are formed locally in the CNS and are believed to exert a number of actions, including effects on neuronal development and modulation of NMDA and GABA receptor functions (Charalampopoulos et al., 2006; Melcangi and Panzica, 2006; Maninger et al., 2009). Metabolism of DHEA and related steroids varies in different tissues (Akwa et al., 1993; Jellinck et al., 2001; Tang et al., 2006). In reproductive organs, where DHEA is an important sex hormone precursor, its metabolism mainly leads to the formation of estradiol and testosterone. CYP7B1-mediated metabolism of DHEA, which appears to be quantitatively important in CNS cells, results in the formation of hydroxyderivatives with still largely unknown functions. Metabolism of CNS steroids also may be different depending on brain region and/or cell type. In cells of rat hippocampus, CYP7B1-mediated hydroxylation is reported to be the predominant pathway for DHEA metabolism (Jellinck et al., 2001). Other metabolites that can be formed from DHEA in the CNS include DHEA-sulfate and androstenedione. Cells of the nervous system consist of neurons and glial cells (Fex Svenningsen et al., 2003; Jessen, 2004; Dhandapani and Brann,
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2007). The main glial cell types in the CNS are oligodendrocytes and astrocytes. Interaction between glial cells and neurons is essential for normal CNS function. Among several other actions, glial cells play an important role for maintenance of appropriate levels of neurotransmitters and other compounds in the neuronal environment (Jessen, 2004). Astrocytes, which express a number of steroidogenic enzymes, are important for brain neurosteroidogenesis (Zwain and Yen, 1999). Pathways involving astrocytes have also been proposed to affect neuronal survival (Dhandapani and Brann, 2007). Previous reports by us and other investigators have identified CYP7B1 as a target for estrogen regulation in cells of kidney and liver (Tang et al., 2006, 2008; Yamamoto et al., 2006). There is an increasing amount of data suggesting potential links between CYP7B1-mediated actions and functions of the CNS. For this reason we have investigated whether estrogen might have any effects on CYP7B1 also in the brain. In the present study we report strong suppression of CYP7B1 by estrogens in primary cultures of rat CNS cells, particularly in astrocytes. The results of the current investigation indicate that estrogens may increase the levels of DHEA, and possibly other neurosteroids in the CNS, via effects on CYP7B1-mediated metabolism. 2. Materials and methods 2.1. Materials Materials for cell culturing were obtained from Sigma–Aldrich (Stockholm, Sweden), BD Biosciences (Stockholm, Sweden) and Invitrogen (Stockholm, Sweden). Unlabeled steroids were purchased from Sigma-Aldrich (Stockholm, Sweden). Radiolabeled [1,2,6,7-3H(N)] dehydroepiandrosterone (94.5 Ci/mmol) and [4-14C]17b-estradiol (52 mCi/mmol) were obtained from Perkin Elmer (Upplands Va¨sby, Sweden). All remaining chemicals were of analytical grade and purchased from commercial sources. 2.2. Animals and cell cultures The study was approved by the regional ethics committee for research on animals in Uppsala (Sweden) and carried out in accordance with the policy of the Society for Neuroscience. The animals were obtained from Scanlab (Sollentuna, Sweden) or Taconic (Ry, Denmark). The aim concerning cell cultures was to test pure astrocyte cultures, cultures containing all types of glial cell present in the nervous system, and cultures which included neurons as well as glial cells. For this purpose we used three different types of cell cultures. The first types of cell cultures were made according to the method previously described by McCarthy and de Vellis (1980). In brief, cultures were prepared from the entire brain of newborn Sprague–Dawley rat pups (postnatal day 1 or 2, male and female) and placed in a 100 mm Petri dish with cold Leibovitz’s L-15 medium (Invitrogen, Stockholm, Sweden). Brain tissue was mechanically dissociated through a glass Pasteur pipette and passed through a 70 mm nylon cell strainer (BD, Stockholm, Sweden) to remove all large fragments. Dissociated cells were washed twice in cold L-15 and centrifuged for 10 min at 1000 rpm to remove debris. After centrifugation the cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Stockholm, Sweden) supplemented with 10% fetal calf serum, (FCS; Invitrogen, Stockholm, Sweden) 0.3% L-glutamine (Invitrogen, Stockholm, Sweden) and 1% penicillin–streptomycin (Sigma–Aldrich, Stockholm, Sweden). Cells were then plated at a density of 2 105 cells/cm3 on poly-L-lysinecoated 75 cm2 cell Falcon cell culture flasks (BD, Stockholm, Sweden) or in 12 or 24 well dishes. After 7–9 days the cultures flasks were placed in a shaker incubator (Innova 40, New Brunswick Scientific, Edison, NJ, USA) and shaken at 200 rpm with a 1.5-in stroke diameter at 37 8C for 18 h to rid the cultures of microglia and oligodendrocytes. Both these cell types detach during the procedure and are removed when the cell culture medium is aspirated. The adhering astrocytes were then washed with fresh medium, to remove all non-adhesive cells, scraped from the bottom of the flask, and diluted with fresh media. The astrocyte suspension was mechanically dissociated using a Pasteur pipette and again filtered through a 70 mm nylon cell strainer (BD Stockholm, Sweden). A total cell count determining the number of astrocytes was done as described above. The astrocytes were plated on poly-L-lysine coated 12- or 24-well dishes at a density of 2 105 cells per/cm3 and left to grow to confluency for one week. The cell cultures containing all types of glia, that were grown in 12 or 24 well dishes, were processed when reaching confluency, after 7–9 days. The third type of cultures, containing all types of cells of the developing nervous system, including neurons, was made as described previously (Fex Svenningsen et al., 2003). Primary cerebellar cell cultures were prepared from Sprague–Dawley
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rat embryos, on day 17 (E17). Pregnant female Sprague–Dawley rats were sacrificed by CO2. The uterus containing the embryos was removed from the adult rat and placed in a 100 mm Petri dish with sterile cold Leibovitz’s (L-15) medium (Invitrogen, Stockholm, Sweden). The embryos were decapitated and the brain was removed from the cranium. The cerebellum was then removed from the back of the brain. The meninges were removed and the cerebellar tissue was transferred to a new dish containing sterile L-15 medium and kept on ice. The tissue was mechanically dissociated through a glass Pasteur pipette and filtered through a 70 mm nylon cell strainer (BD Stockholm, Sweden) to remove remaining cell clusters. Dissociated cells were washed in L-15 and centrifuged at 1000 rpm to remove debris. The cells were re-dissociated in Neurobasal cell culturing medium supplemented with 1% penicillin/streptomycin, 2% B27 (Invitrogen, Stockholm, Sweden), and 0.3% glutamine. The total and viable cell counts were determined by direct microscopic counting, using trypan blue (Sigma–Aldrich, Stockholm, Sweden) staining in a Bu¨rker chamber. The cultures were plated in 12 or 24 well dishes and left to grow for two weeks to reach a stage similar to the cells prepared from newborn rat pups. 2.3. Assay of enzymatic activities The cells were cultured in DMEM supplemented with 10% (v/v) fetal calf serum on 12- or 24-well tissue culture plates. Confluent CNS co-cultures or astrocyte cultures were incubated in the presence or absence of 17b-estradiol, estrone, propylpyrazoletriol or diarylpropionitrile (0.01–1 mM) for 4–24 h and 7ahydroxylation of DHEA was assayed by HPLC as previously described (Tang et al., 2006; Pettersson et al., 2008). The cell cultures were incubated for 6 h with 3 H-labeled DHEA (10 mM, 0.5 mCi), dissolved in DMSO, followed by extraction of the cell media with ethyl acetate. The organic phase was collected, evaporated with N2, and subjected to HPLC on a 125 mm 4 mm LiChrosphere RP 18 column (5 mm; Merck) using methanol/water as the mobile phase. Elution of labeled steroids was monitored by a Radiomatic 150TR Flow Scintillation Analyzer (Hewlett-Packard, Stockholm, Sweden). The elution system consisted of methanol/water (50:50, v/v) for 10 min followed by a linear gradient of 50–100% methanol for the next 10 min and finally 100% methanol for 5 min. The 7a-hydroxymetabolite was found almost exclusively in the cell media, similarly to the results in our previously reported work using other cell types (Norlin et al., 2000). The potential metabolism of estradiol in astrocyte cultures was assayed by incubation with 14C-labeled 17b-estradiol (1 mM, 0.1 mCi) for 24 h and analysis of the organic phase by silica gel thin layer chromatography (TLC) with chloroform/ ethyl acetate/acetone 60:20:10 (v/v/v) as the mobile phase (Tang et al., 2006). Cell culture samples harvested immediately after addition of estradiol, corresponding to an incubation time of 0 h, were used as controls. Unlabeled estradiol, estrone and 2hydroxyestradiol were used as references and developed together with the radiolabeled samples. The TLC-plate was scanned for localization of the radioactive products, using a Berthold Tracemaster 20 TLC scanner (Berthold/Frieske GmbH, Karlsruhe-Durlach, Germany), followed by exposure to iodine vapors (o/n) to visualize the unlabeled steroids. The retention times of reference compounds were compared with those of the sample plate. 2.4. Analysis of mRNA levels Quantitation of CYP7B1 mRNA in astrocytes, treated with estradiol, was performed by real time RT-PCR. RNA was isolated from cell cultures using an RNeasy Mini kit (Qiagen, Sollentuna, Sweden) and reversed transcribed to cDNA by a Reverse Transcription System (Promega Biotech AB, Nacka, Sweden). The real time PCR analysis was performed with the iQ SYBR Green Supermix (Bio-Rad, Stockholm, Sweden) using an iQ5 Real-Time PCR Detection System (Bio-Rad, Stockholm, Sweden) in accordance with the manufacturer’s recommendations (Lundqvist et al., 2010). The sequences of the primers used were: forward primer, 50 -TAGGACTAAACCACAGTCGC-3, and reverse primer, 50 -TGCAGCCTTATTCCGCTA-30 (Hirayama et al., 2006). The final primer concentration was 0.2 mM and the amplification was performed using the following PCR conditions: 95 8C for 10 min followed by 35 cycles of 94 8C for 1 min, 62 8C for 1 min, and 72 8C for 1 min. Actin was used as the endogenous control. The relative mRNA expression levels were calculated according to the comparative CT (DDCT) method, comparing the Ct values of the samples of interest with the non-treated samples, while simultaneously normalizing both to an endogenous control (in this case actin). All real time RT-PCR data were normalized to the endogenous control. The relative mRNA expression was expressed as percent of the mRNA levels in samples treated with ethanol (vehicle). In some experiments, semiquantitative RT-PCR was used to analyze CYP7B1 mRNA in astrocytes. In these experiments, the PCR primers for CYP7B1 were those described previously (Martin et al., 2001). Rat GAPDH was used as the internal control. 2.5. Other methods Analysis of statistical significance was performed using ANOVA in Microsoft Excel. p Values <0.05 were considered statistically significant. Protein concentrations in cell homogenates were determined by the method of Lowry et al. (1951).
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3. Results 3.1. Estradiol suppresses the 7a-hydroxylation of DHEA in mixed CNS cultures Many studies have suggested important physiological and pharmacological roles for estrogens and other sex hormones in the CNS (Melcangi and Panzica, 2006; Dhandapani and Brann, 2007; Arnold and Beyer, 2009). Although several potential mechanisms for these effects have been proposed, the function(s) of estrogens in CNS cells remain unclear. In this study, we measured the effects of estradiol on CYP7B1-mediated metabolism of DHEA in primary rat CNS cultures. CYP7B1-mediated 7a-hydroxylation is one of the main pathways for metabolism of DHEA in the CNS (Akwa et al., 1993; Jellinck et al., 2001). Addition of estradiol to CNS co-cultures, containing neurons, astrocytes and oligodendrocytes, resulted in significant suppression of CYP7B1-mediated DHEA 7a-hydroxylation (Fig. 1). Estradiol suppressed CYP7B1-mediated metabolism of DHEA in rat fetal cerebellar co-cultures by 50% 13 (Fig. 1A). Similar results were obtained using neonatal rat glial cultures, implicating that this reaction may occur primarily in glial cells (Fig. 1B). 3.2. CYP7B1-mediated expression and activity in primary cultures of rat astrocytes From previously published studies with material from rat brain, it appears that CYP7B1, the enzyme responsible for DHEA 7ahydroxylation, is predominantly expressed in astrocytes (Akwa et al., 1993; Zhang et al., 1997). To verify this in our cultures, we assayed CYP7B1 mRNA in astrocytes obtained from the co-cultures [()TD$FIG]
of neonatal rat brain. As expected, this experiment showed high levels of CYP7B1 mRNA in astrocytes (data not shown). In addition, primary cultures of astrocytes alone were able to metabolize DHEA into its 7a-hydroxyderivative at a significant rate. The 7ahydroxylase activity towards DHEA in our experiments was 0.8 0.3 nmol/mg h. Astrocytes are considered to be important for steroidogenesis in the CNS. These cells have been reported to have a number of functions, including production of neuroactive substances and regulatory effects on neuronal survival (Paixa˜o and Klein, 2010; Zwain and Yen, 1999; Dhandapani and Brann, 2007). It has also been suggested that astrocytes may play an important role for estradiol-mediated neuroprotection (Dhandapani and Brann, 2007). 3.3. Estrogens suppress the 7a-hydroxylation of DHEA in primary astrocyte cultures To study regulatory effects on DHEA metabolism in cultures of astrocytes alone we treated neonatal primary cultures of astrocytes with estradiol prior to analysis of CYP7B1-mediated DHEA hydroxylation. As shown in Fig. 2, treatment with estradiol (1 mM) for 24 h strongly suppressed CYP7B1-mediated hydroxylase activity in astrocyte cultures (by 66% 8). A similar suppressive effect, although weaker, was found with shorter treatment times (4– 6 h). Lower concentrations of estradiol (10–100 nM) did not show consistent effects on CYP7B1 activity, in astrocytes or in mixed CNS cultures (data not shown). To examine whether the added estradiol remained in an unmetabolized form throughout the incubation period we assayed estradiol metabolism in astrocytes using thin layer chromatography. These experiments showed that 50–75% (varying in different experiments) of the estradiol remained unmetabolized after 24 h. Very little, if any, formation of 2- or 4-hydroxymetabolites from estradiol was observed under these conditions. The main metabolite formed from estradiol was a compound with the same retention time as estrone. We then carried out experiments with estrone, to study if this estrogen also would affect CYP7B1-mediated DHEA metabolism. Treatment of astrocytes with estrone significantly suppressed CYP7B1 hydroxylase activity (by 42% 13), although the effect was not quite as strong as that of estradiol (Fig. 2). 3.4. Diarylpropionitrile suppresses the 7a-hydroxylation of DHEA in primary astrocytes To study potential effects of synthetic estrogen receptor agonists, we also carried out experiments with propylpyrazoletriol
[()TD$FIG]
Fig. 1. Effect of treatment with estradiol (E2) (1 mM) on CYP7B1-mediated 7ahydroxylation of DHEA in (A) rat fetal cerebellar co-cultures and (B) neonatal rat glial cultures. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of 3–5 experiments. *Statistically significant difference compared to control (p < 0.01).
Fig. 2. Effect of treatment with 1 mM of estradiol (E2) or estrone on CYP7B1mediated 7a-hydroxylation of DHEA in primary cultures of rat astrocytes. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of 6–10 experiments. *Statistically significant difference compared to control (p < 0.01).
[()TD$FIG]
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Fig. 3. Effect of treatment with of propylpyrazoletriol (PPT) (1 mM), an agonist of ERa, or diarylpropionitrile (DPN) (1 mM), an agonist of ERb, on CYP7B1-mediated 7a-hydroxylation of DHEA in primary cultures of rat astrocytes. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of five experiments. *Statistically significant difference compared to control (p < 0.01).
(PPT), an agonist of ERa, and diarylpropionitrile (DPN), an agonist of ERb. Interestingly, addition of 1 mm of DPN significantly suppressed CYP7B1-mediated DHEA hydroxylation whereas PPT (in the same concentration) did not (Fig. 3). The suppressive effect of DPN on CYP7B1-mediated activity was in the same order of magnitude as that of estradiol. 3.5. Estradiol suppresses the CYP7B1 mRNA levels in primary astrocytes To obtain more information on the effects of estradiol in astrocytes, we measured CYP7B1 mRNA levels in rat primary cultures of such cells, in the presence or absence of estradiol, using real time RT-PCR. In accordance with the data from analysis of CYP7B1-mediated catalytic activity, treatment with estradiol significantly suppressed the CYP7B1 mRNA levels in astrocytes (Fig. 4). About 30% suppression (33% 20) of CYP7B1 mRNA by estradiol treatment was observed under the conditions employed. 4. Discussion Our study reports significant suppressive effects by estrogens on CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA levels in rat CNS cultures. CYP7B1-mediated catalysis is one of the main pathways for the metabolism of DHEA in the CNS and effects
[()TD$FIG]
Fig. 4. Effect of treatment with 1 mM of estradiol (E2) on the CYP7B1 mRNA levels in primary cultures of rat astrocytes. Treatment and analysis of mRNA levels were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of seven experiments. *Statistically significant difference compared to control (p < 0.01).
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on CYP7B1 should therefore strongly influence the levels of this neurosteroid (Akwa et al., 1993; Rose et al., 1997; Jellinck et al., 2001). DHEA, which is produced by astrocytes in vivo, has been implicated in a number of processes essential for CNS function, including modulation of the actions of several neurotransmitter receptors and activation of kinase signaling important for cellular viability (Charalampopoulos et al., 2006; Melcangi and Panzica, 2006; Maninger et al., 2009). Some of the many proposed functions for this steroid include effects on neuronal development, modulation of immune function and an important role for neuronal survival. Decreased levels of DHEA have been associated with neuronal dysfunction and neurodegeneration. Abnormal levels of DHEA and/or the sulfated form DHEA-sulfate have also been implicated in neuropsychiatric and neurodegenerative conditions (Maninger et al., 2009). Several previous studies have indicated estrogen-related effects in the CNS, in physiological as well as in pharmacological concentrations, and the potential of estrogen replacement therapy to decrease the risk of dementia has been discussed for many years (Vedder et al., 1999; Brann et al., 2007). Estrogen and estrogen receptor modulators are reported to exert neurotrophic and neuroprotective actions in humans as well as animals (Melcangi and Panzica, 2006; Brann et al., 2007; Arnold and Beyer, 2009). Estrogens have, for instance, been shown to protect neurons against oxidative stress. Furthermore, an increased risk for Alzheimer’s disease has been linked to single nucleotide polymorphisms (SNPs) in the CYP19A1 gene, which is necessary for estradiol production. A number of different mechanisms are reported to mediate estrogen action in the CNS, e.g. modulation of calcium flux, increased activation of survival proteins, altered glutamate release and several others (Melcangi and Panzica, 2006; Brann et al., 2007; Arnold and Beyer, 2009). From the current data it seems that estrogens may also affect the levels of DHEA in CNS cells. The role(s) of CYP7B1 in the CNS have not been fully elucidated. High CYP7B1 mRNA levels are reported in the hippocampus, the region considered most important for memory and learning (Rose et al., 1997; Yau et al., 2003). Interestingly, some patients with Alzheimer’s disease are reported to have altered levels of CYP7B1 and/or 7a-hydroxy-DHEA (Attal-Khemis et al., 1998; Yau et al., 2003). In addition, recent studies have linked a motor-neuron degenerative disease, hereditary spastic paraplegia, to mutations in the coding region of human CYP7B1 (Schu¨le et al., 2009). We recently reported that the Akt/PI3K (phosphoinositide 3-kinase) cascade, a signaling pathway important for cell survival, can affect CYP7B1 transcription, also suggesting a possible connection between CYP7B1 action and neuroprotective events (Tang et al., 2008). Several other steroids present in the CNS are metabolized by CYP7B1, including pregnenolone, which is the immediate precursor to progesterone (Rose et al., 1997). Both progesterone and pregnenolone are reported to have neuroprotective properties (Melcangi and Panzica, 2006). It is possible that regulatory effects on CYP7B1 activity and/or expression may affect the levels also of other steroids of importance for CNS function. From the results of this and previous studies, we conclude that CYP7B1 expression and activity is high in rat astrocytes (Akwa et al., 1993; Zhang et al., 1997). It has been reported that expression of human CYP7B1 is predominant in neurons (Trap et al., 2005), suggesting a difference in cellular localization for this enzyme between humans and rats. The present results indicate that CYP7B1-mediated metabolism and expression in rat brain is significantly suppressed by estrogens. These findings are different compared to previous data concerning estrogen-dependent regulation of CYP7B1 in kidney-
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and liver-derived cell lines, where estradiol upregulates the CYP7B1 gene, at least in the presence of estrogen receptors (Tang et al., 2006, 2008). This indicates that estrogen-mediated regulation of CYP7B1 action could be tissue-specific and/or involve different cellular mechanisms. In the present study we also observed suppressive effects on CYP7B1 by an ERb agonist. This finding suggests that ERb might be important for the effects observed, although other mechanisms, either transcriptional or post-transcriptional, cannot be excluded at the present stage. In summary, neuroprotective effects by estradiol and other estrogens are reported in several studies and have been proposed to involve effects on astrocytes. In view of the present data we propose that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the DHEA levels by suppression of its metabolism. Acknowledgements The present work was supported by grants from the Swedish Research Council Medicine, the Swedish Brain Foundation and Gyllenstiernska Krapperupsstiftelsen (Sweden). References Akwa, Y., Sananes, N., Gouezou, M., Robel, P., Baulieu, E.E., Le Goascogne, C., 1993. Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density. J. Cell Biol. 121, 135–143. Arnold, S., Beyer, C., 2009. Neuroprotection by estrogen in the brain: the mitochondrial compartment as presumed therapeutic target. J. Neurochem. 110, 1–11. Attal-Khemis, S., Dalmeyda, V., Michot, J.L., Roudier, M., Morfin, R., 1998. Increased total 7a-hydroxy-dehydroepiandrosterone in serum of patients with Alzheimer’s disease. J. Gerontol. A: Biol. Sci. Med. Sci. 53, B125–132. Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., Khan, M.M., 2007. Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72, 381–405. Charalampopoulos, I., Alexaki, V.I., Tsatsanis, C., Minas, V., Dermitzaki, E., Lasaridis, I., Vardouli, L., Stournaras, C., Margioris, A.N., Castanas, E., Gravanis, A., 2006. Neurosteroids as endogenous inhibitors of neuronal cell apoptosis in aging. Ann. N. Y. Acad. Sci. 1088, 139–152. Dhandapani, K.M., Brann, D.W., 2007. Role of astrocytes in estrogen-mediated neuroprotection. Exp. Gerontol. 42, 70–75. Fex Svenningsen, A˚., Shan, W.S., Colman, D.R., Pedraza, L., 2003. Rapid method for culturing embryonic neuron-glial cell cocultures. J. Neurosci. Res. 72, 565–573. Hirayama, T., Honda, A., Matsuzaki, Y., Miyazaki, T., Ikegami, T., Doy, M., Xu, G., Lea, M., Salen, G., 2006. Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol. Hepatology 44, 602–611. Jellinck, P.H., Lee, S.J., McEwen, B.S., 2001. Metabolism of dehydroepiandrosterone by rat hippocampal cells in culture: possible role of aromatization and 7hydroxylation in neuroprotection. J. Steroid Biochem. Mol. Biol. 78, 313–317.
Jessen, K.R., 2004. Glial cells. Int. J. Biochem. Cell. Biol. 36, 1861–1867. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Lundqvist, J., Norlin, M., Wikvall, K., 2010. 1a,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells. Biochim. Biophys. Acta 1801, 1056–1062. Maninger, N., Wolkowitz, O.M., Reus, V.I., Epel, E.S., Mellon, S.H., 2009. Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Front. Neuroendocrinol. 30, 65–91. Martin, C., Bean, R., Rose, K., Habib, F., Seckl, J., 2001. cyp7b1 catalyses the 7ahydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate. Biochem. J. 355, 509–515. McCarthy, K.D., de Vellis, J., 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890–902. Melcangi, R.C., Panzica, G.C., 2006. Neuroactive steroids: old players in a new game. Neuroscience 138, 733–739. Norlin, M., Andersson, U., Bjo¨rkhem, I., Wikvall, K., 2000. Oxysterol 7a-hydroxylase activity by cholesterol 7a-hydroxylase (CYP7A). J. Biol. Chem. 275, 34046– 34053. Paixa˜o, S., Klein, R., 2010. Neuron-astrocyte communication and synaptic plasticity. Curr. Opin. Neurobiol. 20, 466–473. Pettersson, H., Holmberg, L., Axelson, M., Norlin, M., 2008. CYP7B1-mediated metabolism of dehydroepiandrosterone and 5a-androstane-3b,17b-diol-potential role(s) for estrogen signaling. FEBS J. 275, 1778–1789. Rose, K.A., Stapleton, G., Dott, K., Kieny, M.P., Best, R., Schwarz, M., Russell, D.W., Bjo¨rkhem, I., Seckl, J., Lathe, R., 1997. Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7a-hydroxy dehydroepiandrosterone and 7a-hydroxy pregnenolone. Proc. Natl. Acad. Sci. U. S. A. 94, 4925–4930. Schu¨le, R., Brandt, E., Karle, K.N., Tsaousidou, M., Klebe, S., Klimpe, S., Auer-Grumbach, M., Crosby, A.H., Hubner, C.A., Schols, L., Deufel, T., Beetz, C., 2009. Analysis of CYP7B1 in non-consanguineous cases of hereditary spastic paraplegia. Neurogenetics 10, 97–104. Tang, W., Eggertsen, G., Chiang, J.Y., Norlin, M., 2006. Estrogen-mediated regulation of CYP7B1: a possible role for controlling DHEA levels in human tissues. J. Steroid Biochem. Mol. Biol. 100, 42–51. Tang, W., Pettersson, H., Norlin, M., 2008. Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling. J. Steroid Biochem. Mol. Biol. 112, 63–73. Trap, C., Nato, F., Chalbot, S., Kim, S.B., Lafaye, P., Morfin, R., 2005. Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization. J. Neuroimmunol. 159, 41–47. Vedder, H., Anthes, N., Stumm, G., Wurz, C., Behl, C., Krieg, J.C., 1999. Estrogen hormones reduce lipid peroxidation in cells and tissues of the central nervous system. J. Neurochem. 72, 2531–2538. Yamamoto, Y., Moore, R., Hess, H.A., Guo, G.L., Gonzalez, F.J., Korach, K.S., Maronpot, R.R., Negishi, M., 2006. Estrogen receptor a mediates 17a-ethynylestradiol causing hepatotoxicity. J. Biol. Chem. 281, 16625–16631. Yau, J.L., Rasmuson, S., Andrew, R., Graham, M., Noble, J., Olsson, T., Fuchs, E., Lathe, R., Seckl, J.R., 2003. Dehydroepiandrosterone 7-hydroxylase CYP7B: predominant expression in primate hippocampus and reduced expression in Alzheimer’s disease. Neuroscience 121, 307–314. Zhang, J., Akwa, Y., el-Etr, M., Baulieu, E.E., Sjo¨vall, J., 1997. Metabolism of 27-, 25and 24-hydroxycholesterol in rat glial cells and neurons. Biochem. J. 322 (Pt 1), 175–184. Zwain, I.H., Yen, S.S., 1999. Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology 140, 3843–3852.
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism A˚sa Fex Svenningsen a,1, Grzegorz Wicher a,b, Johan Lundqvist b, Hanna Pettersson b, Mikael Corell a, Maria Norlin b,* a b
Departments of Neuroscience, University of Uppsala, Sweden Department of Pharmaceutical Biosciences, University of Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 November 2010 Received in revised form 17 December 2010 Accepted 20 January 2011 Available online 12 February 2011
The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor b, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Steroid Hydroxylation Neuroprotection Glia Primary culture
1. Introduction The steroid-hydroxylating enzyme CYP7B1 is involved in metabolism of several physiological steroids in the CNS and elsewhere (Rose et al., 1997; Martin et al., 2001; Pettersson et al., 2008). This enzyme has been associated with a number of processes, including neuronal viability, endocrine signaling, cholesterol homeostasis and effects on memory and cognition (Rose et al., 1997; Jellinck et al., 2001; Yau et al., 2003; Pettersson et al., 2008; Schu¨le et al., 2009). Altered CYP7B1 levels and/or function have been linked to inflammation, malignancy and neurodegenerative conditions. Although expressed in many tissues in humans and animals,
Abbreviations: CYP, cytochrome P450; DHEA, dehydroepiandrosterone; DMEM, Dulbeccos’s modified Eagle’s medium; DPN, diarylpropionitrile; ER, estrogen receptor; PPT, propylpyrazoletriol; TLC, thin layer chromatography. * Corresponding author at: Department of Pharmaceutical Biosciences, Division of Biochemistry, University of Uppsala Box 578, S-751 23 Uppsala, Sweden. Tel.: +46 18 471 4331; fax: +46 18 558778. E-mail address: [email protected] (M. Norlin). 1 Present address: Institute of Molecular Medicine, Department of Neurobiology Research, University of Southern Denmark, Odense, Denmark. 0197-0186/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.01.024
CYP7B1 was first isolated from rodent brain (Rose et al., 1997). Wellknown substrates for CYP7B1 include the neurosteroids dehydroepiandrosterone (DHEA) and pregnenolone, which are formed locally in the CNS and are believed to exert a number of actions, including effects on neuronal development and modulation of NMDA and GABA receptor functions (Charalampopoulos et al., 2006; Melcangi and Panzica, 2006; Maninger et al., 2009). Metabolism of DHEA and related steroids varies in different tissues (Akwa et al., 1993; Jellinck et al., 2001; Tang et al., 2006). In reproductive organs, where DHEA is an important sex hormone precursor, its metabolism mainly leads to the formation of estradiol and testosterone. CYP7B1-mediated metabolism of DHEA, which appears to be quantitatively important in CNS cells, results in the formation of hydroxyderivatives with still largely unknown functions. Metabolism of CNS steroids also may be different depending on brain region and/or cell type. In cells of rat hippocampus, CYP7B1-mediated hydroxylation is reported to be the predominant pathway for DHEA metabolism (Jellinck et al., 2001). Other metabolites that can be formed from DHEA in the CNS include DHEA-sulfate and androstenedione. Cells of the nervous system consist of neurons and glial cells (Fex Svenningsen et al., 2003; Jessen, 2004; Dhandapani and Brann,
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2007). The main glial cell types in the CNS are oligodendrocytes and astrocytes. Interaction between glial cells and neurons is essential for normal CNS function. Among several other actions, glial cells play an important role for maintenance of appropriate levels of neurotransmitters and other compounds in the neuronal environment (Jessen, 2004). Astrocytes, which express a number of steroidogenic enzymes, are important for brain neurosteroidogenesis (Zwain and Yen, 1999). Pathways involving astrocytes have also been proposed to affect neuronal survival (Dhandapani and Brann, 2007). Previous reports by us and other investigators have identified CYP7B1 as a target for estrogen regulation in cells of kidney and liver (Tang et al., 2006, 2008; Yamamoto et al., 2006). There is an increasing amount of data suggesting potential links between CYP7B1-mediated actions and functions of the CNS. For this reason we have investigated whether estrogen might have any effects on CYP7B1 also in the brain. In the present study we report strong suppression of CYP7B1 by estrogens in primary cultures of rat CNS cells, particularly in astrocytes. The results of the current investigation indicate that estrogens may increase the levels of DHEA, and possibly other neurosteroids in the CNS, via effects on CYP7B1-mediated metabolism. 2. Materials and methods 2.1. Materials Materials for cell culturing were obtained from Sigma–Aldrich (Stockholm, Sweden), BD Biosciences (Stockholm, Sweden) and Invitrogen (Stockholm, Sweden). Unlabeled steroids were purchased from Sigma-Aldrich (Stockholm, Sweden). Radiolabeled [1,2,6,7-3H(N)] dehydroepiandrosterone (94.5 Ci/mmol) and [4-14C]17b-estradiol (52 mCi/mmol) were obtained from Perkin Elmer (Upplands Va¨sby, Sweden). All remaining chemicals were of analytical grade and purchased from commercial sources. 2.2. Animals and cell cultures The study was approved by the regional ethics committee for research on animals in Uppsala (Sweden) and carried out in accordance with the policy of the Society for Neuroscience. The animals were obtained from Scanlab (Sollentuna, Sweden) or Taconic (Ry, Denmark). The aim concerning cell cultures was to test pure astrocyte cultures, cultures containing all types of glial cell present in the nervous system, and cultures which included neurons as well as glial cells. For this purpose we used three different types of cell cultures. The first types of cell cultures were made according to the method previously described by McCarthy and de Vellis (1980). In brief, cultures were prepared from the entire brain of newborn Sprague–Dawley rat pups (postnatal day 1 or 2, male and female) and placed in a 100 mm Petri dish with cold Leibovitz’s L-15 medium (Invitrogen, Stockholm, Sweden). Brain tissue was mechanically dissociated through a glass Pasteur pipette and passed through a 70 mm nylon cell strainer (BD, Stockholm, Sweden) to remove all large fragments. Dissociated cells were washed twice in cold L-15 and centrifuged for 10 min at 1000 rpm to remove debris. After centrifugation the cells were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Stockholm, Sweden) supplemented with 10% fetal calf serum, (FCS; Invitrogen, Stockholm, Sweden) 0.3% L-glutamine (Invitrogen, Stockholm, Sweden) and 1% penicillin–streptomycin (Sigma–Aldrich, Stockholm, Sweden). Cells were then plated at a density of 2 105 cells/cm3 on poly-L-lysinecoated 75 cm2 cell Falcon cell culture flasks (BD, Stockholm, Sweden) or in 12 or 24 well dishes. After 7–9 days the cultures flasks were placed in a shaker incubator (Innova 40, New Brunswick Scientific, Edison, NJ, USA) and shaken at 200 rpm with a 1.5-in stroke diameter at 37 8C for 18 h to rid the cultures of microglia and oligodendrocytes. Both these cell types detach during the procedure and are removed when the cell culture medium is aspirated. The adhering astrocytes were then washed with fresh medium, to remove all non-adhesive cells, scraped from the bottom of the flask, and diluted with fresh media. The astrocyte suspension was mechanically dissociated using a Pasteur pipette and again filtered through a 70 mm nylon cell strainer (BD Stockholm, Sweden). A total cell count determining the number of astrocytes was done as described above. The astrocytes were plated on poly-L-lysine coated 12- or 24-well dishes at a density of 2 105 cells per/cm3 and left to grow to confluency for one week. The cell cultures containing all types of glia, that were grown in 12 or 24 well dishes, were processed when reaching confluency, after 7–9 days. The third type of cultures, containing all types of cells of the developing nervous system, including neurons, was made as described previously (Fex Svenningsen et al., 2003). Primary cerebellar cell cultures were prepared from Sprague–Dawley
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rat embryos, on day 17 (E17). Pregnant female Sprague–Dawley rats were sacrificed by CO2. The uterus containing the embryos was removed from the adult rat and placed in a 100 mm Petri dish with sterile cold Leibovitz’s (L-15) medium (Invitrogen, Stockholm, Sweden). The embryos were decapitated and the brain was removed from the cranium. The cerebellum was then removed from the back of the brain. The meninges were removed and the cerebellar tissue was transferred to a new dish containing sterile L-15 medium and kept on ice. The tissue was mechanically dissociated through a glass Pasteur pipette and filtered through a 70 mm nylon cell strainer (BD Stockholm, Sweden) to remove remaining cell clusters. Dissociated cells were washed in L-15 and centrifuged at 1000 rpm to remove debris. The cells were re-dissociated in Neurobasal cell culturing medium supplemented with 1% penicillin/streptomycin, 2% B27 (Invitrogen, Stockholm, Sweden), and 0.3% glutamine. The total and viable cell counts were determined by direct microscopic counting, using trypan blue (Sigma–Aldrich, Stockholm, Sweden) staining in a Bu¨rker chamber. The cultures were plated in 12 or 24 well dishes and left to grow for two weeks to reach a stage similar to the cells prepared from newborn rat pups. 2.3. Assay of enzymatic activities The cells were cultured in DMEM supplemented with 10% (v/v) fetal calf serum on 12- or 24-well tissue culture plates. Confluent CNS co-cultures or astrocyte cultures were incubated in the presence or absence of 17b-estradiol, estrone, propylpyrazoletriol or diarylpropionitrile (0.01–1 mM) for 4–24 h and 7ahydroxylation of DHEA was assayed by HPLC as previously described (Tang et al., 2006; Pettersson et al., 2008). The cell cultures were incubated for 6 h with 3 H-labeled DHEA (10 mM, 0.5 mCi), dissolved in DMSO, followed by extraction of the cell media with ethyl acetate. The organic phase was collected, evaporated with N2, and subjected to HPLC on a 125 mm 4 mm LiChrosphere RP 18 column (5 mm; Merck) using methanol/water as the mobile phase. Elution of labeled steroids was monitored by a Radiomatic 150TR Flow Scintillation Analyzer (Hewlett-Packard, Stockholm, Sweden). The elution system consisted of methanol/water (50:50, v/v) for 10 min followed by a linear gradient of 50–100% methanol for the next 10 min and finally 100% methanol for 5 min. The 7a-hydroxymetabolite was found almost exclusively in the cell media, similarly to the results in our previously reported work using other cell types (Norlin et al., 2000). The potential metabolism of estradiol in astrocyte cultures was assayed by incubation with 14C-labeled 17b-estradiol (1 mM, 0.1 mCi) for 24 h and analysis of the organic phase by silica gel thin layer chromatography (TLC) with chloroform/ ethyl acetate/acetone 60:20:10 (v/v/v) as the mobile phase (Tang et al., 2006). Cell culture samples harvested immediately after addition of estradiol, corresponding to an incubation time of 0 h, were used as controls. Unlabeled estradiol, estrone and 2hydroxyestradiol were used as references and developed together with the radiolabeled samples. The TLC-plate was scanned for localization of the radioactive products, using a Berthold Tracemaster 20 TLC scanner (Berthold/Frieske GmbH, Karlsruhe-Durlach, Germany), followed by exposure to iodine vapors (o/n) to visualize the unlabeled steroids. The retention times of reference compounds were compared with those of the sample plate. 2.4. Analysis of mRNA levels Quantitation of CYP7B1 mRNA in astrocytes, treated with estradiol, was performed by real time RT-PCR. RNA was isolated from cell cultures using an RNeasy Mini kit (Qiagen, Sollentuna, Sweden) and reversed transcribed to cDNA by a Reverse Transcription System (Promega Biotech AB, Nacka, Sweden). The real time PCR analysis was performed with the iQ SYBR Green Supermix (Bio-Rad, Stockholm, Sweden) using an iQ5 Real-Time PCR Detection System (Bio-Rad, Stockholm, Sweden) in accordance with the manufacturer’s recommendations (Lundqvist et al., 2010). The sequences of the primers used were: forward primer, 50 -TAGGACTAAACCACAGTCGC-3, and reverse primer, 50 -TGCAGCCTTATTCCGCTA-30 (Hirayama et al., 2006). The final primer concentration was 0.2 mM and the amplification was performed using the following PCR conditions: 95 8C for 10 min followed by 35 cycles of 94 8C for 1 min, 62 8C for 1 min, and 72 8C for 1 min. Actin was used as the endogenous control. The relative mRNA expression levels were calculated according to the comparative CT (DDCT) method, comparing the Ct values of the samples of interest with the non-treated samples, while simultaneously normalizing both to an endogenous control (in this case actin). All real time RT-PCR data were normalized to the endogenous control. The relative mRNA expression was expressed as percent of the mRNA levels in samples treated with ethanol (vehicle). In some experiments, semiquantitative RT-PCR was used to analyze CYP7B1 mRNA in astrocytes. In these experiments, the PCR primers for CYP7B1 were those described previously (Martin et al., 2001). Rat GAPDH was used as the internal control. 2.5. Other methods Analysis of statistical significance was performed using ANOVA in Microsoft Excel. p Values <0.05 were considered statistically significant. Protein concentrations in cell homogenates were determined by the method of Lowry et al. (1951).
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3. Results 3.1. Estradiol suppresses the 7a-hydroxylation of DHEA in mixed CNS cultures Many studies have suggested important physiological and pharmacological roles for estrogens and other sex hormones in the CNS (Melcangi and Panzica, 2006; Dhandapani and Brann, 2007; Arnold and Beyer, 2009). Although several potential mechanisms for these effects have been proposed, the function(s) of estrogens in CNS cells remain unclear. In this study, we measured the effects of estradiol on CYP7B1-mediated metabolism of DHEA in primary rat CNS cultures. CYP7B1-mediated 7a-hydroxylation is one of the main pathways for metabolism of DHEA in the CNS (Akwa et al., 1993; Jellinck et al., 2001). Addition of estradiol to CNS co-cultures, containing neurons, astrocytes and oligodendrocytes, resulted in significant suppression of CYP7B1-mediated DHEA 7a-hydroxylation (Fig. 1). Estradiol suppressed CYP7B1-mediated metabolism of DHEA in rat fetal cerebellar co-cultures by 50% 13 (Fig. 1A). Similar results were obtained using neonatal rat glial cultures, implicating that this reaction may occur primarily in glial cells (Fig. 1B). 3.2. CYP7B1-mediated expression and activity in primary cultures of rat astrocytes From previously published studies with material from rat brain, it appears that CYP7B1, the enzyme responsible for DHEA 7ahydroxylation, is predominantly expressed in astrocytes (Akwa et al., 1993; Zhang et al., 1997). To verify this in our cultures, we assayed CYP7B1 mRNA in astrocytes obtained from the co-cultures [()TD$FIG]
of neonatal rat brain. As expected, this experiment showed high levels of CYP7B1 mRNA in astrocytes (data not shown). In addition, primary cultures of astrocytes alone were able to metabolize DHEA into its 7a-hydroxyderivative at a significant rate. The 7ahydroxylase activity towards DHEA in our experiments was 0.8 0.3 nmol/mg h. Astrocytes are considered to be important for steroidogenesis in the CNS. These cells have been reported to have a number of functions, including production of neuroactive substances and regulatory effects on neuronal survival (Paixa˜o and Klein, 2010; Zwain and Yen, 1999; Dhandapani and Brann, 2007). It has also been suggested that astrocytes may play an important role for estradiol-mediated neuroprotection (Dhandapani and Brann, 2007). 3.3. Estrogens suppress the 7a-hydroxylation of DHEA in primary astrocyte cultures To study regulatory effects on DHEA metabolism in cultures of astrocytes alone we treated neonatal primary cultures of astrocytes with estradiol prior to analysis of CYP7B1-mediated DHEA hydroxylation. As shown in Fig. 2, treatment with estradiol (1 mM) for 24 h strongly suppressed CYP7B1-mediated hydroxylase activity in astrocyte cultures (by 66% 8). A similar suppressive effect, although weaker, was found with shorter treatment times (4– 6 h). Lower concentrations of estradiol (10–100 nM) did not show consistent effects on CYP7B1 activity, in astrocytes or in mixed CNS cultures (data not shown). To examine whether the added estradiol remained in an unmetabolized form throughout the incubation period we assayed estradiol metabolism in astrocytes using thin layer chromatography. These experiments showed that 50–75% (varying in different experiments) of the estradiol remained unmetabolized after 24 h. Very little, if any, formation of 2- or 4-hydroxymetabolites from estradiol was observed under these conditions. The main metabolite formed from estradiol was a compound with the same retention time as estrone. We then carried out experiments with estrone, to study if this estrogen also would affect CYP7B1-mediated DHEA metabolism. Treatment of astrocytes with estrone significantly suppressed CYP7B1 hydroxylase activity (by 42% 13), although the effect was not quite as strong as that of estradiol (Fig. 2). 3.4. Diarylpropionitrile suppresses the 7a-hydroxylation of DHEA in primary astrocytes To study potential effects of synthetic estrogen receptor agonists, we also carried out experiments with propylpyrazoletriol
[()TD$FIG]
Fig. 1. Effect of treatment with estradiol (E2) (1 mM) on CYP7B1-mediated 7ahydroxylation of DHEA in (A) rat fetal cerebellar co-cultures and (B) neonatal rat glial cultures. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of 3–5 experiments. *Statistically significant difference compared to control (p < 0.01).
Fig. 2. Effect of treatment with 1 mM of estradiol (E2) or estrone on CYP7B1mediated 7a-hydroxylation of DHEA in primary cultures of rat astrocytes. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of 6–10 experiments. *Statistically significant difference compared to control (p < 0.01).
[()TD$FIG]
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Fig. 3. Effect of treatment with of propylpyrazoletriol (PPT) (1 mM), an agonist of ERa, or diarylpropionitrile (DPN) (1 mM), an agonist of ERb, on CYP7B1-mediated 7a-hydroxylation of DHEA in primary cultures of rat astrocytes. Treatment and analysis of catalytic activity were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of five experiments. *Statistically significant difference compared to control (p < 0.01).
(PPT), an agonist of ERa, and diarylpropionitrile (DPN), an agonist of ERb. Interestingly, addition of 1 mm of DPN significantly suppressed CYP7B1-mediated DHEA hydroxylation whereas PPT (in the same concentration) did not (Fig. 3). The suppressive effect of DPN on CYP7B1-mediated activity was in the same order of magnitude as that of estradiol. 3.5. Estradiol suppresses the CYP7B1 mRNA levels in primary astrocytes To obtain more information on the effects of estradiol in astrocytes, we measured CYP7B1 mRNA levels in rat primary cultures of such cells, in the presence or absence of estradiol, using real time RT-PCR. In accordance with the data from analysis of CYP7B1-mediated catalytic activity, treatment with estradiol significantly suppressed the CYP7B1 mRNA levels in astrocytes (Fig. 4). About 30% suppression (33% 20) of CYP7B1 mRNA by estradiol treatment was observed under the conditions employed. 4. Discussion Our study reports significant suppressive effects by estrogens on CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA levels in rat CNS cultures. CYP7B1-mediated catalysis is one of the main pathways for the metabolism of DHEA in the CNS and effects
[()TD$FIG]
Fig. 4. Effect of treatment with 1 mM of estradiol (E2) on the CYP7B1 mRNA levels in primary cultures of rat astrocytes. Treatment and analysis of mRNA levels were carried out as described in Section 2. Controls were treated with vehicle (ethanol). Error bars represent the standard deviation of the means of seven experiments. *Statistically significant difference compared to control (p < 0.01).
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on CYP7B1 should therefore strongly influence the levels of this neurosteroid (Akwa et al., 1993; Rose et al., 1997; Jellinck et al., 2001). DHEA, which is produced by astrocytes in vivo, has been implicated in a number of processes essential for CNS function, including modulation of the actions of several neurotransmitter receptors and activation of kinase signaling important for cellular viability (Charalampopoulos et al., 2006; Melcangi and Panzica, 2006; Maninger et al., 2009). Some of the many proposed functions for this steroid include effects on neuronal development, modulation of immune function and an important role for neuronal survival. Decreased levels of DHEA have been associated with neuronal dysfunction and neurodegeneration. Abnormal levels of DHEA and/or the sulfated form DHEA-sulfate have also been implicated in neuropsychiatric and neurodegenerative conditions (Maninger et al., 2009). Several previous studies have indicated estrogen-related effects in the CNS, in physiological as well as in pharmacological concentrations, and the potential of estrogen replacement therapy to decrease the risk of dementia has been discussed for many years (Vedder et al., 1999; Brann et al., 2007). Estrogen and estrogen receptor modulators are reported to exert neurotrophic and neuroprotective actions in humans as well as animals (Melcangi and Panzica, 2006; Brann et al., 2007; Arnold and Beyer, 2009). Estrogens have, for instance, been shown to protect neurons against oxidative stress. Furthermore, an increased risk for Alzheimer’s disease has been linked to single nucleotide polymorphisms (SNPs) in the CYP19A1 gene, which is necessary for estradiol production. A number of different mechanisms are reported to mediate estrogen action in the CNS, e.g. modulation of calcium flux, increased activation of survival proteins, altered glutamate release and several others (Melcangi and Panzica, 2006; Brann et al., 2007; Arnold and Beyer, 2009). From the current data it seems that estrogens may also affect the levels of DHEA in CNS cells. The role(s) of CYP7B1 in the CNS have not been fully elucidated. High CYP7B1 mRNA levels are reported in the hippocampus, the region considered most important for memory and learning (Rose et al., 1997; Yau et al., 2003). Interestingly, some patients with Alzheimer’s disease are reported to have altered levels of CYP7B1 and/or 7a-hydroxy-DHEA (Attal-Khemis et al., 1998; Yau et al., 2003). In addition, recent studies have linked a motor-neuron degenerative disease, hereditary spastic paraplegia, to mutations in the coding region of human CYP7B1 (Schu¨le et al., 2009). We recently reported that the Akt/PI3K (phosphoinositide 3-kinase) cascade, a signaling pathway important for cell survival, can affect CYP7B1 transcription, also suggesting a possible connection between CYP7B1 action and neuroprotective events (Tang et al., 2008). Several other steroids present in the CNS are metabolized by CYP7B1, including pregnenolone, which is the immediate precursor to progesterone (Rose et al., 1997). Both progesterone and pregnenolone are reported to have neuroprotective properties (Melcangi and Panzica, 2006). It is possible that regulatory effects on CYP7B1 activity and/or expression may affect the levels also of other steroids of importance for CNS function. From the results of this and previous studies, we conclude that CYP7B1 expression and activity is high in rat astrocytes (Akwa et al., 1993; Zhang et al., 1997). It has been reported that expression of human CYP7B1 is predominant in neurons (Trap et al., 2005), suggesting a difference in cellular localization for this enzyme between humans and rats. The present results indicate that CYP7B1-mediated metabolism and expression in rat brain is significantly suppressed by estrogens. These findings are different compared to previous data concerning estrogen-dependent regulation of CYP7B1 in kidney-
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and liver-derived cell lines, where estradiol upregulates the CYP7B1 gene, at least in the presence of estrogen receptors (Tang et al., 2006, 2008). This indicates that estrogen-mediated regulation of CYP7B1 action could be tissue-specific and/or involve different cellular mechanisms. In the present study we also observed suppressive effects on CYP7B1 by an ERb agonist. This finding suggests that ERb might be important for the effects observed, although other mechanisms, either transcriptional or post-transcriptional, cannot be excluded at the present stage. In summary, neuroprotective effects by estradiol and other estrogens are reported in several studies and have been proposed to involve effects on astrocytes. In view of the present data we propose that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the DHEA levels by suppression of its metabolism. Acknowledgements The present work was supported by grants from the Swedish Research Council Medicine, the Swedish Brain Foundation and Gyllenstiernska Krapperupsstiftelsen (Sweden). References Akwa, Y., Sananes, N., Gouezou, M., Robel, P., Baulieu, E.E., Le Goascogne, C., 1993. Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density. J. Cell Biol. 121, 135–143. Arnold, S., Beyer, C., 2009. Neuroprotection by estrogen in the brain: the mitochondrial compartment as presumed therapeutic target. J. Neurochem. 110, 1–11. Attal-Khemis, S., Dalmeyda, V., Michot, J.L., Roudier, M., Morfin, R., 1998. Increased total 7a-hydroxy-dehydroepiandrosterone in serum of patients with Alzheimer’s disease. J. Gerontol. A: Biol. Sci. Med. Sci. 53, B125–132. Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., Khan, M.M., 2007. Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72, 381–405. Charalampopoulos, I., Alexaki, V.I., Tsatsanis, C., Minas, V., Dermitzaki, E., Lasaridis, I., Vardouli, L., Stournaras, C., Margioris, A.N., Castanas, E., Gravanis, A., 2006. Neurosteroids as endogenous inhibitors of neuronal cell apoptosis in aging. Ann. N. Y. Acad. Sci. 1088, 139–152. Dhandapani, K.M., Brann, D.W., 2007. Role of astrocytes in estrogen-mediated neuroprotection. Exp. Gerontol. 42, 70–75. Fex Svenningsen, A˚., Shan, W.S., Colman, D.R., Pedraza, L., 2003. Rapid method for culturing embryonic neuron-glial cell cocultures. J. Neurosci. Res. 72, 565–573. Hirayama, T., Honda, A., Matsuzaki, Y., Miyazaki, T., Ikegami, T., Doy, M., Xu, G., Lea, M., Salen, G., 2006. Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol. Hepatology 44, 602–611. Jellinck, P.H., Lee, S.J., McEwen, B.S., 2001. Metabolism of dehydroepiandrosterone by rat hippocampal cells in culture: possible role of aromatization and 7hydroxylation in neuroprotection. J. Steroid Biochem. Mol. Biol. 78, 313–317.
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