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Endocrinology, doi:10.1210/en.2007-0645
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Endocrinology Vol. 149, No. 1 320-328
Copyright © 2008 by The Endocrine Society

Dehydroepiandrosterone Induces a Neuroendocrine Phenotype in Nerve Growth Factor-Stimulated Chromaffin Pheochromocytoma PC12 Cells

Christian G. Ziegler, Flavie Sicard, Peter Lattke, Stefan R. Bornstein, Monika Ehrhart-Bornstein and Alexander W. Krug

Medical Clinic III (C.G.Z., F.S., S.R.B., M.E.-B., A.W.K.), Carl Gustav Carus University Hospital, University of Dresden, and Institute of Clinical Chemistry and Laboratory Medicine (P.L.), Carl Gustav Carus University Hospital, 01307 Dresden, Germany

Address all correspondence and requests for reprints to: Dr. Med. Alexander W. Krug, University Hospital Carl Gustav Carus, Medical Clinic III, University of Dresden, Fetscherstraβe 74, 01307 Dresden, Germany. E-mail: alexander.krug{at}uniklinikum-dresden.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex, which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders, a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. Nerve growth factor (NGF)-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein-2. We examined whether DHEA may stimulate the cells toward a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence but not absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells toward a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE INNER layer of the adrenal cortex, the zona reticularis, high concentrations of androgens, especially dehydroepiandrosterone (DHEA) are produced. In adult adrenal glands, cells of the zona reticularis are interwoven with catecholamine-producing medullary chromaffin cells, suggesting paracrine interactions (1, 2, 3). Interestingly, DHEA levels show an age-dependent decline accompanied by a decrease in adrenal medulla function. DHEA also is the major product of the adrenal premordium during fetal development. During adrenogenesis, sympathoadrenal progenitor cells from the neuroectoderm move into this adrenal premordium and come into close contact with DHEA-producing fetal adrenal cells. Once there, they acquire the features of neuroendocrine catecholamine-producing cells (4, 5).

In humans congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency leads to hyperandrogenism. We have demonstrated that 21-OH deficiency is accompanied by severe chromaffin dysplasia and dysfunction in mice (6) and humans (7, 8). In the mouse 21-OH knockout model restoration of 21-OH by gene transfer could reverse the androgen excess as well as the structural, biochemical, and endocrine adrenomedullary alterations (9). However, the exact mechanisms how androgens may influence the chromaffin system are not known.

Based on this evidence, in this study, we focused on analyzing in a more specific way the effect of DHEA on chromaffin cells on a molecular and mechanistic level with respect to cell survival processes based on the cell number and differentiation processes. Using the PC12 cell line, which was derived from a tumor of the adrenal medulla, we used a well-characterized in vitro model of mitotic and proliferation competent chromaffin cells. PC12 cells exhibit many properties of chromaffin cells, including catecholamine synthesis, storage, and secretion (10). On the other hand, after administration of nerve growth factor (NGF), the cells differentiate into a neuronal phenotype (11). Therefore, this cell line is the most suitable chromaffin-derived cell model available to study the mechanisms of DHEA action on the chromaffin system on a molecular level. At the same time, the use of a cell line allows to exclude unknown influences on the chromaffin cells as from endothelial cells or cortical cells in primary culture or animal models.

In their natural environment, adrenal medulla (progenitor) cells are exposed to a variety of different factors, including growth factors and adrenal androgens, which influence proliferation of these cells. Only recently we demonstrated a role for DHEA in the proliferation of primary bovine adrenomedullary chromaffin cells. DHEA inhibited proliferation induced by epidermal growth factor and leukemia inhibitory factor (12) as well as NGF-mediated cell proliferation in serum-deprived PC12 cells (13).

In a first step, we asked whether DHEA might influence the NGF-induced neuronal differentiation process in PC12 cells. In a next step, we examined the effect of DHEA, alone and together with NGF, on neuroendocrine differentiation parameters. Our results provide in vitro evidence that DHEA shifts NGF-stimulated chromaffin PC12 cells toward a neuroendocrine phenotype. In a further step, we aimed at elucidating the molecular basis of NGF-DHEA interaction. The ERK1/2 MAPK pathway is well known to be involved in cellular differentiation and proliferation processes, and ERK1/2 activation is necessary for NGF-mediated cell differentiation in PC12 cells (14). We therefore examined the influence of DHEA on NGF-induced ERK1/2 activation, providing evidence that DHEA suppresses NGF-stimulated activation of this signaling cascade in PC12 cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat PC12 cells were sustained in Keighn’s modified Ham’s F-12 medium (American Type Culture Collection, Manassas, VA) with 2 mM [SCAP];l-glutamine and 1500 mg/liter sodium bicarbonate supplemented with 15% horse serum, 2.5% bovine calf serum and 20 U/ml penicillin-streptomycin (Life Technologies, Inc., Gaithersburg MD) in a humidified 5% CO2-95% O2 atmosphere at 37 C. The culture medium was changed every 2 d. Before the experiments, cells were grown in this culture medium for 72 h and then left in serum-free medium for the next 12–16 h. DHEA and the growth factor NGF were dissolved in ethanol and 10 mmol/liter sodium acetate (pH 5), respectively. In some experiments we also used the MAPK kinase (MEK) inhibitor U0126 (10 µmol/liter), which was dissolved in dimethylsulfoxide (DMSO; Calbiochem, Darmstadt, Germany).

The final volume of ethanol or DMSO in each well, including controls, was 0.01% for all assays performed. To rule out a potential influence of the solvent on the cellular parameters assessed, we conducted the corresponding control experiments. The solvent did not have an effect.

We determined the cell numbers using a Coulter counter (Mölab, Hilden, Germany) after 24 and 48 h treatment of PC12 cells with or without NGF (2–200 ng/ml) and with or without DHEA at 10–6 to 10–10 mol/liter, respectively; 40 µl of cell suspension were diluted in 9960 µl of Celloton buffer. The number of cells was determined in Giga-parts per liter.

Cytotoxicity assay
PC12 cells were seeded in white 96-well plates at a concentration of 5000 cells per 0.32 cm2. To evaluate a possible cytotoxic effect of DHEA, we used a CytoTox 96 nonradioactive cytotoxicity assay (Promega, Mannheim, Germany), which measures lactate dehydrogenase activity, according to the manufacturer’s instructions.

Neurite outgrowth
Single cell PC12 suspensions were plated in 200 mm2 wells at a density of 2 x 104 cells/well in serum-free culture medium and then treated with NGF and DHEA at doses of 2–200 ng/ml and 10–6 to 10–10 mol/liter, respectively. After 24 and 48 h incubation, the percentage of cells showing neurite outgrowth was determined by light microscopy. Cells with one or more neurites whose lengths were at least twice the diameter of the cell body were scored as positive. Neurite outgrowth was determined from at least three different regions of interest in three independent experiments (15, 16).

HPLC
To measure dopamine concentrations in culture medium, catecholamines were extracted by solid-phase extraction after 24 and 48 h stimulations of cells with or without NGF (20 ng/ml) and with or without DHEA (10–6 to 10–10 mol/liter), respectively. The cell medium samples were processed according to the sample preparation protocol for urine (Chromsystems, Munich, Germany). Briefly, 6 ml neutralization buffer and 100 µl internal standard (3,4-dihydroxy-benzylamine) were added to 3 ml cell medium. This mixture was run on a sample clean-up column by vacuum. The eluate was discarded. Subsequently the solid-phase extraction column was washed twice with pure water (10 ml) and eluted with 6 ml elution buffer by vacuum. Finally, the eluate was collected and acidified with 180 µl 5 N HCL. An aliquot of the purified eluate was applied to an HPLC reverse-phase column, and dopamine concentrations were measured using an electrochemical detector (Bio-Rad, Munich, Germany). Dopamine measurements were quantified in correlation to the added internal standard.

Western blot analysis
This was performed as described previously (17). Briefly, PC12 cells were washed twice in ice-cold PBS and lysed in ice-cold cell lytic mammalian cell lysis/extraction reagent (Sigma Aldrich, Munich, Germany) containing 1% protease inhibitor cocktail (Sigma). Cell lysates were matched for protein content, and equal amounts of protein were loaded on each lane, separated by SDS-PAGE, and transferred to nitrocellulose membrane. After blocking, membranes were immunostained with rabbit anti-phospho-ERK1/2 antibody (1:1000; Cell Signaling, Danvers, MA), rabbit anti-synaptosome-associated protein of 25 kDa (SNAP-25) antibody (1:2000; Synaptic Systems, Goettingen Germany), mouse anti-vesicle-associated membrane protein-2 (VAMP-2) antibody (1:1000, Synaptic Systems), goat anti-chromogranin A (CgA) antibody (1:1000; Santa Cruz, Heidelberg, Germany), and rabbit anti-β-actin antibody (1:1000; Santa Cruz). Bound primary antibody was detected using a Western Breeze detection kit (Invitrogen, Karlsruhe, Germany) with the appropriate secondary antibody. Chemoluminescence signals were read with the GeneGnome chemoluminescence detector (Syngene, Frederick, MD).

Quantification of ERK1/2 phosphorylation by ELISA
This was performed as described by us previously, in a slightly modified way (13, 17, 53). We performed fast-activated cell-based ELISA (FACE; ActifMotive, Rixensart, Belgium). Cells were seeded in 96-well plates at a concentration of 20,000 cells per 0.32 cm2. After reaching approximately 80% confluence, the cells were serum starved for 24 h. Subsequently cells were equilibrated in 1x HEPES-Ringer solution (130.0 mmol/liter NaCl, 5.4 mmol/liter KCl, 1.0 mmol/liter CaCl2, 1.0 MgCl2, 1.0 mmol/liter NaH2PO4, 10 mmol/liter HEPES, and 5 mmol/liter glucose (pH 7.4) at 37 C for 30 min and then stimulated in the same buffer plus the respective vehicle (ethanol or DMSO) with or without the hormones and growth factors of interest. Immediately afterward, cells were fixed with 8% formaldehyde in PBS for 20 min at room temperature and washed three times for 5 min with 0.1% Triton X-100 in PBS. Endogenous peroxidase was quenched for 20 min with freshly prepared 1% H2O2 and 0.1% azide. Cells were washed again three times in the same buffer, blocked by 10% fetal calf serum in PBS/Triton X-100 for 1 h and finally incubated overnight with the primary antibody (phosphorylated ERK; Cell Signaling; 1:1000) in PBS/Triton X-100 containing 5% BSA at 4 C under gentle shaking. The next day, cells were washed three times with PBS/Triton X-100 and incubated with the secondary antibody (antirabbit horseradish peroxidase-linked IgG; Cell Signaling; 1:3000) in PBS/Triton X-100 containing 5% BSA for 1 h at room temperature and then washed three times in PBS/Triton X-100 for 5 min and 10 min with PBS. After that, cells were incubated in 50 µl of chemoluminescence working solution (ActifMotive) in the dark. Chemoluminescence signals were read within 10 min using a Mithras multireader (Berthold Technologies, Bad Wildberg, Germany). Once chemoluminescence had been read, cells were washed twice in PBS/Triton X-100 and twice in PBS. The wells were air dried for 5 min at room temperature, and stained with 100 µl of crystal violet solution (25% in PBS) for 30 min at room temperature. Cells were subsequently washed three times in PBS, and 100 µl of 1% sodium dodecyl sulfate solution were added, and the plate was incubated on a shaker for 1 h at room temperature. Finally, absorbance was measured at 595 nm. Chemoluminescence signals were normalized to the protein content in each well as determined by crystal violet staining. This allows to normalize ERK1/2 phosphorylation to total protein content in each well.

Statistics
Data are presented as means ± SEM. Significance of differences was tested by ANOVA with Bonferroni’s as a secondary test. Differences were considered significant at values of P < 0.05. Cells from at least two different passages were used for each experimental series; n represents the number of cells or tissue culture dishes investigated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The PC12 clone is not viable in serum-free medium, with most of the cells dying within the first days after serum withdrawal. However, with NGF present, the cells undergo mitosis and differentiate into a neuronal phenotype, indicated by neurite outgrowth and expression of neuronal marker proteins such as SNAP-25 and VAMP-2/synaptobrevin (18, 19, 20, 21, 22). In a recent study, we demonstrated that at concentrations of 10–6 mol/liter or higher, DHEA inhibited NGF-mediated cell survival in serum-deprived PC12 cells (13). Figure 1Go additionally demonstrates that DHEA alone had no effect on PC12 cell number, compared with serum-deprived control cells. The effect was not due to DHEA-induced cell necrosis because DHEA alone or in combination with NGF did not exert a cytotoxic effect in PC12 cells in the concentrations used, as indicated by the lactate dehydrogenase-release method. DHEA-stimulated cells did not release more lactate dehydrogenase than unstimulated control cells, demonstrating that DHEA is not cytotoxic on PC12 cells in our experimental setting.


Figure 1
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  		FIG. 1. Cell numbers determined using a Coulter counter after 24 h (A) and 48 h (B) treatment of PC12 cells with NGF (20 ng/ml) and 10–6 mol/liter DHEA alone and in combination, respectively. DHEA (10–6 mol/liter) significantly reduced NGF-mediated cell survival, whereas DHEA alone did not have any effect on cell number. In this investigation (A and B) and in an earlier study (13 ), we have shown that DHEA concentrations less than 10–6 mol/liter had no significant effect on NGF-stimulated cell survival in our experimental setting; DHEA (10–6 to 10–10 mol/liter) alone did not affect cell number; n = 6–10 for all plotted values. CON, Control.

 
One possible explanation for the DHEA-mediated decrease of NGF-induced cell survival is a DHEA-induced shift toward differentiation. Therefore, one of our aims was to find out whether DHEA influences the NGF-mediated neuronal differentiation process. We assessed cellular parameters of neuronal differentiation, such as neurite outgrowth and the expression of SNAP-25 and VAMP-2 neuronal marker proteins. We found 10–6 mol/liter DHEA to cause a significant reduction in NGF-induced neurite outgrowth after 24 and 48 h and 10–7 mol/liter DHEA after 48 h (Fig. 2Go). Accordingly, expression of SNAP-25 and VAMP-2 were reduced by DHEA as indicated by Western blot analysis (Fig. 3Go). Taken together, our results show an inhibitory effect of DHEA on NGF-mediated neuronal differentiation in PC12 cells.


Figure 2
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  		FIG. 2. Neurite outgrowth of PC12 cells after 24 h (A) and 48 h (B) treatment with NGF (2–200 ng/ml) and DHEA (10–6 to 10–10 mol/liter). A and B, As expected, NGF induced neurite outgrowth of the cells. This effect was significantly reduced by 10–6 mol/liter DHEA after 24 and 48 h. DHEA (10–7 mol/liter) reduced NGF-mediated neurite outgrowth after 48 h; lower DHEA concentrations (10–8 to 10–10 mol/liter) had no significant effect on NGF-mediated neurite outgrowth. DHEA alone (10–6 to 10–10 mol/liter) did not induce neurite outgrowth; n = 9–25 for all plotted values. C, Representative bright-field microscopy images demonstrating the reduction of NGF (20 ng/ml)-induced neurite outgrowth (red arrows) by 10–6 mol/liter DHEA in PC12 cells. CON, Control.

 

Figure 3
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  		FIG. 3. Western blot analysis demonstrated that NGF (2–200 ng/ml) increased the expression of SNAP-25 (A) and VAMP-2/synaptobrevin (B) after 24 h treatment. This effect was reduced by DHEA. DHEA (10–6 mol/liter) alone did not significantly influence expression of SNAP-25 and VAMP-2 under our experimental conditions. Each blot is a representative blot of three to six different experiments shown above densitometric analysis. Values are given as means ± SEM as a percentage of control (100%). ODs were determined by GeneSnap software from SynGene; n = 3–6 for all plotted values. Values were considered significant at: *, P < 0.05 vs. control and §, P < 0.05 vs. 20 ng/ml NGF.

 
We then tested whether DHEA might direct PC12 cells toward a neuroendocrine phenotype. Figure 4Go summarizes the results: DHEA strongly stimulated catecholamine release from NGF-treated cells after 24 h, whereas NGF and DHEA alone had no significant effect on catecholamine release, compared with control cells (Fig. 4AGo). The influence of NGF and DHEA on expression of CgA, a neuroendocrine marker protein, is shown in Fig. 4BGo. NGF-treated cells express decreased CgA levels, compared with control cells, whereas addition of DHEA to NGF showed a tendency to increased CgA levels (P > 0.05). Interestingly, cells incubated with DHEA alone showed a notable increase in CgA protein, compared with control cells, supporting the notion of a suppressive effect of NGF on the neuroendocrine marker protein CgA.


Figure 4
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  		FIG. 4. A, Dopamine release from PC12 cells after 24 h treatment with NGF (20 ng/ml) and DHEA (10–6 to 10–10 mol/liter) alone and in combination. HPLC measurements demonstrated that NGF (20 ng/ml) alone did not significantly increase dopamine release (P > 0,05). However, 10–6 mol/liter DHEA strongly stimulated catecholamine release from NGF-stimulated (20 ng/ml) cells. NGF and DHEA alone, in all concentrations used, did not have any significant effect, compared with control cells. B, Expression of neuroendocrine marker protein CgA after 24 h treatment with NGF (20 ng/ml) and DHEA (10–6 mol/liter) alone and in combination. NGF inhibited CgA expression, whereas addition of DHEA showed a tendency to increase CgA protein (P > 0,05). DHEA alone clearly increased CgA levels, compared with control cells. The blot shown is a representative blot of four independent experiments used for densitometric analyses. Values are given as means ± SEM as a percentage of control (CON; 100%). ODs were determined by GeneSnap software from SynGene; n = 3–5 for all plotted values. Values were considered significant at: *, P < 0.05 vs. control.

 
The MAPKs ERK1/2 are crucially involved in cellular processes such as proliferation and differentiation (13, 23, 24, 25, 26, 27). ERK1/2 activation is recognized as being required for neuronal differentiation in PC12 cells (14, 28, 29, 30). Therefore, another aim was to determine whether DHEA might interfere with NGF-mediated ERK1/2 signaling. Figure 5Go shows the dependence of NGF-induced ERK1/2 phosphorylation in PC12 cells on time and concentration. Semimaximal ERK1/2 phosphorylation was reached at approximately NGF 20 ng/ml. Addition of 10–6 mol/liter DHEA to NGF (20 ng/ml) significantly reduced ERK1/2 activation as evaluated by ELISA (Fig. 6Go, A and B). The effect described was not observed with DHEA concentrations less than 10–6 mol/liter (Fig. 6BGo). Western blot analysis confirmed these findings (Fig. 6CGo). Figure 6AGo illustrates that DHEA leads to a rightward shift of the NGF dose-response curve with a reduction of the maximal NGF effect. Interestingly, 10–6 mol/liter DHEA in the absence of NGF induced a statistically not significant but moderate phosphorylation of ERK 1/2. As expected, MEK inhibitor UO126 prevented NGF-mediated up-regulation of SNAP-25 (Fig. 7Go).


Figure 5
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  		FIG. 5. Time and dose dependence of ERK1/2 phosphorylation (pERK) in PC12 cells as determined by ELISA; n = 9–12 for every plotted value.

 

Figure 6
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  		FIG. 6. Phosphospecific ERK1/2 ELISA (A and B) and Western blot analysis (C) after 5 min treatment of PC12 cells with NGF and DHEA as indicated. A, The NGF-mediated dose-response curve (2–200 ng/ml) is shifted to the right by costimulation with DHEA (10–6 mol/liter). B, Dose dependency of the inhibitory effect of DHEA on NGF-mediated (20 ng/ml) ERK1/2 phosphorylation (pERK; n = 9–18 for all plotted values). C, Western blot analysis confirmed the inhibitory effect of DHEA (10–6 mol/liter) on NGF-induced ERK1/2 phosphorylation.

 

Figure 7
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  		FIG. 7. Western blot analysis demonstrated that the NGF (20 ng/ml)-induced up-regulation of the neuronal marker protein SNAP-25 was blocked by the MEK inhibitor U0126 (10–5 mol/liter) after 24 h treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that DHEA decreases NGF-induced cell survival and shifts the cells toward a neuroendocrine phenotype in the presence of NGF in our in vitro chromaffin cell model. The PC12 cell line is widely used to study adrenal catecholamine metabolism, chromaffin cell proliferation, and differentiation processes as well as the underlying signaling events (2, 14, 31). We recently demonstrated that a combinatorial code of growth factors and neuropeptides can define the differentiation process in PC12 cells (32). Our in vitro results support the hypothesis of cortical-medullary interactions within the developing and adult adrenal gland.

The adrenal gland is composed of two embryologically different tissues, the mesodermally derived adrenal cortex and the neuroectodermally derived medulla. Within the adrenal gland, chromaffin cells and their progenitors are exposed to a wide variety of growth factors and hormones, including adrenal androgens such as DHEA. DHEA also is the major product of the adrenal premordium during fetal development, when chromaffin progenitors acquire the features of neuroendocrine catecholamine-producing cells (4). Even in adulthood, a subpopulation of proliferation-competent chromaffin cells exists throughout life (33). DHEA has been identified as an important regulator for the proliferation of neuronal stem cells (31, 34, 35). DHEA also has been shown to protect neurones against excitatory amino acid-induced neurotoxicity (36) and to increase neurogenesis in the adult rodent hippocampus in vivo (35). Decreased DHEA plasma levels have been associated with neuronal degeneration and dysfunction (37). Moreover, DHEA also affects growth factor-induced mitogenesis and proliferation of vascular smooth muscle cells (38). Recent evidence suggests an antiapoptotic effect of DHEA on chromaffin cells (31). Thus, it is tempting to speculate that DHEA may also play a role in adrenal gland development, chromaffin cell differentiation processes, and tumorigenesis by influencing the neighboring chromaffin progenitor cells in the adrenal medulla.

The anatomy of the adult adrenal gland is characterized by a tight proximity of catecholamine-producing medullary cells and the DHEA-secreting zona reticularis, suggesting paracrine interactions starting in embryogenesis and extending throughout the whole life span (1). Only recently we could show in primary cultures of bovine adrenomedullary chromaffin cells that growth factor-stimulated chromaffin cell proliferation is inhibited by DHEA (12).

According to the traditional concept, glucocorticoids are considered to be the major determining factors defining the commitment of chromaffin phenotype and suppressing neuronal differentiation (39). This contrasts with recent findings from glucocorticoid receptor-deficient mice, which develop normal numbers of chromaffin cells, indicating that glucocorticoid signaling is not essential for forming the neuroendocrine phenotype in chromaffin cells (39, 40). However, no chromaffin tissue is formed in steroidogenic factor-1-deficient mice that are devoid of any steroidogenic tissue (41). Furthermore, we observed a marked adrenomedullary dysplasia with ectopic chromaffin cells located beneath the organ capsule in the steroidogenic factor-1 haplotype with almost normal glucocorticoid levels (42). Moreover, patients with frequently occurring 21-OH deficiency and hyperandrogenism also demonstrate severe chromaffin cell dysfunction (7, 8), and the age-dependent decline in DHEA plasma levels is correlated with a steady decrease in catecholamine levels. Restoring 21-OH activity by gene transfer in the 21-OH knockout model reverses androgen excess as well as morphological and functional adrenomedullary alterations. Taken together, these observations suggest that factors other than glucocorticoids, such as DHEA produced by the neighboring adrenocortical cells, may be involved in chromaffin cell development and differentiation.

Bearing in mind the inhibitory effect of DHEA on NGF-stimulated cell survival, we were curious as to whether this effect on reduction of the cell number might be due to the induction of differentiation. We could demonstrate that DHEA inhibited NGF-induced neuronal differentiation processes in PC12 cells, as indicated by a reduction in neurite outgrowth as well as SNAP-25 and VAMP-2 expression. DHEA in all concentrations used (10–6 to 10–10 mol/liter) in the absence of NGF did not have a significant effect on neurite outgrowth, compared with control cells, indicating that activation of the cells by NGF is necessary for DHEA to mediate the inhibitory effect on neurite outgrowth. Our results contrast the findings described by Compagnone and Mellon (43). In their study, the authors could demonstrate that DHEA in low concentrations of 10–9 mol/liter caused neurite outgrowth in primary cultures of mouse embryonic neocortical neurons. However, this difference may be attributed to the different cell system used originating from different tissue and developmental context. Furthermore, culture conditions in our study are different from that used by Compagnone and Mellon (43). In earlier studies DHEA has been shown to protect PC12 cells against serum deprivation-induced apoptosis, which contrasts our results (31, 51). This, again, could be due to cell culture conditions. Higher sensitivity of our cell clone to NGF might possibly be due to higher cell passage, accompanied by a lesser sensitivity to DHEA (44). This is supported by our observation that NGF induces neuronal differentiation relatively fast, within 24 h.

CgA is a member of the granin protein family, one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells (45). CgA is a major component of chromaffin vesicles, and a specific depletion of CgA expression by antisense RNAs in PC12 cells leads to a profound loss of secretory granule formation (46). Because DHEA enhanced CgA expression, it consequently promoted the neuroendocrine phenotype of the cells by influencing secretory granule formation. In addition, DHEA increased the secretion of dopamine from NGF-treated cells, an effect that was not observed with DHEA alone. The strongest effect of DHEA was observed with a DHEA concentration of 10–6 mol/liter. In vivo, similarly high DHEA concentrations are possible locally within the adrenal, making an effect on chromaffin cells likely.

To date, the exact physiological role of DHEA and a specific intracellular receptor have not been defined (47, 48). According to their classical method of action, steroid hormones act via intracellular receptors, which act as ligand-dependent transcription factors (49, 50). In addition to this effector mechanism, rapid, so-called nongenomic effects of various steroids have been described. Plasma membrane-associated receptors are postulated to mediate these steroid effects also involved in important physiological and pathological processes. Liu and Dillon (48) described a plasma membrane binding side on endothelial cells, suggesting the existence of a specific DHEA receptor coupled to G{alpha}I2 and G{alpha}I3 that can activate endothelial nitric oxide synthase in these cells. Only recently a DHEA-specific binding site on the plasma-membrane of PC12 cells was described, which is coupled to G-proteins and considered to mediate the neuroprotective effect of DHEA (51). However, these findings do not rule out the possibility of a coexisting intracellular receptor for DHEA. In various systems, DHEA exerts its effects after conversion into estrogens and androgens. A recent study has ruled out this possibility for PC12 cells (31).

Our findings support a rapid signaling mechanism because DHEA interfered with NGF-induced ERK1/2 activation within minutes. ERK1/2 MAPKs are known to be involved in neuronal stem cell differentiation and proliferation processes (14, 52), and ERK1/2 activation has been associated with nongenomic effects of DHEA in vascular cells (53). A rapid modulating interaction with ERK1/2 phosphorylation has been demonstrated for other steroid hormones such as aldosterone. This effect seems to be dependent on activation of c-src kinases (54). Interestingly, c-src also seems to play a role in rapid DHEA action in PC12 cells (51), possibly representing a common mechanism in rapid steroid hormone action.

Taken together, our data show that DHEA inhibits NGF-mediated neuronal differentiation in PC12 cells and shifts the cells toward a neuroendocrine phenotype in the presence of NGF. Moreover, our data suggest that DHEA mediates this effect by interfering with NGF-induced ERK1/2 MAPK activation. Interestingly, DHEA administration to PC12 cells without NGF moderately increased ERK1/2 phosphorylation. This contrasts the inhibitory DHEA effect in the presence of NGF. However, this is not a contradiction. Activation and/or inhibition of the ERK cascade by a specific factor can be induced by multiple signaling mechanisms, depending on cell type and contextual factors. Most importantly, the ability of different signaling pathways to activate ERK with specific time courses and within distinct cellular locations may lead to different functional cellular responses. This depends on the pathway that is predominantly activated (55, 56). Thus, interference of DHEA with a preactivated, NGF-driven ERK1/2 signaling cascade may be mediated by a different signaling mechanism than activation of ERK by DHEA in the absence of NGF and, consequently, lead to a distinct cellular response.

These results provide experimental in vitro evidence to support the hypothesis that DHEA may be involved in the neuroendocrine differentiation and function of chromaffin cells in the adrenal medulla.


   Acknowledgments
 
We are very thankful to Simone Sperber and Martina Kohl for their technical assistance.


   Footnotes
 
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 655-A6 "Collaborative Research Centre 655 Dresden: Cells into tissues: stem cell and progenitor commitment and interactions during tissue formation" (to M.E.-B. and S.R.B.) and Meddrive grants (to A.W.K. and C.G.Z.) from the University of Dresden.

Disclosure Statement: The authors of this manuscript have nothing to disclose.

First Published Online September 20, 2007

Abbreviations: CgA, Chromogranin A; DHEA, dehydroepiandrosterone; DMSO, dimethylsulfoxide; MEK, MAPK kinase; NGF, nerve growth factor; 21-OH, 21-hydroxylase; SNAP-25, synaptosome-associated protein of 25 kDa; VAMP-2, vesicle-associated membrane protein-2.

Received May 15, 2007.

Accepted for publication September 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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