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G protein-associated, specific membrane binding sites mediate the neuroprotective effect of dehydroepiandrosterone

Ioannis Charalampopoulos^*, Vassilia-Ismini Alexaki, Iakovos Lazaridis^*, Erene Dermitzaki, Nicolaos Avlonitis, Christos Tsatsanis, Theodora Calogeropoulou, Andrew N. Margioris, Elias Castanas and Achille Gravanis^*,1

Departments of
^* Pharmacology,
Experimental Endocrinology and
Clinical Chemistry, School of Medicine, University of Crete, Heraklion, Greece; and
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Athens, Greece

¹Correspondence: Dept. of Pharmacology, School of Medicine, University of Crete, P.O. Box 2208, Heraklion 71003, Greece. E-mail: gravanis{at}med.uoc.gr

SPECIFIC AIM

The brain and the adrenals produce the neuroactive steroid dehydroepiandrosterone (DHEA) and its sulfate ester DHEAS. DHEA protects rat hippocampal neurons against NMDA-induced excitotoxicity. DHEA and DHEAS were shown to exert most of their actions on neural cells at micromolar concentrations, modulating NMDA and GABA_A receptors. We recently reported that DHEA and DHEAS at 1 nM, also protect NMDA and GABA_A receptor negative neural crest-derived PC12 rat sympathoadrenal cells against apoptosis, activating within minutes the prosurvival factors NF-B and CREB, two upstream effectors of antiapoptotic Bcl-2 proteins (PNAS 2004, 101:8209). At nanomolar concentrations, these neurosteroids acutely stimulate the secretion of catecholamines via induction of the depolymerization and disassembly of submembrane actin cytoskeleton.

The exact nature of the receptor systems mediating the rapid and potent actions of DHEA on PC12 cells is unknown. The rapid onset of DHEA and DHEAS actions on PC12 cells supports the hypothesis that a membrane component might be implicated in the action of these neuroactive steroids. Thus, the aim of the present work was to test the hypothesis that DHEA exerts its antiapoptotic and cytoprotective actions via specific membrane binding sites.

PRINCIPAL FINDINGS

1. Membrane-impermeable conjugate DHEA-BSA protects PC12 cells against serum deprivation-induced apoptosis in a pertussis toxin-reversible manner
In the present study, we report that DHEA chemically coupled to macromolecule bovine serum albumin (BSA) (DHEA-BSA), and thus is in a form unable to enter into the cell, protected PC12 cells from serum deprivation-induced apoptosis, with an efficacy similar to this of DHEA and DHEAS. Apoptosis was reduced to 48.7 ± 1.0% of parallel controls (cells cultured in serum-free medium and exposed to the vehicle) compared with 46.0 ± 2.8% and 52.6 ± 2.9% for DHEA and DHEAS respectively (Fig. 1 A). The antiapoptotic effect of DHEA-BSA was dose dependent (IC₅₀ of 1.53±0.28 nM), and similar to that of DHEA (Fig. 1B ).

View larger version (16K): [in this window] [in a new window]   Figure 1. DHEA-BSA protected PC12 cells against serum deprivation-induced apoptosis in a pertussis toxin (PTX) reversible manner. Cells were cultured either in complete media (serum supplemented) or serum-free media supplemented with 1% BSA and containing various steroids. Apoptosis was quantified and depicted as % of control (serum-free media in the absence of steroids). Values represent mean ± SD of 3 separate experiments each performed in triplicate (*P<0.05). A) Cells cultured for 24 h in serum-free medium in the absence or presence of 10–7 M DHEA, DHEAS, or DHEA-BSA in the absence or the presence of 10–6 M PTX. B) Cells cultured for 24 h in serum-free medium containing DHEA or DHEA-BSA ranging from 10–11 to 10–5 M.

Our hypothesis for the involvement of a membrane-mediated DHEA action was further supported by experiments showing that protection of PC12 cells against apoptosis by DHEA/DHEAS and DHEA-BSA was significantly reversed by 10^–6M of pertussis toxin (PTX), suggesting the involvement of G protein-coupled binding sites (Fig. 1A ). DHEA increased the specific binding of [³⁵S]GTPS, in a dose-dependent manner, resulting to a 20% increase of specific [³⁵S]GTPS binding with 10^–7 M of DHEA.

Western blot analysis of serum deprivation-induced suppression of antiapoptotic Bcl-2 and Bcl-xL protein levels in PC12 cells showed that exposure of cells for 12 h to 10^–7 M of DHEA-BSA prevented this effect, restoring Bcl-2 and Bcl-xL proteins to levels comparable to those of serum supplementation, and similar to those of DHEA or DHEAS. The ability of DHEA/DHEAS and DHEA-BSA to prevent serum deprivation suppression of Bcl-2/Bcl-xL proteins was completely reversed by 10^–6M of pertussis toxin.

2. DHEA binds with high affinity on isolated membranes from PC12, human chromaffin, and rat hippocampal cells
As depicted in Fig. 2 A, [³H]DHEA bound on isolated membranes from PC12 cells with high affinity (K_D 0.9 nM) and low capacity (21.1 fmol/mg protein). Binding was rapid at 37°C and was completed within 30 min (Fig. 2B ). Human chromaffin cell membranes also exhibited a specific binding for [³H]DHEA, with an apparent affinity of 0.1 nM and a binding capacity of 35.3 fmol/mg protein. Similarly, [³H]DHEA bound specifically to rat hippocampal cell membranes with an apparent affinity of 0.9 nM and a binding capacity of 93.6 fmol/mg protein.

View larger version (15K): [in this window] [in a new window]   Figure 2. DHEA binds with high affinity on isolated membranes from PC12 cells. Membranes were incubated for 30 min in the presence of [3H]DHEA concentrations varying from 0.1 to 20 nM in the presence or absence of 500 nM molar excess of DHEA for the nonspecific or total binding, respectively. Specific binding is presented. The KD and Bmax values for the membrane binding sites were determined from Scatchard plots (presented in inserts), based on saturation bindings. To carry out time course bindings, membranes were incubated for different intervals (5–120 min) with 5 nM [3H]DHEA in the presence or absence of 500 nM molar excess of DHEA for the nonspecific or total binding, respectively. Specific binding is presented. Samples were in all experiments in triplicates (B).

Incubation of PC12 cells with the fluorescent analog of DHEA-BSA (DHEA-BSA-FITC) resulted in a specific spot-like membrane fluorescent staining. No intracellular staining was observed, confirming that DHEA-BSA conjugate was not entering into the cell. We have confirmed the presence of specific membrane sites for DHEA by flow cytometric analysis of intact whole PC12 cells stained with DHEA-BSA-FITC.

3. Selectivity of DHEA membrane binding sites: androgens and glucocorticoids displace [³H]DHEA binding on isolated PC12 cell membranes
Displacement experiments of [³H]DHEA showed that both unlabeled DHEA and DHEAS competed for [³H]DHEA binding on isolated PC12 cell membranes. Pregnane allopregnanolone (ALLO), synthetic estrogen diethylstilbestrol (DES), and the progesterone analog ORG2058 (ORG) failed to displace [³H]DHEA from its binding sites at concentrations ranging from 1 pM to 1 µM. In contrast, a significant displacement was documented by the active androgens testosterone (TESTO) and dehydrotestosterone (DHT), (IC₅₀: 14,2 and 13.6 nM) and glucocorticoids corticosterone (CORT) and dexamethasone (DEX) (IC₅₀: 9,9 and 9.3 nM).

4. The antiapoptotic effects of DHEA are prevented by androgens and glucocorticoids
Our findings suggest that androgens and glucocorticoids may interact with the membrane DHEA binding sites, although with a 10-fold lower affinity than DHEA. However, previous data have shown that these two steroids were ineffective in protecting PC12 cells from serum deprivation-induced apoptosis. Priming of serum-deprived cells for 30 min with 10^–6 M of TESTO/DHT or CORT/DEX, then exposure to the combination of DHEA with the steroids resulted in a complete block of the antiapoptotic effect of DHEA. Androgens and glucocorticoids also reversed the ability of DHEA to prevent serum deprivation suppression of Bcl-2/Bcl-xL antiapoptotic proteins and phosphorylation of prosurvival Src kinase.

CONCLUSIONS AND SIGNIFICANCE

We previously reported that DHEA and DHEAS protect NMDA and GABA_A receptor negative neural crest-derived PC12 cells against serum deprivation-induced apoptosis at very low concentrations (1 nM) via the antiapoptotic Bcl-2 proteins. We now report that membrane impermeable conjugated DHEA-BSA protected PC12 cells against serum deprivation-induced apoptosis with an apparent IC₅₀ of 1.5 nM in a manner similar to that of unconjugated DHEA/DHEAS (1.8 nM), strongly suggesting the involvement of specific membrane binding sites. DHEA-BSA was efficiently mimicking DHEA/DHEAS actions on antiapoptotic Bcl-2 proteins by preventing their suppression by serum deprivation.

We now present experimental evidence suggesting the involvement of Gi protein in the DHEA- and DHEA-BSA-induced protection of PC12 cells against serum deprivation-induced apoptosis since their beneficial effect in preventing apoptosis and inducing antiapoptotic Bcl-2 proteins were abolished in the presence of 10^–6M pertussis toxin (PTX). DHEA increased the specific binding of [³⁵S]GTPS on PC12 cell membrane preparations, in a dose-dependent manner. Our findings support further previous observations by Liu and Dillon associating membrane DHEA binding in human and bovine endothelial cells to Gi proteins. Based on recent findings showing phosphorylation-activation of Src tyrosine kinase by direct interaction with Gi, this group hypothesized that DHEA may indeed activate Src kinase, by a Gi-dependent pathway. Our findings support their observations and overall hypothesis. Indeed, both DHEA and DHEA-BSA acutely increased (within 5 min of exposure) the phosphorylation of Src, an effect that was reversed by the Gi inhibitor PTX. It is of note that activation of the Src-protein kinase C (PKC) pathway induces NF-B activity and PC12 cell survival. Previous studies have demonstrated that atypical PKC activation of Src plays a critical role in NF-B-dependent transcription, by directly regulating IKK, enhancing thus NF-B activity and survival of PC12 cells in a serum-free environment. Their observation is in line with our recently published data demonstrating that DHEA protects PC12 cells against serum deprivation-induced apoptosis via activation of another member of the PKC family (the PKC) and NF-B, two prosurvival upstream effectors of the antiapoptotic Bcl-2 proteins (Fig. 3 ).

View larger version (23K): [in this window] [in a new window]   Figure 3. Hypothetical mechanism of the neuroprotective actions of DHEA. Our data suggest that DHEA binds with high affinity to membrane sites, activating within minutes the cascade Gi proteins/Src/PKC/NFB, resulting finally in the induction of the antiapoptotic Bcl-2 proteins.

Several structurally related steroids did not displace [³H]DHEA binding. The synthetic glucocorticoid DEX and the androgen DHT showed a 10-fold lower affinity for the DHEA membrane binding sites. However, both steroids were deprived of any protective antiapoptotic effect on PC12 cells. The data stated above give credit to the hypothesis that the former may act as endogenous antagonists of DHEA. Priming of PC12 cells for 30 min with DEX or DHT, then their exposure to DHEA with a molar excess of DEX or DHT completely reversed the protective effects of DHEA, as well as its stimulatory effect on the antiapoptotic Bcl-2 proteins and on prosurvival Src phosphorylation. These findings suggest that part of the neurotoxic effects of glucocorticoids and testosterone might be attributed to their antagonist effect on the neuroprotective effect of endogenous DHEA. The decline of brain DHEA levels during aging and in Alzheimer’s disease might exacerbate this phenomenon, rendering neurons more vulnerable to glucocorticoid and androgen toxicity. Glucocorticoid neurotoxicity becomes more pronounced in aged subjects since cortisol levels in the CSF are increasing in the course of normal aging as well as in relatively early stages of Alzheimer’s disease.

FOOTNOTES

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