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

Abstract

The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects NMDA-/GABAA-receptor negative neural crest-derived PC12 cells from apoptosis. We now report that membrane-impermeable DHEA-BSA conjugate replaces unconjugated DHEA in protecting serum-deprived PC12 cells from apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the prosurvival factor Src and induction of the anti-apoptotic protein Bcl-2 and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated PC12 cell membranes revealed saturation within 30 min and binding of DHEA with a Kd of 0.9 nM. A similar binding activity was detectable in isolated membranes from rat hippocampus and from normal human adrenal chromaffin cells. The presence of DHEA-specific membrane binding sites was confirmed by flow cytometry and confocal laser microscopy of DHEA-BSA-FITC stained cells. In contrast to estrogens and progestins, glucocorticoids and androgens displaced DHEA from its membrane binding sites but with a 10-fold lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents acted as pure antagonists, blocking the antiapoptotic effect of DHEA as well as the induction of Bcl-2 proteins and Src kinase activation. In conclusion, our findings suggest that neural crest-derived cells possess specific DHEA membrane binding sites coupled to G proteins. Binding to these sites confers neuroprotection.

DHEA-Bodipy—A functional fluorescent DHEA analog for live cell imaging

“…DHEA-Bodipy binds rapidly to plasma membranes of living PC12 cells. Similar results were obtained by Charalampopoulos et al with DHEA-BSA-FITC, a membrane impermeable fluorescein isothiocyanat labeled bovine serum albumin conjugate of DHEA (Charalampopoulos et al, 2006). In contrast to DHEA-BSA-FITC, DHEA-Bodipy is nonpolar similar to DHEA.…”

“…In PC12 cells, DHEA has been reported to bind to specific plasma membrane binding sites (Charalampopoulos et al, 2006). We incubated living PC12 cells with 1 µM DHEA-Bodipy.…”

“…Recent studies show that DHEA protects rat chromaffin cells and PC12 rat pheochromocytoma cells against serum-deprivation induced apoptosis (Charalampopoulos et al, 2004). In PC12 cells, DHEA binds to and activates a Gα coupled receptor that in turn leads to an induction of anti-apoptotic Bcl proteins (Charalampopoulos et al, 2006) and to an activation of Akt kinase (Charalampopoulos et al, 2008). Besides, several other receptors have been proposed as possible DHEA receptors, for example the pregnane X receptor (PXR), estrogen receptor α (ERα) and β and the sigma-1 receptor subtype (Goodwin et al, 2002;Maurice et al, 2006;Webb et al, 2006).…”

“…These kinases activate the prosurvival transcription factors CREBP and NF-kappaB which stimulate the expression of antiapoptotic Bcl-proteins Bcl-2 and Bcl-xL. In addition, PKC phosphorylates Bcl-2 which is necessary for its antiapoptotic function (Charalampopoulos et al, 2006;Charalampopoulos et al, 2004). Moreover, DHEA induces phosphorylation of Akt kinase which inactivates pro-apoptotic Bad (Charalampopoulos et al, 2008).…”

Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact

“…Non-genomic steroid actions have been reported for most steroids including glucocorticoids (1)(2)(3)(4), progesterone (5), estrogens (6,7), androgens (8,9), and neurosteroids (10). For a recent review, see (11).…”