Dehydroepiandrosterone and allopregnanolone protect sympathoadrenal medulla cells against apoptosis via antiapoptotic Bcl-2 proteins

Abstract

The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEA sulfate (DHEAS), and allopregnanolone (Allo), produced by the CNS and the adrenals, appear to exert a protective effect in hippocampal and cortical neuron ischemia- and excitotoxicity-induced injury. We hypothesized that they may also play a protective role on the adrenal medulla, an important part of the sympathetic nervous system, and the tissue adjacent to their primary site of production. DHEA, DHEAS, and Allo protected rat chromaffin cells and the rat pheochromocytoma PC12 cell line, an established model for the study of adrenomedullary cell apoptosis and survival, against serum deprivation-induced apoptosis. Their effects were time- and dose-dependent, with EC50 1.8, 1.1, and 1.5 nM, respectively. The antiapoptotic effect of DHEA DHEAS and Allo was compared to that of a long list of structurally related compounds and was found to be structure-specific, confined mainly to conformation 3beta-OH-Delta5 for androstenes and 3alpha-OH for pregnanes. Indeed, 3-keto, Delta4, or C7 hydroxylated androstenes and 3beta pregnanes were ineffective. The prosurvival effect of DHEA(S) and Allo was N-methyl-D-aspartate-, GABAA-, sigma1-, or estrogen receptor-independent. It involved the antiapoptotic Bcl-2 proteins, their role being sine qua non for their action because Bcl-2 antisense oligonucleotides reversed their effects. Finally, DHEA(S) and Allo activated cAMP response element-binding protein and NF-kappaB, upstream effectors of antiapoptotic Bcl-2 protein expression. They also activated the antiapoptotic kinase PKCalpha/beta, a posttranslational activator of Bcl-2 protein. Our findings suggest that decline of DHEA(S) and Allo during aging or stress may leave the adrenal medulla unprotected against proapoptotic challenges.

Figures

Fig. 1.

DHEA(S) and Allo protected rat chromaffin cells in culture against serum deprivation-induced apoptosis. Freshly isolated rat chromaffin cells were cultured either in complete or serum-free media containing 10–7 M DHEA, DHEAS, or Allo for various time periods (2–48 h). Apoptosis was quantified by the APOPercentage assay. Values represent mean ± SD of three independent experiments. Each measurement was performed in triplicate (*, P < 0.05).

Fig. 2.

DHEA(S) and Allo protected PC12 rat pheochromocytoma cells against serum deprivation-induced apoptosis. Cells were cultured either in complete media (serum-supplemented) or serum-free media containing steroids as per protocol. (A) Time course. Cells cultured for various time periods (2–48 h) in serum-free medium in the absence or presence of 10–7 M DHEA, DHEAS, or Allo. Apoptosis was quantified by the APOPercentage assay, depicted as OD. Values represent mean ± SD of six separate experiments, each performed in triplicate (*, P < 0.05). (B) Dose response. Cells cultured for 24 h in serum-free medium containing different steroid concentrations ranging from 10–11 to 10–5 M. Apoptosis was quantified by the APOPercentage assay, depicted as percent of serum-free parallel controls. Values represent mean ± SD of three separate experiments, each performed in triplicate (*, P < 0.05). (C) Representative FACS analysis. Cells were cultured in 24-well plates (106 cells per well) for 24 h in serum-free media in the absence or presence of 10–7 M DHEA, DHEAS, or Allo. The x axis represents Annexin V-FITC, whereas the y axis represents the number of events. A total of 10,000 cells were assigned per treatment. (D) Cells were cultured for 24 h in serum-free media with various steroids at 10–7 M in the absence or the presence of 10–5 M of estrogen receptor antagonist ICI 182,780 or the σ1R antagonist haloperidol. Apoptosis was quantified by the APOPercentage assay, depicted as OD. Values represent mean ± SD of four separate experiments, each performed in triplicate (*, P < 0.05).

Fig. 3.

DHEA(S) and Allo induced the expression of the antiapoptotic Bcl-2 proteins in serum-deprived PC12 cells. Cells were cultured for 2–12 h either in complete or serum-free media containing 10–7 M DHEA, DHEAS, or Allo. Cellular extracts containing total mRNA or total proteins were collected, and levels of Bcl-2 and Bcl-xL mRNAs (A) and proteins (B) were measured either by semiquantitative RT-PCR or by Western blot, as described in Materials and Methods and normalized per actin mRNA or protein content, respectively. Values represent mean ± SD of three independent experiments (*, P < 0.05).

Fig. 4.

Antisense oligonucleotides against the Bcl-2 transcript reversed the cytoprotective effects of DHEA(S) and Allo on serum-deprived PC12 cells. Cells were cultured for 24 h in serum-free media containing 10–7 M DHEA, DHEAS, or Allo in the absence or the presence of 50 μmol per liter of nonsense (NS) or antisense (AS) 20-mer phosphorothioate oligodeoxynucleotides directed toward the translation-initiation site of the Bcl-2 transcript. Apoptosis was quantified by the APOPercentage assay (A) and the Bcl-2 proteins by Western blot (B). Values represent the mean ± SD of three independent experiments (*, P < 0.05); S, steroid; 1, Bcl-2; 2, actin).

Fig. 5.

DHEA(S) and Allo activated transcription factors NF-κB and CREB in serum-deprived PC12 cells. Cells were cultured in complete or serum-free media containing 10–7 M DHEA, DHEAS, or Allo. (A) Representative EMSA assay, performed with 1 μg of nuclear extract of cells treated with steroids for 60 min, combined with 5 × 105 cpm of a 32P-labeled double-stranded DNA probe representing the NF-κB-binding site. Similar results were obtained in two additional independent experiments. (B) Confocal laser-scanning microscopy of cells grown on chamber slides and treated with steroids for 1 h were fixed and incubated with a rabbit anti-p65 NF-κB antibody and visualized by using FITC-conjugated secondary anti-rabbit polyclonal antibody. Cells were analyzed by using confocal laser-scanning microscopy (×400). (C) Western blot analysis of cells treated with steroids for 60 min, with antibodies specific for phosphorylated (pCREB) and total CREB (tCREB). Values are expressed as pCREB/tCREB ratio and represent mean ± SD of three independent experiments (*, P < 0.05).

Fig. 6.

DHEA(S) and Allo induced the phosphorylation of PKC in serum-deprived PC12 cells. Cells were cultured for 10–30 min either with complete or serum-free media, containing 10–7 M DHEA, DHEAS, or Allo. Western blot analysis was performed by using antibodies specific for phosphorylated (pPKC) and total (tPKC) conventional PKCα/β. Values are expressed as pPKC/tPKC ratio and represent mean ± SD of three independent experiments (*, P < 0.05).