Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3 ',5 '-monophosphate and p53 activation in immortalized human granulosa cells: Involvement of Bcl-2

Glucocorticoid hormones are known to enhance gonadotropin/cAMP-induced ster oidogenesis in rat and human granulosa cells. As glucocorticoids induce apo ptosis in numerous cell types, we investigated the role of glucocorticoids in the control of apoptosis in immortalized human granulosa cells (HO-23) t ransfected with a temperature-sensitive mutant of p53 (Val(135)). When HO-2 3 were incubated with forskolin in the presence or absence of dexamethasone (Dex) at 32 or 37 C, progesterone production was higher by 4- and 8-fold i n the presence of Dex at 37 or 32 C, respectively (P < 0. 01). The expressi on of adrenodoxin (ADX), which is an intrinsic part of the cytochrome P450 side-chain cleavage enzyme system, remained the same in the presence or abs ence of Dex in forskolin-stimulated cells. Dex reduced apoptosis (to 33% of control) in cultures after activation of p53 by shifting the temperature f rom 37 to 32 C. Moreover, Dex suppressed apoptosis induced by serum depriva tion (to 40% of control) or forskolin stimulation (to 28% and 40% at 37 and at 32 C, respectively). The protective effect of Dex on cAMP-, p53-, and s erum deprivation-induced apoptosis was confirmed by both 4',6-diamido-2-phe nylindole hydrochloride DNA staining and terminal deoxynucleotidyltransfera se-mediated dUTP nick end labeling with an ED50 of 7 nM Dex. Hydrocortisone showed a similar antiapoptotic effect. The protective effect of glucocorti coids against apoptosis was completely abolished by RU486 when cells were c oincubated with 10 nM Dex and 10-100 nM RU486. The protection against apopt osis by glucocorticoid involved a sharp elevation in intracellular levels o f Bcl-2 (3-7.6 fold; P < 0.01). In contrast to the effect of Dex in the pre vention of apoptosis in HO-23 granulosa cells, Dex dramatically stimulated apoptosis by 3-fold in LTR-6 myeloid leukemia cells expressing the same tem perature-sensitive mutant (Val(135) p53) and the same amount of glucocortic oid receptor-alpha. Forskolin did not stimulate apoptosis when incubated wi th these cells. However, it augmented by 1.2 fold the p53-induced apoptosis in cells shifted from 37 to 32 C. Dex further enhanced apoptosis by 1.9-fo ld in p53-activated cultures (32 C). Incubation of the cells with Dex drama tically reduced Bcl-2 levels to 15% of control at 37 C (P < 0.01) or 32 C i n the presence or absence of forskolin (P < 0.01). Our data suggest that gl ucocorticoids exert a protective effect against induced apoptosis in immort alized granulosa cells and a stimulatory effect on apoptosis in myeloid leu kemia cells. Moreover, modulation of Bcl-2 levels plays an important role i n mediating the glucocorticoid effect on cell survival. The opposite effect of glucocorticoids on Bcl-2 levels in the two cell lines may be due to the different ontogeneses of the two cell types: epithelial for granulosa cell s vs. mesenchymal for myeloid cells studied in the present work.