Differential regulation of apoptosis-associated genes by estrogen receptor alpha in human neuroblastoma cells.

PURPOSE: The neuroendocrinology of female sex hormones is of great interest for a variety of neuropsychiatric disorders. In fact, estrogens and estrogen receptors (ERs) exert neuromodulatory and neuroprotective functions. Here we investigated potential targets of the ER subtype alpha that may mediate neuroprotection and focused on direct modulators and downstream executors of apoptosis. METHODS: We employed subclones of human neuroblastoma cells (SK-N-MC) stably transfected with one of the ER subtypes, ERalpha or ERbeta. Differences between the cell lines regarding the mRNA expression levels were examined by qPCR, changes on protein levels were examined by Western Blot and immunocytochemistry. Differences concerning apoptosis induction were analysed by cell survival assays which included primary rat neurons. RESULTS: In this report we show a potent protection against apoptosis-stimuli in ERalpha expressing cells compared to controls lacking ERalpha. In fact, almost a complete silencing of Caspase 3 expression in SK-ERalpha cells compared to SK-01 control transfected cells was observed. In addition, prosurvival bcl2, bag1 and bag3 expression was highly up-regulated in the presence of ERalpha. CONCLUSION: Taken together, we identified Caspase 3, BAG1 and BAG3 as key targets of ERalpha in neuronal cells that may play a role in ERalpha-mediated neuroprotection.

Oestrogen receptor subtype-specific repression of calpain expression and calpain enzymatic activity in neuronal cells--implications for neuroprotection against Ca-mediated excitotoxicity.

Calpains represent a superfamily of Ca2+-activated cysteine-proteases, which are important mediators of apoptosis and necrosis. In the brain, m-calpain and micro-calpain, the two ubiquitous calpain-isoforms, are strongly activated in neurones after an excitotoxic Ca2+ influx occurring, for example, during cerebral ischemia. Because oestrogen and its receptors (ERalpha/ERbeta) can exert neuroprotective activity, we investigated their influence on expression of calpains and their endogenous inhibitor, calpastatin. We found that ectopic expression of ERalpha in human neuroblastoma SK-N-MC cells led to a ligand-independent constitutive down-regulation of m-calpain accompanied by an up-regulation of micro-calpain expression. Up-regulation of micro-calpain was reversed in the presence of oestrogen, which, in turn, could be blocked by co-treatment with the oestrogen-receptor antagonist ICI 182,780. Expression of calpastatin was not altered, either in the absence or in the presence of oestrogen. Additional studies revealed that ERalpha-expressing cells exhibited decreased calpain enzymatic activity and increased survival when cells were exposed to the Ca2+ ionophore, ionomycin. Since all investigated effects could be observed exclusively in the presence of ERalpha, but not ERbeta, and since the effects are reduced when ERalpha and ERbeta are co-expressed, our data suggest a novel ER subtype-specific neuroprotective action by repressing calpain expression and calpain activity under conditions of a massive Ca2+ influx.

Functional interaction of estrogen receptor alpha and caveolin isoforms in neuronal SK-N-MC cells.

Estrogen receptors (ERs) are expressed in neuronal cells and exhibit a wide variety of activities in the central nervous system. The actions of ERs are regulated in a hormone-dependent manner as well as by a number of co-activators and -repressors. A recently identified co-activator of ERalpha is caveolin-1 which has been shown to mediate the ligand-independent activation of this steroid receptor. In the present study we have demonstrated that neuronal SK-N-MC cells lacking functional ERalpha show high levels of caveolin-1/-2 specific transcripts and proteins. Ectopic expression of ERalpha in SK-N-MC cells leads to the transcriptional suppression of caveolin-1 and -2 genes. This silencing event is accompanied by changes in the methylation pattern of the caveolin-1 promoter. Certain CpG dinucleotides were methylated in the caveolin-1 promoter region of the SK-ERalpha cells whereas the same sites were non-methylated in control SK-N-MC cells, implicating a gene silencing mechanism including hypermethylation of DNA. In addition, inhibitors of methyltransferases or histone deacetylases, enzymes involved in the establishment and maintenance of silenced chromatin status, partially restored caveolin transcription in SK-ERalpha cells. In conclusion, our observations provide a possible mechanism of negative feedback regulation of ERalpha co-activator caveolin by the steroid receptor itself in this cellular model.

Estrogen receptor alpha-mediated silencing of caveolin gene expression in neuronal cells.

Estrogen receptors (ER alpha/ER beta) are expressed in neuronal cells and exhibit a variety of activities in the central nervous system. ER activity is regulated in a ligand-dependent manner and by co-regulatory factors. Caveolin-1 is a recently identified co-activator of ER alpha mediating the ligand-independent activation of this steroid receptor. Here the influence of ERs on caveolin expression in human neuroblastoma SK-N-MC cells as well as in rodent brain was investigated. We found that ectopic expression of ER alpha in SK-N-MC cells (SK-ER alpha) leads to a ligand-independent transcriptional suppression of caveolin-1/-2 genes. This suppression is specifically mediated by ER alpha and not ER beta because ER beta counteracts the observed caveolin-silencing process. Interestingly, decreased caveolin expression in SK-ER alpha is accompanied by changes in the methylation pattern of caveolin promoters. The analysis of selected promoter regions of the human caveolin-1 gene showed that certain CpG dinucleotides were hypermethylated in SK-ER alpha cells, whereas the same sites were unmethylated in control, ER beta-, and ER alpha/beta co-expressing SK-N-MC cells. Inhibition of DNA methylation or histone deacetylation led to partial re-expression of caveolin-1/-2 genes in SK-ER alpha. In vivo analysis revealed a down-regulation of caveolin-1 expression after long term estrogen exposure in certain regions of the mouse brain. In conclusion, we have shown for the first time that ER alpha and not ER beta silences caveolin-1/-2 expression in an epigenetic fashion in neuronal cells. The observed mechanism of gene silencing by ER alpha may have implications for the transcriptional regulation of further ER alpha target genes.