Steroidogenic acute regulatory protein expression and pregnenolone synthesis in rat astrocyte cultures.

ABSTRACT

Neurosteroids are steroids synthesised by brain cells. The molecular mechanism of neurosteroidogenesis from cholesterol has not yet been revealed. We studied the potential role of the steroidogenic acute regulatory (StAR) protein in neurosterodogenesis by using rat brain astrocytes. The novelty of the study is that regulation of StAR is described in primary cultures from embryonic mesencephalon and cerebellum regions of the brain. Dibutyryl cyclic AMP (dbcAMP) treatment increased StAR protein expression in astrocyte cultures. This was observed in immunoblots of mitochondrial fractions and by immunocytochemistry. Dual-labelling showed that the cyclic AMP-induced increase in StAR immunofluorescence was localised to mitochondria. In addition, mitochondrial cytochrome P450-side chain cleavage enzyme was demonstrated with a specific antibody, indicating the potential for pregnenolone production in these cells. Radioimmunoassay on ether-extracted conditioned media of control and dbcAMP treated cells demonstrated pregnenolone production by mesencephalic and cerebellar astrocyte cultures. Furthermore, 24-h pregnenolone levels, in the presence of inhibitors of further pregnenolone metabolism, were significantly increased by dbcAMP exposure. A murine StAR promoter-luciferase fusion plasmid was activated by dbcAMP in transiently transfected mesencephalic and cerebellar astrocytes. These novel results indicate that cyclic AMP signalling can regulate StAR expression and pregnenolone production in brain astrocytes, and provide additional insight into the role of StAR in neurosteroidogenesis.