Effects of ACTH and cAMP on steroidogenic acute regulatory protein and P450 11β-hydroxylase messenger RNAs in rainbow trout interrenal cells: Relationship with in vitro cortisol production

Abstract

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane, thereby making the molecule available for cholesterol side-chain cleavage enzyme, which carries out the first conversion in the steroidogenic pathway. In mammals, StAR controls this rate limiting step in steroidogenesis, and both StAR protein and StAR mRNA levels become rapidly elevated in response to tropic hormone stimulation. The relationship between StAR gene expression and steroid production in fish has not yet been well explored. We investigated the relationship between adrenocorticotropic hormone (ACTH)- and cAMP-stimulated cortisol production in vitro and levels of StAR transcripts in interrenal cells of rainbow trout. To assess the effect of ACTH on mRNA levels of a downstream steroidogenic enzyme, we also investigated the effects of ACTH on transcripts encoding 11β hydroxylase (P450 11β). In a series of experiments, juvenile rainbow trout head kidney tissue containing interrenal cells was incubated with either ACTH or dibutyryl cyclic AMP (dbcAMP). Cortisol in incubation media were measured by radioimmunoassay and total RNA was isolated from the tissue for Northern analysis or for quantitative real-time PCR. Incubation of tissue with 150 ng/mL ACTH for 1–18 h induced a progressive increase in cortisol accumulation in media, but StAR mRNA levels increased modestly and mostly insignificantly over 18 h, irrespective of treatment. Exposure of tissue for 18 h to 5, 150, 500 or 1500 ng ACTH/mL resulted in a strong increase in cortisol production, with a peak response (15-fold increase over controls) achieved with 150 ng/mL ACTH. Although there was a trend towards a dose–response effect, mean StAR mRNA levels were only significantly affected by the highest concentration of ACTH used (1500 ng/mL), which induced a less than 2-fold increase in StAR transcripts. However, there was a significant linear relationship between StAR mRNA levels and ACTH-induced cortisol accumulation in media ( p < 0.001, r 2 = 0.55). Incubation of tissue with 5 mM dbcAMP for 6 or 18 h induced large increases in cortisol accumulation in media over controls, but had no significant effect on StAR mRNA levels. By contrast, ACTH induced a clear dose-dependent increase in P450 11β transcripts, with 150 ng/mL ACTH inducing an 8-fold increase in levels compared to control; nonetheless, only a weak correlation existed between transcript levels and ACTH-induced cortisol secretion ( p < 0.003, r 2 = 0.26). Thus, despite the relatively high degree of conservation of StAR proteins in vertebrates, we have been unable to demonstrate that a rapid, acute increase in transcription of the StAR gene is the dominant mechanism supporting flow of cholesterol to the mitochondria during acute increases in cortisol production in rainbow trout. The strong stimulation of P450 11β gene transcription by ACTH probably enhances biosynthetic capacity during longer term chronic ACTH stimulation.