The regulation of steroid 11 [beta]-hydroxylase gene expression in cultured bovine adrenocortical cells

Abstract

Second messenger systems play an important role in regulating gene expression. Adrenocorticotropin (ACTH) acting through cAMP dependent protein kinase is known to induce the adrenal cytochrome P450 steroidogenic enzymes, 11~-hydroxylase and 17a-hydroxylase by increasing transcription of the genes for these enzymes. In this thesis, activators of Ckinase and IGF-I were studied to examine their effects on enzyme activity and gene expression. IGF-I has an important function in llP-hydroxylase expression. An absence of IGF-1 results in a reduced level of both cAMP-stimulated 11~hydroxylase enzyme activity and mRNA. This effect was specific for 11~- · hydroxylase, as 17a-hydroxylase mRNA levels and enzyme activity were not affected in the absence of IG F-1. Since these enzymes were regulated differently by IGF-1, it was possible they were. regulated differently by A-kinase activating agents. Although the agents stimulated both 17a-hydroxylase and llP-hydroxylase, llP-hydroxylase activity was more sensitive to cAMP levels, and declined at cAMP concentrations_ above 100 μM, suggesting that A-kinase may downregulate in the presence of excess cAMP. _Although some steroids serve as negative regulators of 11~hydroxylase activity; it was unknown whether these effects were transcriptional. In the _presence of androstenedione, enzyme activity was inhibited, bt1st mRNA levels remained unchanged, suggesting a posttranscriptional 8:Ction. In the absence of ascorbate, which prevents oxidative damage. to. llf3-hydroxylase, the enzyme activity was decreased; mRNA levels- were unaffected, suggesting that ascorbate also acts posttranscriptionally. Effects of protein kinase C activation were analyzed using the phorbol ester, TPA. Although TPA alone had no effect, it decreased the levels of cAMP induced mRNA and enzyme activity of llf3-hydroxylase. Angiotensin II had similar effects.