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

      Naseeruddin, Syed Ahmed; Department of Cell and Molecular Biology (1989-11)
      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.