Abstract
Optimal steroid hormone output from the adrenal cortex depends on the peptide hormone ACTH which elicits its effect by increasing the intracellular levels of the second messenger cAMP, and a subsequent activation of the cAMP-dependent protein kinase. An important aspect of ACTH action is the ability of this hormone to increase transcription of a group of genes that encodes the steroid hydroxylases, enzymes that catalyze the conversion of cholesterol to biologically active steroids. A general feature of these genes is that they depend on atypical cAMP-responsive sequences not found in other genes that are regulated by this second messenger.
A major focus of this thesis was to investigate the molecular mechanisms underlying cAMP-induced transcription of the gene encoding cytochrome P450 17alpha-hydroxylase (CYP17). This enzyme is necessary for syntbesis of cortisol and adrenal androgens. CYP17 contains two sequences in its promoter region that mediate cAMP-responsiveness on reporter genes when transfected into steroidogenic cells. These sequences, which are designated cAMP-responsive sequences 1 and 2 (CRS1 and CRS2), display differences with regard to nucleotide sequence and protein interactions. CRS1 and CRS2 are also differently regulated by the protein kinase C signal transduction pathway, since treatment of adrenocortical cells with phorbol esters represses cAMP-induced transcription mediated by CRS1, but has no effect on transcription conferred by CRS2. The mouse VL30 elements constitute a family of retrotransposons that are highly inducible by various physiological stimuli, and in steroidogenic tissues, VL30 expression is induced in response to tropic hormones.
We identified DNA regulatory elements that mediated the ACTH-dependent transcription of a VL30 retrotransposon in mouse adrenal cells. These elements were related to classical cAMP regulatory elements (CREs) but exhibited differences in protein binding both with respect to affinity and specificity, suggesting that steroidogenic-cell specific gene expression in response to cAMP can be mediated both through DNA sequences related to classical CREs as well as through atypical cAMP-responsive sequences present in the steroid hydroxylase genes. Taken together, the results presented in this thesis provide new insights into the molecular aspects of ACTH action on steroid hormone biosynthesis, and also unravel novel aspects of cAMP-dependent gene regulation.
