The human adrenal cortex is a complex endocrine gland that secrets mineralocorticoids and glucocorticoids. These steroids arise from morphologically and biochemically distinct zones of the adrenal cortex. The long range objective of the proposed research is to define the mechanisms that lead to and control zonation of the human adrenal. There is formidable evidence that aldosterone synthase (P450c18) concentrates in the zona glomerulosa and that zona fasciculata cells express 11beta- hydroxylase (P450c11), 17alpha-hydroxylase (P450c17), but no P450c18. The lack of an appropriate in vitro model system that maintains zona glomerulosa function has limited our understanding of the mechanisms controlling the zone-specific expression of these enzymes and their role in the norman and pathophysiology of the adrenal. We have overcome this obstacle by the development of a strain of NCI-H295 cells that will differentially express P450c18, P450c11, and P430c17 in a zone-specific manner. In addition, we have demonstrated that the H295 cells represent the first adrenocortical cell line which has maintained the ability to produce aldosterone and respond to angiotensin II. We will utilize these cells to define the biomolecular regulation of adrenal zonation.
In Specific Aim 1, the principal second messenger systems regulating zone- specific steroidogenesis and the expression of the mRNAs for P450c18, P450c11, and P450c17 will first be determined. Due to the centripetal nature of adrenocortical blood flow, the role of intra-adrenal factors (i. e., insulin-like growth factors, transforming growth factor betas and steroid hormones) in zone-specific steroidogenesis and expression of P450c18, P450c11, and P450c17 will also be determined. The zone-specific expression of steroidogenic enzymes is most likely due to regulation of gene transcription. Thus, the 5'-genetic regions responsible for the activation or inhibition of transcription of P450c18 and P450c17 will be determined in Specific Aim 2, using transfection assays of chimeric gene constructs into H295 cells. These experiments will help to determine the specific consensus sequences involved in the zone-specific expression of these enzymes. By way of preliminary studies, we find that AII is importance in the regulating the zone-specific production of steroids and differential expression of steroidogenic enzymes. The selected expression of the type 1 angiotensin II receptor (AT1-R) in the zona glomerulosa may be instrumental in causing zonation. Therefore, in Specific Aim 3, the endocrine and paracrine mechanisms regulating H295 cell expression of the AT1-R will be defined by monitoring angiotensin II binding and the level of AT1-R mRNA by northern analysis. Taken together the information gained from these studies will help define the fundamental mechanisms controlling zonation in the mammalian adrenal.
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