Rainey Lab Research

I. Regulation of Zonation of the Mammalian Adrenal Gland

BACKGROUND: The human adrenal cortex acts as a compound endocrine gland that secretes both mineralocorticoids and glucocorticoids. These steroids arise from distinct zones of the adrenal cortex that have both morphologic and biochemical differences (Fig. 1).

Neither the origin of the stem cells of the cortical zones nor the mechanisms leading to the zone-specific production of steroids is clearly defined.

The functional zonation of the adrenal cortex can be traced to the zone-specific expression of the enzymes involved in steroid biosynthesis (Fig 2).

This is particularly true for aldosterone synthase (CYP11B2), 11beta-hydroxylase (CYP11B1) and 17alpha-hydroxylase (CYP17).  Our laboratory focuses on the regulated expression of these enzymes to better define the molecular mechanisms leading to the zonation of the adrenal cortex.

GOALS OF THIS PROJECT:
Our First Goal is to extend our previous studies directed at defining the regulatory elements in the 5’-flanking region of aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) genes. These studies will complete the definition of the trans-acting factors responsible for glomerulosa specific expression and fasciculate repression of CYP11B2. Focus will be placed on the role of nuclear receptor, NURR1 (NR4A2), which stimulates CYP11B2 promoter activity, is up-regulated by angiotensin II and is expressed primarily in the zona glomerulosa.

Goal Two will be to determine the mechanisms responsible for the lack of CYP17 seen in the glomerulosa.  Preliminary studies indicate that zonation results from multiple mechanisms including angiotensin II increased AP-1 transcription factors and WNT signaling. The WNT downstream factors responsible for CYP17 repression and the mechanisms of AP-1 repression of CYP17 transcription will be defined.

Goal Three will extend our ongoing adrenal cell culture studies to the in vivo situation using bacterial artificial chromosome bacterial (BAC) transgenesis to target zona glomerulosa with an inhibitor of NURR1. The murine adrenal, like the human adrenal, limits expression of CYP11B2 to the zona glomerulosa making it an excellent model to define the factors regulating aldosterone production. We will also use this strategy to produce a glomerulosa-CRE mouse that will be an important tool for further study of genes that may regulate glomerulosa function.

SIGNIFICANCE: Hyperaldosteronism results from the disruption of zonation, and particularly aberrant CYP11B2 expression in nodular hyperplasia and aldosterone-producing adenomas. The proposed studies will provide a detailed understanding of the molecular mechanisms regulating CYP11B2 expression within the adrenal and should provide insight into the diseases associated with aberrant expression.

II. The Molecular Cause of Adrenarche and Mechanisms Controlling Adrenal Androgen Production

The human adrenal cortex produces aldosterone, cortisol and the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S). The processes regulating aldosterone and cortisol synthesis are well defined; however, the mechanisms regulating the production of DHEA-S remain elusive. Adrenarche refers to the onset of DHEA and DHEA-S production from the adrenal zona reticularis that can be detected around 6 years of age (Fig 3).

The phenotypic result of adrenarche is pubarche or the development of axillary and pubic hair that occurs in both girls and boys at about age 8 to 9.  The phenomenon of adrenarche is unique to human beings and to some old world primates, and a reversal of adrenarche appears to occur in the aging process (Fig 3).  Premature and exaggerated adrenarche can be indicative of future onset of adult diseases, thus increasing the clinical relevance of adrenarche. The physiological triggers of adrenarche and the physiologic role(s) of DHEA-S remain speculative.  However, the biochemical pathways that define adrenarche have been characterized in detail, and the appearance of key enzymes and cofactors in the adrenal zona reticularis track with the progression of adrenarche.

Here we take the novel approach of focusing on the mechanisms that regulate the development of the DHEA-S-secreting phenotype that is associated with the zona reticularis. We propose to define the mechanisms that regulate adrenal cell expression of two proteins that directly impact the ability of the adrenal reticularis to produce DHEA-S. The enzyme 3β-hydroxysteroid dehydrogenase (HSD3B2) has the unique ability to remove precursors from the pathway leading to DHEA-S thereby inhibiting adrenal androgen biosynthesis. HSD3B2 is expressed at high levels in the cortisol-producing cells of the adrenal but is not expressed in the DHEAS-producing cells of the adrenal reticularis. Our first goal is to focus on the transcription of the gene encoding HSD3B2 with the objective of defining the reason for its absence in DHEAS-producing cells. Adrenarche is also associated with an increase in 17 alpha hydroxylase and particularly 17,20 lyase activity in part due to an increase in adrenal expression of cytochrome b5 at adrenarche. Our second goal will focus on mechanisms regulating transcription of the gene encoding cytochrome b5.  By focusing on the mechanisms regulating HSD3B2 and cytochrome b5, we hope to provide novel information on the regulation of the human adrenal reticularis and its production of DHEA-S.

III. Genetic Causes of Endocrine Hypertension

There is growing evidence that chronic inappropriate elevations in circulating aldosterone occur leading to renal, cardiovascular and other pathologic complications. Primary aldosteronism is a major cause of endocrine hypertension and has been proposed to affect ~6% of the hypertensive population. The most common causes of primary aldosteronism are aldosterone-producing adenoma (APA) and nodular hyperplasia, which occur in part due to the disruption of the tightly regulated and site-specific expression of CYP11B2 (aldosterone synthase). The best defined cause of hyperaldosteronism is glucocorticoid-suppressible hyperaldosteronism, which results from unequal meiotic crossing-over between CYP11B1 and CYP11B2, yielding a chimeric gene with a 5’ end corresponding to CYP11B1 and a 3’ end corresponding to CYP11B2. Such a gene encodes an enzyme with aldosterone synthase activity, but the gene is expressed in the zona fasciculata and is regulated by ACTH rather than ANG II. These findings demonstrate that DNA sequences within the 5’ ends of these genes mediate their zone-specific expression. In contrast to the comparatively well-understood etiology of glucocorticoid-suppressible hyperaldosteronism, the mechanisms leading to the autonomous hypersecretion of aldosterone in APA have not been defined. The suppressed renin levels seen in patients with primary aldosteronism suggest that APA are not ANG II driven. Several studies have demonstrated that adenomas maintain ANG II receptors and steroid-metabolizing enzymes, including CYP11B2. These two markers of the adrenal glomerulosa continue to be expressed even when the adenoma is found deep within the adrenal cortex suggesting a loss of the normal mechanisms which inhibit expression of CYP11B2 outside of the glomerulosa or an autonomous activation of signaling pathways that maintain its expression.

We have developed an international collaboration to determine the alterations in adrenal cell phenotype that caused APA. These include Dr. Paul Stewart (Birmingham, UK) a world leader in mineralocorticoid-induced hypertension and Franco Montero (Paudua, Italy) who is one of the world’s leading scientist in studying APA and has a large bank of normal and pathologic adrenal tissues. In addition we are working closely with Hironobu Sasano and Takashi Suzuki (Sendai Japan), who are experts on endocrine pathology and immunohistochemistry. With these collaborators, we have started using microarray comparison of the gene expression profile of normal adrenal and APA. We have found several candidate genes that could cause aberrant CYP11B2 expression and aldosterone production. We are currently connecting our array data to adrenal cell physiology. We believe this connection will provide important insights into mechanisms that underlie a significant cause of hypertension.