Adrenal zona glomerulosa targeting in transgenic mice

Abstract

The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11 B2)-. CYP11 B2 is primarily expressed in the zona glomerulosa (ZG). of the adrenal cortex. Adrenocortical, expression of CYP11 B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Con$iderable in vitro analyses have been performed towards defining the mechanisms that control CYP11 B2 expression. Previous studies from our laboratory and others have identified · several cis-regulatory elements on the 5' · flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11 B2 gene transcription. Moreover, key· transcription factors that bind these cis-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP 11 B2 gene. However, thus far, all evidence is based on in. vitro analyses of transcriptional regulation, which does not always depict in vivo_occurrences. To initiate our in vivo assessment of CYP1182 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP1182 genes, which interestingly revealed high sequence similarity in the · 5' flanking promoter region of the CYP1182 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP1182 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11 b2 promoter driving expression of a Lacz reporter gene.· Importantly, 4 founder Tg mouse lines revealed Lacz expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed Lacz mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of Lacz exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of Lacz properly restricted to adrenal ZG, we concluded that regions required for Cyp11 b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment. will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly · and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroid.ogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.