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Adrenal Zona Glomerulosa Targeting in Transgenic Mice Jeniel Parmar

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Adrenal Zona Glomerulosa Targeting in Transgenic Mice Jeniel Parmar Adrenal Zona Glomerulosa Targeting in Transgenic Mice By Jeniel Parmar Submitted to the Faculty of the School of Graduate Studies of the Medical College of Georgia in partial fulfillment of the Requirements of the Degree of Doctor Philosophy December, 2009 V Adrenal Zona Glomerulosa Targeting in Transgenic Mice This dissertation is submitted by Jeniel Parmar and has been examined and approved by an appointed committee of faculty of the School of Graduate Studies of the Medical College of Georgia. The signatures which appear below verify the fact that all the required changes have been incorporated and that the thesis/dissertation has received final approval with reference to content, form, and accuracy of presentation. This dissertation is therefore in partial fulfillment of the requirement for the degree of Doctor of Philosophy. Date Major Advisor - William E. Rainey, 8 Dept. Chair - R Clinton Webb, PhD Dean, School of Gr te Studies - Gretchen Caughman, PhD VI JENIEL PARMAR Adrenal Zona Glomerulosa Targeting in Transgenic Mice (Under the guidance of WILLIAM E. RAINEY II~ Ph.D.) 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. I 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 cis­ regulatory 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. II 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. III INDEX WORDS: Adrenal cortex, aldosterone, aldosterone synthase, transgenic mice, Lacz, cell lines, in vitro model systems. NOMENCLATURE GUIDLINES: Throughout this dissertation, names of all human genes and proteins will be fully capitalized (e.g., CYP11 B2, CYP11 B1, and CYP17)-. The names of all rodent genes and proteins will be depicted with only the first letter capitalized (e.g., Cyp11 b2, Cyp11 b1, and Cyp17). IV TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................ IX LIST OF FIGURES ......................................................................X LIST OF TABLES ........................................................................XI I LIST OF ABBREVIATIONS ........................................ ■- •• ___ •••••••••••••• XI U I. INTRODUCTION A. Statement of the Problem: CYP1182 Gene Regulation ........ _.. 1 1) Specific Aim I ............................................................... 3 2) Review of Literature and Rationale .............................. 4 · B. Statement of the Problem: A Paucity of Adrenal Cell Lines .... 20 1) Specific Aim 11 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 22 2) Review of Literature and Rationale .............................. 23 II. MANUSCRIPTS· A. Peer Reviewed Articles ........................................................... 30 1) "Gene targeting to the mouse adrenal zona glomerulosa" VII 2) "Development of an ACTH Responsive Human Adrenocortical Carcinoma Cell Line" 8. Invited Review Article ............................................................. 76 "Comparisons of Adrenocortical Cell Lines as in vitro Test Systems" C. Book Chapter ........................................................................ 107 "Adrenocortical Cell Lines" Ill. DISCUSSION A. Specific Aim I: CYP1182 Gene Regulation ........................... 146 B. Specific Aim II: A Paucity of Adrenal Cell Lines .................... 155 IV. BIBLIOGRAPHY ............................. ~ ................................... 162 VIII ACKNOWLEDGEMENTS I wish to express my sincerest gratitude to Dr. William E. Rainey for taking me under his wing and guiding me throughout my graduate career. Seldom does one have the opportunity to learn from a true advisor. I deeply value the wisdom and scientific expertise that he bestowed upon me. I will miss his enthusiasm and ceaseless support. The experience will surely never be forgotten. I am indebted to Dr. Tsugio Seki for his kind technical as well as advisory support. I would also like to thank my committee members Dr. Edward Inscho for never letting me forget about the kidneys, Dr. Wendy Bollag for regularly reminding me about the importance of primary cell cultures, and Dr. Lawrence Layman for teaching me the value of statistical analyses. My appreciation is extended to the readers of my thesis Dr. Michael Edwards and Dr. Celso Gomez-Sanchez for willingly provide selfless support. I would like to thank the faculty of the department of Physiology at the Medical College of Georgia for providing me with an exceptional education. I would also like to thank the past and present Rainey lab members and our department management including Anthony, Jennifer, Daniel, Claudia, Rebecca, Nick, Bobbie, Kathy, Faye, Valerie, and Brenda, who have all been a distinguished part of my life in the past several years. Finally, a special word of appreciation must be extended to my family. I would like to thank my parents for their seemingly inexhaustible faith and understanding· during my education. I would like to thank my best friend and brother, Hiren, and his wife, Jigna, for their eternal support and their cheerful and enthusiastic endorsements. I would also like to thank my friend, companion and wife, Ami, for her love and compassion. IX LIST OF FIGURES Figure Page 1. Phylogenetic an.cestral lineages ............................................................. 5 2. H&E stained mouse adrenocortical tissue ................................................. 10 3. Major adrenocortical steroidogenic pathways ............................................ 12 4. Glucocorticoid suppressible hyperaldosteronism model ..........- ..................... 16 5. Cyp11 b2 evolutionary conserved regions (ECR) ....................................... 18 6. Cyp11 b2 ECR analysis and Tg construct (manuscript A 1) ........................... 53 7. Cyp11 b2 transcript quantity (manuscript A1 } ......· .........................•............
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