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The Identification and Characterization of Estrogen

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The Identification and Characterization of Estrogen Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 2009 The deI ntification and Characterization of Estrogen Receptors in the Mouse and Human Lens and Their Role in Cataract Development Mary Rachel Kirker Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Kirker, M. (2009). The deI ntification and Characterization of Estrogen Receptors in the Mouse and Human Lens and Their Role in Cataract Development (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/750 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. THE IDENTIFICATION AND CHARACTERIZATION OF ESTROGEN RECEPTORS IN THE MOUSE AND HUMAN LENS AND THEIR ROLE IN CATARACT DEVELOPMENT A Dissertation Submitted to the Graduate School of Pharmaceutical Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By M. Rachel Kirker December 2009 Copyright by M. Rachel Kirker 2009 THE IDENTIFICATION AND CHARACTERIZATION OF ESTROGEN RECEPTORS IN THE MOUSE AND HUMAN LENS AND THEIR ROLE IN CATARACT DEVELOPMENT By M. Rachel Kirker Approved March 27, 2009 ________________________________ ________________________________ Vicki L. Davis, Ph.D. Jane E. Cavanaugh, Ph.D. Dissertation Chair Committee Member Assistant Professor of Pharmacology Assistant Professor of Pharmacology Graduate School of Pharmaceutical Sciences Graduate School of Pharmaceutical Science Duquesne University Duquesne University Pittsburgh, Pennsylvania Pittsburgh, Pennsylvania ________________________________ ________________________________ Carmen M.H. Colitz, D.V.M., Ph.D. Kyle W. Selcer, Ph.D. Committee Member Committee Member Diplomate, ACVO Associate Professor of Biological Sciences Animal Eye Specialty Clinic Bayer School of Natural and Aquatic Animal Eye Care Environmental Sciences Adjunct Associate Professor Duquesne University North Carolina State University and Pittsburgh, Pennsylvania The Ohio State University ________________________________ ________________________________ Paula A. Witt‐Enderby David A. Johnson, Ph.D. Committee Member Associate Professor of Pharmacology and Professor of Pharmacology and Toxicology Toxicology Graduate School of Pharmaceutical Sciences Director of Graduate Studies Duquesne University Graduate School of Pharmaceutical Sciences Pittsburgh, Pennsylvania Duquesne University Pittsburgh, Pennsylvania ________________________________ J. Douglas Bricker, Ph.D. Dean of Mylan School of Pharmacy and Graduate School of Pharmaceutical Sciences Duquesne University Pittsburgh, Pennsylvania iii ABSTRACT THE IDENTIFICATION AND CHARACTERIZATION OF ESTROGEN RECEPTORS IN THE MOUSE AND HUMAN LENS AND THEIR ROLE IN CATARACT DEVELOPMENT By M. Rachel Kirker December 2009 Dissertation supervised by Vicki L. Davis, Ph.D. The increased risk of age‐related cataracts in postmenopausal women and studies in animal models suggest that estrogen may have a protective role in the lens. However, very little is known regarding the role of estrogen and its receptors in the lens. To begin unraveling the estrogen signaling mechanism in the lens, the following aims were investigated: 1) to determine if estrogen receptors are expressed in the lens, [125I]‐17β‐estradiol binding and mRNA expression of ERα, ERβ and GPR30 were examined in the mouse and human lens; 2) to determine if the loss of ERα and/or ERβ receptors will induce spontaneous development of cataracts and to examine their role in an inducible iv cataract model; and 3) to identify estrogen‐regulated genes in the lens that influence cataract development in the ER∆3‐induced cataract model. High‐ affinity, saturable binding sites for 17β‐estradiol were identified in the nuclear, cytosolic, and membrane fractions of the mouse and human lens. Additionally, detectable binding in the membrane fraction and expression of GPR30 mRNA in the mouse and human lens are the first evidence of this novel transmembrane estrogen receptor in the lens of any species. Transcripts for ERα, ERβ, and GPR30 were expressed in the mouse lens which suggests that one or more of these estrogen receptor subtypes are responsible for the binding detected. With the loss of nuclear ER in the lens, spontaneous cataracts did not occur; however, diminished levels or loss of ERα in ER∆3 female mice increased the severity of cortical cataracts with age. These results suggest that ERα in the lens may provide protection against the progression of cataracts in the ER∆3 model. Together with the cataract induction and gene expression studies, six genes were identified to be differentially expressed with estrogen versus vehicle treatment in the lenses of ER∆3 mice. The pax6, tcfap2a, tgfβ2, six3, sox2, and pdgfα genes are known to have a critical role in lens development, proliferation, differentiation and maintenance of lens homeostasis. Therefore, future examination of these genes and their pathways in the lens may contribute to the understanding of the mechanisms of estrogen‐mediated protection of lens transparency. Knowledge v of pathways that function to maintain lens transparency and how estrogen regulated these pathways will assist in the development of estrogen therapies that can be clinically used to delay the onset and/or progression of cataracts. vi DEDICATION To my husband, Herschel, and my parents, Sharon and Ernie Jacobs, who made all this possible with their endless patience and words of encouragement. Without them, I would not be the person I am today. vii ACKNOWLEDGEMENTS I owe thanks to many people, whose assistance was indispensable in completing this project. First among these is Dr. Vicki L. Davis, advisor and committee chair, for her patience and guidance during my tenure at Duquesne. I thank my committee members for their input and support throughout the study. I would like to specifically thank, Drs. Paula Witt‐Enderby and Kyle Selcer for their guidance in the development of the binding studies; Dr. Carmen M.H. Colitz for her many travels to Duquesne University to teach me how to use the slit‐lamp apparatus; and Dr. Jane Cavanaugh for her mentorship over the last year. I want to personally thank, Sheri Rea for her organization skills, without them, I would have never been able to keep track of the hundreds, if not thousands, of animals needed to be generated to complete the Cataract Development Study. Thank you to everyone that I have had a pleasure to work with in the Davis Lab: Sheri Rea, Pangi Johnson, Mary Kotlarczyk, Katie Gallagher, Kathleen Ward, Katie Kelly and Heather Rice. To all my fellow graduate students who have made my experience at Duquesne as pleasurable as it can be. Lastly, I would like to thank my husband, family and friends for their endless support and love; thank you for helping me grow as the person I am today. viii TABLE OF CONTENTS Page Abstract............................................................................................................................. iv Dedication .......................................................................................................................vii Acknowledgements ......................................................................................................viii List of Tables................................................................................................................... xii List of Figures ................................................................................................................. xv List of Abbreviations .................................................................................................... xix Introduction ......................................................................................................................1 Cataracts.................................................................................................................2 Estrogen Receptors.............................................................................................10 Estrogen Receptor Knockout and Transgenic Mouse Models.....................20 ERKO Mouse Models ............................................................................20 ER∆3 Transgenic Mouse Model ...........................................................23 The Crystalline Lens ...........................................................................................28 Retinal Effects on the Lens.................................................................................36 Statement of the Problem...............................................................................................39 Research Objective..............................................................................................39 Hypothesis...........................................................................................................41 Specific Aims.......................................................................................................41 ix Methods............................................................................................................................42 Animal Husbandry.............................................................................................42 ER∆3 Colony........................................................................................................43
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