University of Cincinnati Date: 1/6/2011 I, Jody P. Ebanks , hereby submit this original work as part of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences/Biopharmaceutics. It is entitled: Differential Processing/Degradation of Melanosomes by Epidermal Keratinocytes Student's name: Jody P. Ebanks This work and its defense approved by: Committee chair: R. Randall Wickett Committee member: Tomohiro Hakozaki, PhD Committee member: Raymond Boissy Committee member: Pankaj Desai Committee member: Ana Luisa Kadekaro Committee member: Gerald Kasting 1298 Last Printed:1/13/2011 Document Of Defense Form Differential Processing/Degradation of Melanosomes by Epidermal Keratinocytes A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Division of Pharmaceutical Sciences of the James L. Winkle College of Pharmacy by Jody Patria Ebanks 2011 B.S. Biochemistry, Boston College, Chestnut Hill, MA, 2005 Committee Chair: R. Randall Wickett, Ph.D. ABSTRACT The synthesis and processing of melanosomes, the pigmented organelles of the follicular and interfollicular epidermis, is of major interest in the field of cutaneous biology. Additionally, clarification of the biological and cellular processes of cutaneous pigmentation has several therapeutic and cosmetic based applications, including the alleviation of skin hyperpigmentation. Modification of skin complexion coloration has traditionally been accomplished by inhibition of the rate limiting enzyme of melanogenesis, tyrosinase, or attenuation of melanosome transfer from melanocytes to keratinocytes. The post transfer modification of pigmented melanosomes, the main focus of this dissertation project, provides an attractive and distinct avenue of modulating skin pigmentation. There is currently limited information on how epidermal keratinocyte process recipient melanosomes during terminal differentiation. Furthermore, the variability of melanosomal degradation seen between light and dark skin remains to be clearly established. Therefore, we have developed a novel model system to investigate the degradation of isolated melanosomes by cultured human keratinocytes. Fluorescently labeled and isolated melanosomes, cultured in the presence of light and dark skin derived keratinocyte cultures, were assessed for degradation. The extent of degradation has been qualitatively assessed, using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Within 48 hours of melanosome incorporation, indirect immunofluorescence and confocal microscopy images suggest that light derived keratinocytes may have accelerated melanosome degradation compared to dark keratinocytes. This time dependent decrease in fluorescence was then quantitatively analyzed using flow cytometry analysis. Consistent with the results of the confocal analysis, over a 48 hour time frame, light keratinocytes appear to degrade melanosomes more efficiently than the iii dark skin derived keratinocytes, P=0.039. This methodology offers a novel mechanism to address the differential ability of light and dark keratinocytes to degrade melanosomes. To further delineate the process of melanosome degradation between light and dark skin, we focused on hydrolytic enzymes that have been implicated in epidermal differentiation and potentially melanosome degradation. To investigate this, we performed preliminary microarray analysis on suprabasal epidermis derived from light and dark skin, by laser capture microscopy (LCM). Data analysis of the microarray experimentation showed over-expression of various hydrolytic enzyme genes in the suprabasal epidermal layers, when comparing light to dark skin. Western blot analysis performed to confirm the expression pattern of hydrolytic enzymes from either light or dark skin derived epidermal lysates, demonstrated that cathepsin L2 was reproducibly upregulated in light skin, P=0.048. In addition, immunofluorescence analysis of cathepsin L2 in light and dark foreskin cryosections confirmed this differential expression and demonstrated that this enzyme was expressed throughout the epidermal layers. Biochemical analysis of cathepsin L2 activity in the two complexion types confirms an elevated enzyme activity in light compared to dark skin complexion samples, 1.75 fold higher activity in light skin compared to dark skin, P=0.03. Taken together these results confirm the differential expression of the acid hydrolase cathepsin L2 in light and dark skin at the gene and protein level. These results may have identified a specific acid hydrolase that may play a role in melanosome degradation and pigment processing. iv v ACKNOWLEDGEMENTS In everything I do, I give God thanks for the gifts of talent, persistence, knowledge, and faith that He and only He is capable of endowing unto me. I express the upmost appreciation and love to my Mother and Father for their continual support and belief in me. The sacrifices you have made for me will always be appreciated. I thank my Dad for instilling in me, the value of hard work and dedication. To my Mom, you are the major driving force for me to work harder, your love for me has been steadfast and my love for you cannot be overstated. To my sisters Paige and Alicia you both amaze me at how loving, smart and supportive you continually are. You girls have consistently motivated me to be a good role model and to be more loving. I am proud to be your sister and I cannot wait to attend your graduations! To my brother Patrick, you bring a smile to my face in even the most difficult of situations. I love you more than you will ever know. To my family, I thank you all for supporting me along this journey. I especially thank Debbie and Romeash for their constant words of encouragement and persistent support, you have both constantly believed in me and I thank you for this. I would like to extend my deepest appreciation to Dr. Raymond Boissy for being my mentor for the past five years. Your insight and critical evaluation has been invaluable. I thank you for the vi opportunity to be a part of the Boissy laboratory and allowing me to develop as a student and as a scientist. Special thanks to Dr. R. Randall Wickett and Dr. Gerald Kasting for accepting me into the Cosmetic Science program and supporting me every step of the way throughout this journey. Dr. Wickett, you have shared your exceptional knowledge in cosmetic and skin sciences, laying the foundation for my work. Dr. Kasting, I thank you for challenging me to think critically, a fundamental skill required for success in our field. To Dr. Pankaj Desai, I appreciate your feedback and help throughout this dissertation! To Dr. Tomohiro Hakozaki, I thank you for giving me the chance to work on this research project. Your feedback, critical thoughts, and consistent help were vital to the development of this project. To Dr. Ana Luisa Kadekaro, I cannot explain how much appreciation I have for everything you have done for me for the past several years. Your support and consistent willingness to help me was invaluable, your scientific advice was indispensable, and your kindness unfailing. I would be remiss not to mention the institutions that have provided me with the education and preparation to accomplish this degree. First, I am indebted to Procter and Gamble for providing the support and resources to make this work possible. To the University of Cincinnati, College of Pharmacy/College of Medicine, and to the Graduate School as a whole, I send my appreciation for offering the curriculum and resources necessary for me to complete this degree. My deepest appreciation goes out to Boston College for providing the foundation of my education and teaching me to think critically and how to work diligently. To the BGSA, in particular Amber Evans, Adeola Adeyemo, Tiffany Forde, Vanessa Saunders, Ritch Hall, Teisha Murray, Fedoria Rugless, Sakinah Davis, Eme Amba and of course Dr. vii Bradford Mallory. You guys have become my family over the last few years and I am grateful for every single one of you and for everything we have accomplished together. To my friends in the College of Pharmacy, Jennifer Karr, Rania Ibrahim, Jennifer Davis, Shoná Burkes, Allison Rush, Rachna Gajjar, Amit Kulkarni, Kelly Smith, Divya Samineni, Ganesh Moorthi, Terri Lacount, Shruthi Vaidhyanathan, Poonam Chopra, Sarah Ibrahim, John Anneken, Ganesh Mugundu, Ned Berry, Nirmal Bhide, Nimitha Dave, Nicole Roenker, Courtney Huff, Guarav Tolia, Cheryl Minges. You have all made up a very dynamic group that has helped me in so many ways. To Laura and Greg Temming, you guys have been extremely good friends and a constant support system; I will never forget that! Thanks to Lola Kelly-Smalls for your help as a mentor and friend. Special thanks to the staff in the College of Pharmacy, including but not limited to, Marcia Silver, Donna Taylor and Paula Shaw, I thank you for your assistance and friendly conversations. Dr. Carol Caperelli I thank you for all your help with enzyme kinetics and biochemical assays, I believe Michaelis and Menten would be pleased. I also send my appreciation to Dr. Marty Visscher for all her words of encouragement over the years. Special thanks to Sandy Schwemberger and Andrew Osterburg for all your help with the flow cytometer. Thank you to Birgit Ehmer for all your help with the various microscopy techniques. Your expertise was of great assistance and I appreciate everything you have done to assist me along this journey. I would also like to say a big thank you to Amy Albin for her assistance with the TEM. I am grateful to the wonderful people in the members of the UC Department of Dermatology. I could not have picked a better group of people to work with and eat cake with every month. Dr. viii Zalfa Abdel-Malek, Dr. Diya Mutasim, Dr. Viki Swope, Renny Starner, Dr. Yuki Hashimoto, Dr. Elodie LePape, Amy Roberto, Meghan Lee, Nancy Olson, Jennifer Yang, Josh Jameson, Jared Swope, Christie Alexander, Priya Srivastava, Heather Henry, and Nicole Mosby.