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Regulation of Macrophage Inflammatory Signaling Pathways by AMP-Activated Protein Kinase

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Regulation of Macrophage Inflammatory Signaling Pathways by AMP-Activated Protein Kinase University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2014 Regulation of macrophage inflammatory signaling pathways by AMP-activated protein kinase. Yanfang Zhu University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons Recommended Citation Zhu, Yanfang, "Regulation of macrophage inflammatory signaling pathways by AMP-activated protein kinase." (2014). Electronic Theses and Dissertations. Paper 1649. https://doi.org/10.18297/etd/1649 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS BY AMP-ACTIVATED PROTEIN KINASE By Yanfang “Peipei” Zhu B.Sc. Northwest A & F University, 2007 M.S. University of Louisville, 2011 A Dissertation Submitted to the Faculty of the University of Louisville School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Microbiology and Immunology University of Louisville Louisville, KY May, 2014 Copyright 2014 by Yanfang “Peipei” Zhu All rights reserved. REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS BY AMP-ACTIVATED PROTEIN KINASE By Yanfang “Peipei” Zhu B.Sc. Northwest A & F University, 2007 M.S. University of Louisville, 2011 A Dissertation Approved on April 25, 2014 by the following Dissertation Committee: _________________________________ Jill Suttles _________________________________ Thomas C. Mitchell _________________________________ Robert A. Mitchell _________________________________ Sanjay Srivastava ii DEDICATION Dedicated to my beloved family and friends. iii ACKNOWLEDGMENTS My highest regards go to my mentor, Dr. Jill Suttles. Six years of research training in her lab has provided me one of the most important and valuable experiences in my life, both professionally and personally. Dr. Suttles offered me the most wonderful mentorship and the exciting opportunity to work on the scientific project that inspired me in a life long academic interest. Without her guidance, support, encouragement, and friendship, I could never have come this far on the way of pursuing the truth of nature. My great appreciation also goes to my dissertation committee members, Dr. Thomas C. Mitchell, Dr. Robert A. Mitchell, and Dr. Sanjay Srivastava, for their valuable comments and advice and continuous care to the development of my project. I also want to express my gratitude to the late members of my committee, Dr. Robert D. Stout and Dr. Michael Martin. Bob was our beloved friend and insightful teacher whose lectures motivated me to the macrophage plasticity research. Mike’s profound knowledge in cell signaling made him always the good instructor to go to for suggestions. The scientific achievement of this dissertation study is based on the contribution of every idea and support from all these incredible scientists. It is my great honor and pleasure to work and have my Ph.D. training with them. I would like to thank all the nice people in Jill’s lab for their kind assistance and support with all my experiments. I specially thank Lihua Zhang and her family, to provide me the family-like support both in and out of lab work. I am grateful to all iv the nicest colleagues who have made the six years of training experiences cheerful and have brought me courage through the downside of the road. They are Kim Z. Head, Shunmugavadiv “Meena” Vanchinathan, Ashley Triplett, Kelly C. Caroll, Courtney J. Mitchell, and Jonathan R. Brown. Thank you all for the scientific discussions and technical support, for the laughter and compassionate hugs, and for the friendship that will be remembered and appreciated life-long. I also would like to thank my dearest family and friends. It is never enough to say thank you to my parents Mingshan Zhu and Suyin Sun, for their best love and support even with half of the world between us. You are the greatest blessing I can ever ask for. I want to express my gratitude to my friends and family from the near and the far, Chi Zhang, Pei-hsin Cheng, and my friends and big family in China. You all made me feel beloved and encouraged during my studies in United States and made my life happy and colorful. Lastly, I want to thank Department of Microbiology and Immunology and the Integrated Programs of Biomedical Sciences (IPIBS) program of University of Louisville School of Medicine for the opportunity and financial support for the training of obtaining my Ph.D. degree in immunology. v ABSTRACT REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS BY AMP-ACTIVATED PROTEIN KINASE Yanfang “Peipei” Zhu April 25, 2014 AMP-activated protein kinase, AMPK, is a conserved serine/threonine kinase with a critical function in the regulation of metabolic pathways in eukaryotic cells. Recently, AMPK has been shown to play an additional role as a regulator of inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK activators, or forced expression of a constitutively active form of AMPK, results in polarization to an antiinflammatory phenotype. Additionally, we reported previously that stimulation of macrophages with antiinflammatory cytokines such as IL-10, IL-4 and TGF-β results in rapid activation of AMPK, suggesting that AMPK contributes to the suppressive function of these cytokines. In the current study we investigated the role of AMPK in the regulation of macrophage antiatherogenic functions and demonstrated a new mechanism for AMPK’s role in mediating IL-10-induced gene expression and antiinflammatory effects. The expression of dominant negative (DN-) AMPKα1 in macrophages resulted in a spontaneous obese phenotype in mice associated with fatty vi liver and heart enlargement. The expression of constitutively active (CA-) AMPKα1 in macrophages resulted in decreased IL-6 production but increased expression of ABCA1 and ApoE. Compared to wild-type macrophages, AMPKα1-deficient macrophages failed to express atheroprotective genes including ApoE, LXRα, and ABCA1 in response to IL- 10. Mechanistic studies revealed that IL-10-stimulated wild-type macrophages displayed rapid activation of PI3K and its downstream target Akt, an effect that was not seen in macrophages generated from AMPKα1-deficient mice. Treatment with the PI3K inhibitor LY294002 blocked IL-10’s ability to induce Akt activation but not AMPK activation, suggesting that IL-10-mediated activation of AMPK is independent of PI3K. CA-AMPKα1 macrophages displayed elevated PI3K and CREB activation in response to IL-10 compared to the empty vector transfected macrophages. IL-10 stimulation resulted in increased mTORC1 activity, an Akt downstream target, an effect that was reduced in AMPKα1-deficient mice. IL-10 induced phosphorylation of both Tyr705 and Ser727 residues of STAT3 in an AMPKα1-dependent manner, and these phosphorylation events were blocked by inhibition of CaMKKβ, an upstream activator of AMPK, and by the mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in response to IL-10 observed in AMPKα1-deficient macrophages was accompanied by reduced SOCS3 expression and an inadequacy of IL-10 to suppress LPS-induced proinflammatory cytokine production. Overall, our data demonstrate that AMPKα1 is required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3/SOCS3 antiinflammatory pathways regulating macrophage polarization, a mechanism with broad-reaching applicability in immune homeostasis and in inflammation-associated diseases such as atherosclerosis, autoimmune diseases, and obesity. vii TABLE OF CONTENTS PAGE DEDICATION……………………………………………………………………………iii ACKNOWLEDGEMENTS………………………………………………………………iv ABSTRACT……………………………………………………………………………...vi LIST OF FIGURES……………………………………………………………………… x CHAPTER 1. INTRODUCTION……………………………………………………………………...1 2. AMPK REGULATION OF MACROPHAGE FUNCTION IN ATHEROSCLEROSIS INTRODUCTION……………………………………………………………………….22 MATERIALS AND METHODS………………………………………………………...34 RESULTS………………………………………………………………………………..39 Generation of macrophage-specific CA-AMPKα1 and DN-AMPKα1 mice……39 Macrophage-specific DN-AMPKα1 mice develop spontaneous ………………..40 obesity associated with increased proinflammatory cytokine production ABCA1 expression is increased in macrophage from…………………………...43 macrophage-specific CA-AMPKα1 mice AMPK enhances atheroprotective gene expression induced by…………………48 IL-10 in macrophages AMPK promotes ApoE expression in macrophages……………………………..50 Relationship between PPARγ and AMPK in macrophages……………………...53 DISCUSSION……………………………………………………………………………61 viii 3. AMPK REGULATES IL-10-MEDIATED ANTIINFLAMMATORY SIGNALING PATHWAYS IN MACROPHAGES INTRODUCTION……………………………………………………………………….63 MATERIALS AND METHOD………………………………………………………….69 RESULTS………………………………………………………………………………..72 IL-10 activates AMPK on a time dependent manner……………………..……...72 AMPK is required for IL-10 activation of PI3K/Akt pathway…………………..72 AMPK positively regulates IL-10 activation of Akt/CREB pathway……………83 AMPK positively regulates IL-10 activation of mTORC1/S6K pathway……….85 AMPK is required for
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