ACTIVIN IS CRITICAL FOR THE DEVELOPMENT OF PAIN HYPERSENSITIVITY AFTER INFLAMMATION by PIN XU Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Alison K. Hall Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ____Pin Xu______________________________________________ candidate for the Ph.D. degree *. (signed)__ Gary Landreth______________________________ ___ (chair of the committee) ___Alison Hall____________________________________ ___Jerry Silver___________ ___________ _____ ___ ___Susann Brady-kalnay____________________________ ________________________________________________ ________________________________________________ (date) ____June 5, 2007___________________ *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION This thesis is dedicated to my parents, Hongfa Xu and Ruiyun Pan, and to my husband Chen Liu. iii TABLE OF CONTENTS Page Title Page……………………..…………………………………………………….….…I Typed ETD Sign-off Sheet…………………………………………………….................II Dedication……………………………………………………………………………..…III Table of Contents………………………………………………………..……..……..….IV List of Tables……………………………………………………………………………VII List of Figures…………………………………………………………………….....…VIII Acknowledgements………………………………………………………………………X Abstract…………………………………………………………………………...……...XI Chapter I: General Introduction……………………………………………………………………...1 Chapter II: Activin induces tactile allodynia and increases CGRP upon peripheral inflammation…………………………………………………………………….………35 Abstract…………………………………………………………………………..36 Introduction………………………………………………………………………37 Methods and Materials…………………………………………………………...40 iv Results………………………………………………………………..…………..47 Summary…………………………………………………………………..……..54 Chapter III: Activin acutely sensitizes DRG neurons and induces hyperalgesia via sensitize of TRPV1………………………………………………………………………..…….……72 Abstract…………………………………………………………………….……..73 Introduction………………………………………………………………….……74 Methods and Materials…………………………………………………….……...76 Results………………………………………………………………..…………...80 Summary………………………………………………………………..…….…..83 Chapter IV: Activin regulates CGRP expression in both embryonic and adult DRG neurons …………………………………………………………………………………..94 Abstract…………………………………………………………………….……..95 Introduction……………………………………………………………….………96 Methods and Materials…………………………………………………….……...98 Results………………………………………………………………..…….……101 Summary……………………………………………………………………..….110 v Chapter V: General discussion…………………………………………………….………………..131 Bibliography………………………………………………………….………………...155 vi LIST OF TABLES Table No Page Table 1.1. Family members, Activin A, BMPs and TGFβ signal through some shared receptors and signaling components………………………………………………….33 vii LIST OF FIGURES Figure No. Page Figure 1.1. Sensory neurons respond to signals in skin for early differentiation, plasticity after wound and pain functions………………………………………………………....29 Figure 1.2. Activin and selected TGFβ family members, their receptors and signaling……………………………………………………………………….…..…....31 Figure 2.1. CFA induced inflammation. …………………………………………...…..58 Figure 2.2. Tracer labeled CGRP-IR neurons in the L4 DRG……………………..…...60 Figure 2.3. CGRP expression in different subpopulations of L4 DRG neurons………..62 Figure 2.4. The effect of inflammation on the size distribution of sensory neurons innervating ankle skin……………………………………………………………….......64 Figure 2.5. Central projections of CGRP-IR fibers after localized inflammation………66 Figure 2.6. Activin A or NGF injection induces tactile allodynia…………………..…..68 Figure 2.7 Activin induces CGRP in the innervating DRG neurons………….…….......70 Figure 3.1. Activin A acutely sensitizes the capsaicin induced current in the cultured DRG neurons through the activin type I receptor AcRIB……………………….……....88 Figure 3.2. Activin A causes translocation of PKCδ to cell membrane of rat DRG neurons…………………………………………………………………………………..90 viii Figure 3.3. Activin injection induces thermal hyperalgesia in wildtype, but not TRPV1 null mice……………………………………………………………………….………....92 Figure 4.1. Activin acts with NGF to induce CGRP mRNA in adult DRG cultures…...115 Figure 4.2. Activin effects on CGRP mRNA occur through its own receptor…………117 Figure 4.3. Activin signaling pathways in the adult sensory neurons……………….…119 Figure 4.4. Activin stimulates pSmad2 while NGF stimulates pERK2 in adult sensory neurons……………………………………………………………………….….…….. 122 Figure 4.5. NGF and activin signal through multiple MAPK pathways to regulate CGRP expression………………………………………………………………………….…...124 Figure 4.6. Activin increases CGRP mRNA in E14.5 DRG neuronal culture in a concentration dependent manner…………………………………….…………………126 Figure 4.7. Activin and NGF signaling pathways in embryonic sensory neurons…......128 ix ACKNOWLEGDEMENTS I would like to thank my thesis advisor, Dr. Alison Hall. She taught me with great patience how to do science and design critical experiments. I have learned tremendously from her during my Ph.D. training. Also she set up an excellent model for me as a working mother and scientist. Alison always encourages me to pursue my personal goal and offers her suggestions whenever I meet problems. I am really grateful for her help. I would also like to express my gratitude to members of my thesis committee, Drs. Gary Landreth, Jerry Silver and Susann Brady-Kalnay for all of their supports and great advices on my thesis. I want to thank our collaborators, Dr. Liliana Berti-Mattera (CASE), Dr. Andrew Russo (U. Iowa), Drs. Gerry Oxford and Weiguo Zhu (Stark Res. Inst.) I appreciate the help from people in the Neurosciences department throughout my Ph.D. training. Especially, I want to thank my current labmates, Shibani Mukerji and Ekaterina Katsman, who make the lab life pleasant and I really enjoy our conversations about both scientific topics and personal life. I thank formal Hall lab residents, Bethany Kiernan, Noah Haner and Rebecca Burke, who always offer their help. Finally, I thank my family members for their endless love and support. x Activin is Critical for the Development of Pain Hypersensitivity after Inflammation Abstract By PIN XU Inflammatory pain is a major clinical challenge. The perception of pain is initiated by specialized sensory neurons in the Dorsal Root Ganglia (DRG) called nociceptors. Upon injury, neuropeptides including Calcitonin Gene-Related Peptide (CGRP) are released from nociceptors and the neuropeptide contributes to the development of abnormal pain. The studies in this thesis focus on the role of activin in regulating pain after skin inflammation. In this study, experimental peripheral inflammation was accompanied by increases in activin mRNA and activin immunoreactivity in both keratinocytes and infiltrating immune cells. The number of CGRP containing DRG neurons increased after inflammation, and this increase was among neurons that expressed the lectin IB4 or TrkA. These data indicate that some adult sensory neurons remain plastic and can increase CGRP expression after inflammation or activin administration. Strikingly, activin administration also induced thermal hyperalgesia and mechanical allodynia pain behaviors. I demonstrated that activin induced thermal hyperalgesia involved the sensitization of nociceptors through the Transient Receptor Potential Vallinoid subtype 1 (TRPV1). In addition to changing acute pain behaviors, activin also regulated neuropeptide gene transcription over chronic periods. Activin and nerve growth factor (NGF) worked synergistically to increase CGRP mRNA in adult DRG cultures. xi Biochemical and pharmacological studies indicate that this synergy was initiated by independent stimulation of receptors and intracellular signals, and our data suggest that activin and NGF effects converge on the CGRP promoter to regulate CGRP expression. Taken together, these data demonstrate that increased activin expression after inflammation results in both acute pain behaviors through sensitizing nociceptors and prolonged pain regulation by increasing CGRP expression. xii Chapter I General Introduction 1 Inflammatory pain is a major medical challenge and can be difficult to manage clinically. Pain hypersensitivity is a key sign of inflammation and is not sufficiently controlled with commonly prescribed analgesics. Present studies demonstrate that activin A (activin) regulates a group of specialized sensory neurons sensing nociceptive information. This thesis will focus on the functions of activin in regulating pain sensation. In this section, a general introduction will review nociceptive sensation, neuropeptides important for pain sensation, the regulation of neuropeptides by activin and NGF and current approach for pain management. Perception is mediated by receptors, nervous pathways and specific areas in the brain. Our knowledge of the outside world starts from the senses which do not passively record the surrounding world, but process information via brain activity leading to perception. The sensory experience contains four basic types of information – modality, location, intensity and timing, all of which are encoded in the nervous system by specialized subgroups of neurons. Modality is determined by the type of transmitted energy and the receptors specialized to sense that energy. Sensory receptors transduce unique stimuli and convert them into specific electrical signals, which are conducted to the cortex to produce different perceptions.