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Pathways of Skeletal Muscle Atrophy: HIV as a Model System? Chelsea Bueter, Michelle McKinzey, Chloe Salzmann, Michael Zorniak Department of Biology, Lake Forest College, Lake Forest, Illinois 60045 USA Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Discussion Introduction Paradigm of SMA Hypertrophy vs. Atrophy http://www.nndb.com Introduction Most Studied Pathway Stress PI3K mTOR AKT S6K FOXO p p Atrogin-1 Protein degradation (Nucleus) Protein synthesis ? ? HIV Diabetes ? Cachexia ? Oxidative Stress VPR Protein ? ? ? ? Skeletal Muscle Atrophy Introduction The Past of HIV • Vpr protein stops the cell cycle • Prevents programmed cell death • Increased replication of HIV • AIDS muscle wasting symptom Introduction HIV to SMA? • Vpr secreted like a hormone • Infects other cells and organs • AIDS wasting syndrome in skeletal muscle Introduction Cancer Cachexia • Cachexia is a syndrome in cancer patients • Progressive muscle wasting, weight loss, weakness and fatigue http://www2.msstate.edu/~shbryd/Disorders.html Introduction The Past of Cachexia • Pathway unknown • Cytokines induced muscle wasting • One hypothesis: Muscle wasting in cancer due to increased energy consumption Introduction What is Oxidative Stress? • Cancer, Parkinson’s, Diabetes, and SMA • Free Radicals or Reactive Oxygen Species • Steal electrons to restore valence stability Introduction The Past of Oxidative Stress Goldberg et al, 1986 • Calcium activates protein degradation Appel et al, 1997 • Vitamin E decreases calcium levels • Vitamin E is an anti-oxidant • Thus, oxidative stress calcium levels and activates protein degradation. Introduction Diabetes • Characterized by insulin deficiency or insulin intolerance • Juvenile diabetes (type 1)- genetically linked but also diet linked • Type 2 - middle-aged people-low insulin levels • Leads to many other disorders Introduction The Past of Diabetes • 1993- studies showed that in non-diabetics, insulin levels and activity remained high • Diabetics showed very low insulin levels or activity • Common symptom in diabetics was SMA • Hypothesis= Insulin tolerance may be linked to SMA Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Therapies Discussion HIV ? Skeletal Muscle Atrophy HIV HIV’s Vpr protein • Two Direct Pathways to SMA 1. Insulin Resistance 2. Glucocorticoid Hypersensitivity HIV Effects of Vpr binding Serine/Threonine residues Vpr Vpr 14-3-3 14-3-3 Cdc25 Cdc25 Vpr 14-3-3 Cdc25 HIV Vpr Inhibits Cell Cycle Triggers mitosis machinery Alberts et al 2004 HIV Stopped Cell Cycle Dividing G2/M Dividing G2/M •Vpr stops the cell cycle at G2/M He et al 1995 HIV Vpr inhibits insulin effects on FOXO Serine/Threonine residues on FOXO Vpr Vpr 14-3-3 14-3-3 Cdc25 Cdc25 HIV Effect of Vpr on FOXO •Vpr doesn’t bind FOXO Kino et al 2005 HIV What is insulin supposed to do? Insulin Insulin p FOXO FOXO 14-3-3 FOXO p 14-3-3 14-3-3 FOXO p (Nucleus) 14-3-3 FOXO p HIV Vpr won’t let insulin work! FOXO No FOXO Kino et al 2005 HIV How does Vpr affect glucocorticoid receptors? Vpr LQQLL Kino et al 2000 •Specific LXXLL motif binds GRE •Completely different from ability to arrest cell cyle Vpr as a Co-regulator Kino et al 2000 HIV Vpr as a Co-regulator of GR Vpr GR GRE TFIIB RNA polymerase II TFIID Transcription: enhancing glucocorticoid signal TATA Kino et al 2000 Summary of Vpr & SMA VPR Protein Vpr GRE Vpr 14-3-3 Glucocorticoid FOXO nucleus Atrogin-1 Skeletal Muscle Atrophy Therapies for HIV muscle wasting • Steroid hormone receptor antagonists (RU 486) • Vpr antagonists • Current antiretroviral therapies Cancer Cachexia ? Skeletal Muscle Atrophy Cachexia NF-κB IκB P IκBα NF- κB Nucleus Cachexia Cai et al. Study MISR Mouse MIKK Mouse Constitutively active IκBα Constitutively active IκB Inactive NF-κB Always active NF-κB Cachexia NF-κB Activity • NF-κB activity is high in MIKK mice Cachexia MIKK Mice vs. MISR Mice • MIKK mice have a much lower body mass Cachexia Tumor Activity • Presence of a tumor increases the level of NF-κB activity in wild type mice Cachexia Tumor Necrosis Factor - TNFα • In the presence of IκBα, activity decreases • Without IκBα, inhibitor does not stop production Cachexia What does NF-κB affect? • MURF1 mRNA is much higher in MIKK mice than in MISR mice Cachexia What else does NF-κB affect? • TNF activates NF-κB which causes a decrease in MyoD production Cachexia Troponin in Cardiac Muscle • Troponin-1 is degraded in the presence of MURF1 Cachexia Pathway TNF-α activates NF-κB increases MURF1 decreases MyoD Cachexia Therapy for Cachexia • Salicylate inhibits the NF-κB pathway, preventing muscle loss Oxidative Stress ? Skeletal Muscle Atrophy Oxidative Stress Oxidative Stress Reactive Oxygen Species Calcium Mitochondria Cytochrome c Calpastatin Calpain Caspase-3 Calpastatin NO Sarcomere Unit of Myofibril 20S/26S Proteasome Oxidative Stress Effect of Disuse • Disuse increases oxidative stress Tidball et al, 2002 Oxidative Stress Effect of Oxidative Stress Laser Densitometry • Increase of oxidative stress increases calsequesterin • Calsequestrin sequesters intracellular calcium Hunter et al, 2001 Oxidative Stress Effect of Increased Calcium Type II Diaphragm Muscle Fibers by Immunohistochemistry • Disuse increases calpain and 20S proteasome activity Tidball et al, 2002 Oxidative Stress Calpain Proteolysis • Calcium treatment increases protein cleavage • Protein cleavage can be inhibited by nitric oxide and calpastatin Koh et al, 2000 Oxidative Stress Caspase Activity • Caspases inhibit calpastatin • Calpastatin is a calpain inhibitor Wang et al, 1998 Summary Oxidative Stress Increase Ca2+ Calpain & Caspase 3 activity increases Releases Actomyosin to be degraded in proteasome Skeletal Muscle Atrophy Oxidative Stress Therapies for Oxidative Stress ROS Cell Vitamin E • NO and Calpastatin Transgene • Vitamin E protects against ROS • Vitamin C restores Vitamin E activity Diabetes ? Skeletal Muscle Atrophy Diabetes Insulin levels Ub-ligase E3-α 26 S proteasome Diabetes What activates the Ubiquitinproteasome pathway? • No difference in diabetics without vs. diabetics with acidosis • Acidosis is not a stimulus of ubiquitin dependent atrophy Price et al. 1999 Diabetes Effects of Insulin on Ubproteasome pathway • Less protein degradation in insulin treated muscles • Lower insulin levels leads to activation of Ubproteasome pathway Price et al., 1999 Diabetes Results of Pathway Activation • Levels of Ub-ligase and its coenzyme increase • Amount of Ub-conjugation by these increases Goldberg et al., 1999 Diabetes Proteasome Formation • mRNA for 19 S and 20 S subunits increases • Formation of 26 S proteasome increases Attaix et al., 2004 Diabetes Proteasome Activity • Flourescence in atrophied muscles higher than control muscles • Flourescence is analogous to amount of 26 S proteasome activity Attaix et al., 2004 Summary of Diabetes and SMA Diabetes Insulin decrease/ glucocorticoid increase E3-alpha ubiquitin ligase increases 26 S Proteasome activity increases Skeletal Muscle Atrophy Diabetes Therapy for Diabetes • Treatments for diabetes generally focus on maintaining available insulin levels NOT SMA • Side effect of the insulin treatment, however, is associated with the reverse pathway of atrophy, i.e. hypertrophy Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Discussion ? HIV ? Diabetes ? Cachexia VPR Protein Skeletal Muscle Atrophy Oxidative Stress Vpr VPR Protein Inhibit insulin effects on FOXO Co-activates glucocorticoid receptor Glucocorticoid hypersensitivity Atrogin-1 induction Skeletal Muscle Atrophy Cachexia Cachexia IKK/NFkappa B pathway Murf-1 increase MyoD mRNA decrease Skeletal Muscle Atrophy Oxidative Stress Oxidative Stress Increase Ca2+ Calpain & Caspase 3 activity increases Releases Actomyosin to be degraded in proteasome Skeletal Muscle Atrophy Diabetes Diabetes Insulin decrease/ glucocorticoid increase E3-alpha ubiquitin ligase increases 26 S Proteasome activity increases Skeletal Muscle Atrophy Diabetes Insulin decrease/ glucocorticoid increase E3-alpha ubiquitin increases 26 S Proteasome activity increases HIV Cachexia Oxidative Stress VPR Protein Co-activates glucocorticoid receptor IKK/NFkappa B pathway Murf-1 increase MyoD mRNA decrease Inhibit insulin effects on FOXO Glucocorticoid hypersensitivity Atrogin-1 induction Skeletal Muscle Atrophy Increase Ca2+ Calpain & Caspase 3 activity increases Releases Actomyosin to be degraded in proteasome Acknowledgements Thanks to Dr. D, Sara Herrera, Tammy Hibler, Arun Paul, and Chris Prater