Pathways of Skeletal Muscle Atrophy: HIV as a Model System?
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Transcript of Pathways of Skeletal Muscle Atrophy: HIV as a Model System?
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
Paradigm of SMAHypertrophy vs. Atrophy
http://www.nndb.com
Introduction
Most Studied Pathway
Stress
PI3K
AKT
FOXO
mTOR
Atrogin-1
Protein degradation
Protein synthesis
S6K
(Nucleus)
pp
Introduction
HIV
Skeletal Muscle Atrophy
Diabetes
VPR Protein
Oxidative Stress
Cachexia
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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
http://www2.msstate.edu/~shbryd/Disorders.html
• Cachexia is a syndrome in cancer patients
• Progressive muscle wasting, weight loss, weakness and fatigue
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
Introduction
Presentation Pathway
IntroductionDiseases
HIVCachexiaOxidative StressDiabetes
Therapies Discussion
HIV
?
Skeletal Muscle Atrophy
HIV’s Vpr protein
• Two Direct Pathways to SMA
1. Insulin Resistance
2. Glucocorticoid Hypersensitivity
HIV
Effects of Vpr binding
Vpr
Cdc25
Serine/Threonine residues
Cdc25
14-3-3
14-3-3
Vpr
14-3-3
Vpr
Cdc25
HIV
Vpr Inhibits Cell Cycle
Triggers mitosis machinery
Alberts et al 2004
HIV
Stopped Cell Cycle
G2/MDividing G2/MDividing
HIV
•Vpr stops the cell cycle at G2/M
He et al 1995
14-3-3
Vpr
Vpr inhibits insulin effects on FOXO
Vpr
Cdc25
14-3-3
Cdc25
Serine/Threonine residues on FOXO
HIV
Effect of Vpr on FOXO
HIV
•Vpr doesn’t bind FOXO
Kino et al 2005
What is insulin supposed to do?
FOXO
(Nucleus)
p
Insulin
FOXO p14-3-3
FOXO
Insulin
14-3-3
FOXO p14-3-3
FOXO p
14-3-3
HIV
Vpr won’t let insulin work!
FOXO
No FOXO
HIV
Kino et al 2005
How does Vpr affect glucocorticoid receptors?
Vpr
LQQLL
HIV
•Specific LXXLL motif binds GRE
•Completely different from ability to arrest cell cyle
Kino et al 2000
Vpr as a Co-regulator
Kino et al 2000
Vpr as a Co-regulator of GR
Vpr
TATA
Transcription: enhancing glucocorticoid signal
G R
TFIIB
TFIID
RNA polymerase II
GRE
HIV
Kino et al 2000
Summary of Vpr & SMAVPR Protein
Skeletal Muscle Atrophy
VprGRE
Glucocorticoid
14-3-3
Vpr
FOXOnucleus
Atrogin-1
Therapies for HIV muscle wasting
• Steroid hormone receptor antagonists (RU 486)
• Vpr antagonists
• Current antiretroviral therapies
Cancer Cachexia
Skeletal Muscle Atrophy
?
NF-κB
Nucleus
IκB
NF- κB
IκBα
P
Cachexia
Cai et al. Study
MISR Mouse MIKK Mouse
Constitutively active IκBα
Constitutively active IκB
Cachexia
Inactive NF-κB Always active NF-κB
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
Cachexia Pathway
TNF-α
NF-κB
MURF1 MyoD
activates
decreasesincreases
Therapy for Cachexia
• Salicylate inhibits the NF-κB pathway, preventing muscle loss
Cachexia
Oxidative Stress
Skeletal Muscle Atrophy
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Oxidative StressReactive Oxygen Species
Mitochondria
Caspase-3Calpain
Sarcomere
Unit of Myofibril
20S/26S Proteasome
Calcium
Cytochrome c
CalpastatinCalpastatin
NO
Oxidative Stress
Effect of Disuse
• Disuse increases oxidative stress
Tidball et al, 2002
Oxidative Stress
Effect of Oxidative Stress
• Increase of oxidative stress increases calsequesterin
• Calsequestrin sequesters intracellular calciumHunter et al, 2001
Laser Densitometry
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
Koh et al, 2000
• Calcium treatment increases protein cleavage
• Protein cleavage can be inhibited by nitric oxide and calpastatin
Oxidative Stress
Caspase Activity
• Caspases inhibit calpastatin
• Calpastatin is a calpain inhibitor
Wang et al, 1998
Oxidative Stress
SummaryOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
Therapies for Oxidative Stress
• NO and Calpastatin Transgene• Vitamin E protects against ROS• Vitamin C restores Vitamin E activity
Cell
Vitamin E
ROS
Oxidative Stress
Diabetes
?
Skeletal Muscle Atrophy
Diabetes
Insulin levels
Ub-ligase E3-α
26 S proteasome
What activates the Ubiquitin-proteasome pathway?
• No difference in diabetics without vs. diabetics with acidosis
• Acidosis is not a stimulus of ubiquitin dependent atrophy
Diabetes
Price et al. 1999
Effects of Insulin on Ub-proteasome pathway
• Less protein degradation in insulin treated muscles
• Lower insulin levels leads to activation of Ub-proteasome pathway
Diabetes
Price et al., 1999
Results of Pathway Activation
• Levels of Ub-ligase and its coenzyme increase
• Amount of Ub-conjugation by these increases
Diabetes
Goldberg et al., 1999
Proteasome Formation
• mRNA for 19 S and 20 S subunits increases• Formation of 26 S proteasome increases
Diabetes
Attaix et al., 2004
Proteasome Activity
• Flourescence in atrophied muscles higher than control muscles
• Flourescence is analogous to amount of 26 S proteasome activity
Diabetes
Attaix et al., 2004
Summary of Diabetes and SMADiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
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
Diabetes
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Discussion
HIV
Skeletal Muscle Atrophy
Diabetes
VPR Protein
Oxidative Stress
Cachexia
?
??
Vpr
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Skeletal Muscle Atrophy
Cachexia
Cachexia
Murf-1 increase
IKK/NF-kappa B pathway
MyoD mRNA decrease
Skeletal Muscle Atrophy
Oxidative StressOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
DiabetesDiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
HIV
Skeletal Muscle Atrophy
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Oxidative Stress
Increase Ca2+
Calpain & Caspase 3
activity increases
Releases Actomyosin
to be degraded in proteasome
Cachexia
Murf-1 increase
IKK/NF-kappa B pathway
MyoD mRNA decrease
Diabetes
Insulin decrease/ glucocorticoid
increase
E3-alpha ubiquitin increases
26 S Proteasome activity increases
Acknowledgements
Thanks to Dr. D, Sara Herrera, Tammy
Hibler, Arun Paul, and Chris Prater