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Functional roles of chaperone molecule (HSP70) for
muscle atrophy inhibition: future implication
Taesik Gwaga, Eunjung Kima, Takeshi
Nikawab and Inho Choia
aDivision of Biological Science and Technology,
College of Science and Technology, Yonsei University,
Wonju, Gangwon-Do, Republic of Korea
bDepartment of Nutritional Physiology, Institute of
Health Biosciences, University of Tokushima Graduate
School, Tokushima, Japan
The 10th Japan-Korea Joint Seminar on Space Environment Utilization Research
September 12, 2013
Korea University
(1) a significant decrease in mass or volume of skeletal muscle,
with loss of myofibrillar proteins and tone.
(2) caused by various factors including physical unloading and diseases.
(3) Molecular mechanisms underlying the atrophic process are much shared in the causal states..
Cancer
Bed rest Space microgravity
Spinal injury – denervation
Muscle injury
Inactivity by cast
Cancer
AIDS
Sepsis
Space physiology good solutions the quality of life
Introduction: Factors causing muscle atrophy
Muscle atrophy
Muscle force production ↓
Performance and work ↓
Mission capability ↓
Responsiveness to emergency ↓
Muscle atrophy during prolonged spaceflight
Severe muscle damage by reloading (landing)
Skeletal muscle atrophy
due to unloading
Introduction: Space microgravity causes serious muscle atrophy
Space microgravity Muscle atrophy occurs steadily
despite elaborate exercise program in space.
Adams et al., 2003
We may need another means of countermeasure
to effectively prevent muscle atrophy!
Muscle mass = balance between protein synthesis rate and degradation rate
<source from Hoffmand and Nader, Nature Medicine 10(6):584-585 (2004)>
p70/S6K
Activation of Protein synthesis pathway
Activation of protein breakdown pathway
Exercise /overloading
Spaceflight, bedrest /unloading
Upregulation of Proteases via FoxO
HSP↓
dexamethasone
Introduction: Molecular mechanisms underlying muscle mass regulation
CEL: a quinone methide triterpentene obtained from oriental herbs.
DEX: dexamethasone, a synthetic glucocorticoid that is known to induce muscle atrophy.
Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via
activation of Akt1 and ERK1/2 pathways
A quinine methide triterpene
Heat Shock Protein 72 inducer
Diverse functions: antioxidant activity, neuroprotection, and inhibition of proteasome activity
Previous reports: muscle atrophy prevented by HSP70 overexpression
Celastrol (CEL): general features
Kukreti et al., 2013, Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during
Dexamethasone-mediated Atrophy. Journal of Biological Chemistry. 288:6663-6678
Senf et al., 2008, Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents
skeletal muscle atrophy. FASEB journal : official publication of the Federation of American Societies for
Experimental Biology. 22:3836-3845.
Gehrig et al., 2012, Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.
Nature. 484:394-398.
Cell line: mouse C2C12 myotube
Determination of proper concentration of CEL.
Concentration of CEL used for this study: 1.5 μM for 6 h treatment
Data were obtained from three independent experiments
(each experiment performed in triplicate) and are
presented as mean SEM.
The cells exposed to four doses of CEL for 6 h
Stable dosage
Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via
activation of Akt1 and ERK1/2 pathways
FACS analysis
Data were obtained from three independent experiments
Experimental design
DEX (150µM): time-dependent test
23% loss
in diameter
Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via
activation of Akt1 and ERK1/2 pathways
Myotube diameter affected by CEL and DEX.
White bar = 100 μm
Comparison of the cell diameters among the four groups
Data are presented as the mean SEM. Cell counts: 220 - 250 cells per group.
The symbols a, b, c and d indicate statistical significance among the denoted
groups at P < 0.05 (one-way ANOVA and SNK post hoc multiple comparisons
test).
CEL abolished DEX-induced myotube atrophy
The cell diameter was significantly decreased in myotubes
exposed to 150 μM DEX for ≥12 h
atrophy
hypertrophy
recovery
Data: mean SEM (n = 6). a versus b, P < 0.05 .
Expression of HSP72 upregulated by CEL.
Elevation of HSP72 protein expression by CEL
(A and B) Whole cell lysates subjected to
immunoblot analyses with antibodies rea
cting with total or Ser230-phosphorylate
d HSF1. Mean SEM (n = 6), a versus b
, P < 0.05. (C) Confocal immunofluoresc
ent analysis to visualize the expression a
nd subcellular localization of p-HSF1 in
C2C12 cells. The cells were cultured on
chamber slides and treated as indicated i
n the Methods, and stained with p-HSF1
antibody followed by a secondary antibo
dy conjugated to Rhodamine. The nucleu
s was stained with DAPI (blue). Scale bars = 10 μm.
Upregulation of HSP72 via heat shock factor 1 (HSF1)
transcriptional activity
Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via
activation of Akt1 and ERK1/2 pathways
Anabolic signalling activities upregulated; catabolic signaling activities abolished by celastrol
Anabolic activities compared among the four groups
Data: Mean SEM (n = 6). a versus b, P < 0.05.
Catabolic activities compared among the four groups
Data: Mean SEM (n = 6). *P < 0.05.
Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via
activation of Akt1 and ERK1/2 pathways
HSP72 expression was affected by HSP72 siRNA and CEL in the L6 myotubes
Elevation of HSP72 expression by CEL
Activation of HSF1 by CEL
(A and B) Whole cell lysates
subjected to immunoblot analyses
with Ser230-phosphorylated HSF1.
Mean ± SEM (n = 5). The
symbols a, b and c indicate
statistical significance among the
denoted groups. (C) Confocal
immunofluorescent analysis to
visualize subcellular localization
of HSF1. The cells were stained
with HSF1 antibody followed by a
secondary antibody conjugated to
Alexa488 (green) and DAPI (blue).
White bars = 10 μm.
HSF1 was transactivated and accumulated in the
nucleus by CEL treatment.
HSP72 mRNA and protein expression was upregulated
by CEL treatment despite Hsp72 gene knock down.
248~ 306 fold
Data : mean ± SEM (immunoblot
analyis; n = 6, real-time PCR
analysis; n=4) The symbols a, b, c
and d; significant differences among
the designated groups at P < 0.05.
HSP70 overexpression induced by celastrol abolished the atrophic effect of
HSP siRNA in L6 myotubes
Myotube diameters compared among the four groups.
Data: mean ± SEM (n = 178 -183 cells per group with 3 independent experiments).
The symbols a, b and c indicate significant differences among the denoted groups at P < 0.05.
White bars = 100 µm
The cell diameter was significantly decreased by HSP72 siRNA
atrophy
hypertrophy
recovery
(A) NC siRNA, (B) HSP72 siRNA,
(C) NC siRNA+CEL and (D) HSP72 siRNA+CEL
CEL abolished the atrophic effect of HSP72 siRNA on muscle cell
HSP70 overexpression induced by celastrol abolished the atrophic effect of
HSP siRNA in L6 myotubes
Conclusions
1. The anti-atrophic effect of CEL is mediated by activation of HSF1 and
consequent overexpression of HSP72.
2. HSP overexpression by CEL treatment not only activated the Akt1, S6K and
ERK1/2 signaling pathways but also suppressed FoxO activation, E3 ligase
expression and proteasome activity by DEX in C2C12 myotube.
3. The lack of HSP70 was directly responsible for the L6 myotube atrophy, and
CEL abolished such atrophy of the myotube.
4. An in vivo study on the anti-atrophic potency of celastrol is currently under
way with a direct application of CEL in the presence or absence of siRNA in
the skeletal muscle of a mouse model.
Thank you for your attention!
The 10th Japan-Korea Joint Seminar on Space Environment Utilization Research
Functional roles of chaperone molecule (HSP70) for muscle atrophy inhibition: future implication
Taesik Gwag, Eunjung Kim, Takeshi Nikawa and Inho Choi