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Regulation of the Actin Cytoskeleton in Human Airway Smooth Muscle
Tone
Alex Banathy
Mentors: Dr. Brophy and Dr. Komalavilas
Co-Mentor: Dr. Gamse
What is Asthma? • Chronic lung disease that narrows airways
• Recurring wheezing, coughing, chest tightness and shortness of breath
NIH: National Heart, Lung, and Blood Institute
Asthma’s Significance
• 25 million diagnosed and increasing
• Over 10 million outpatient visits and 40,000 hospitalizations
• 3,400 deaths each year
• Most common chronic illness among children
National Surveillance of Asthma: United States 2001-2010. Centers for Disease Control and Prevention: Vital and Health Statistics
Rappaport H, Bonthapally V (2012) The Direct Expenditures and Indirect Costs Associated with Treating Asthma in the United States. J Aller Ther 3:118
$44 Billion Dollar Problem
Current Forms of Treatment
• Long-term control: Inhaled Corticosteroids and Long-Acting -Agonists – Safe for long term use when combined
– Recent concerns with LABA
– Fluticasone (Flonase), Mometasone (Nasonex)
• Quick relief for attacks: Short-Acting -agonists – Cannot be used regularly
– Albuterol (ProAir), Levalbuterol (Xopenex)
NIH: National Heart, Lung, and Blood Institute
-agonists
• G-protein Coupled Receptor
Penn RB, Benovic JL. Regulation of heterotrimeric G protein signaling in airway smooth muscle. Proc Am Thorac Soc 2008;5:47-57.
PKA
Smooth Muscle Tone Regulation: β2-adrenergic receptor and m3 muscarinic acetylcholine
receptor
Penn RB, Benovic JL. Regulation of heterotrimeric G protein signaling in airway smooth muscle. Proc Am Thorac Soc 2008;5:47-57.
Problems with Long-Term Use
Desensitization
1. Phosphorylation of
the receptor
2. Internalization of
cell-surface receptors
3. Downregulation of
production of new receptors
Rosenbaum DM, Rasmussen SGF, and Kobilka BK. The structure and function of G-protein coupled receptors. Nature 2009; 459: 356-363.
Problems with Long-Term Use
• Polymorphisms of the human 2-adrenoreceptor
• Codon 16: 16% Homozygous Arg-Arg, 37% heterzygous Arg-Gly, 47% Homozygous Gly-Gly
Taylor DR, Drazen JM, Herbison GP, Yandava CN, Hancox RJ, Town GI. Asthma exacerbations during long term beta agonist use: influence of beta(2) adrenoceptor polymorphism. Thorax 2000;55:762-7.
Side Effects
• -Agonist non-tissue specific signaling: increased heart rate, increased blood sugar, and hypokalemia that may invoke cardiac arrhythmias
• Inhaled corticosteroids reduce adrenocortical activity, increase the risk of cataracts, and do not work effectively in smokers
Smooth Muscle Contraction • Cross-Bridge Cycle model well-established
• New evidence points to
requirement of actin
polymerization
Gunst SJ and Zhang W. Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction. Am J Cell Physiol 2008; 295(3):576-587.
New Therapeutics
• Target actin polymerization in airway smooth muscle
• Regulator of actin dynamics: HSP20
– Downstream target of -agonists
– Phosphorylation at Serine 16 induces muscle relaxation
– No effect on myosin light chain phosphorylation or intracellular Ca2+
Alternative Molecular Targets: -agonist Pathway
Decreased intracellular Ca2+
Myosin Contraction
Phosphorylation of Hsp20
Penn RB, Benovic JL. Regulation of heterotrimeric G protein signaling in airway smooth muscle. Proc Am Thorac Soc 2008;5:47-57.
2-AR
AC
Gs
Asthma Pathogenesis and ASM Relaxation
cAMP
PKA
HSP20 P-HSP20
Actin depolymerization
ASM Relaxation
2 – Agonists
P-HSP20
Peptide
Mimetics
Chronic use Of 2 – Agonists
Inflammation
PKA, ARK
phosphorylation
Genetic polymorphism
Komalavilas 2012
Phospho-HSP20 Peptide
• YARAAARQARAWLRRApSAPLPGLK
• 13 amino acid sequence surrounding phosphorylated Serine 16 (red)
• Protein transduction domain from HIV TAT protein (blue)
• Caveolae-dependent
internalization
Flynn CR, Cheung-Flynn J, Smoke CC et al. Internalization and intracellular trafficking of a PTD-conjugated anti-fibrotic peptide, AZX100, in dermal keloid fibroblasts. J
Pharm Sci 2010; 99(7): 3100-3127.
Goal of the Study
• Determine the effects of P20 peptide on ASM relaxation and actin polymerization
• Determine the effects of P20 peptide on actin fiber disruption in human airway smooth muscle cells
Drugs
• Carbachol-Cholinergic agonist that binds to the m3 muscarinic acetylcholine receptor
• Isoproterenol-analog of epinephrine that acts as a β-agonist at the β2-adrenergic receptor
• P20 peptide-phosphomimetic peptide
A B
C D
SF control
P20 peptide
CCH
P20+CCH peptide+CCH
Figure 1: P20 peptide disrupts the formation of stress fibers by the contractile agonist carbachol
Muscle Bath • Measures ring tension
Muscle Bath
0
0.5
1
1.5
2
2.5
0 5 10 15 20
Ten
sio
n (
g)
Time (min)
CCH
P20Peptide+CCHISO+CCH
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 5 10 15 20
Ten
sio
n (
g)
Time (min)
KCl
CCH Prime
A B
C
0
0.5
1
1.5
2
2.5
0 5 10 15 20
Ten
sio
n (
g)
Time (min)
P20 Peptide
ISO
CCH
D
Figure 2: P20 peptide inhibits contraction in pig ASM
G F G F G F G F G F G F
Phalloidin Serum Free
G F G F G F G F G F G F
CCH ISO+CCH
A
B
C D
P20 Peptide +CCH
Figure 3: ISO and P20 peptide decrease the F-actin pool in pig ASM stimulated with CCH
A B
E F
C D
0hr 24hr
G
Figure 4: P20 peptide decreases migration in HASMCs
ProliferationA
bso
rban
ce (
540n
m)
Ser
um F
ree
P20
Pep
tide
PDGF
P20
+PDGF
ISO+P
DGF
0.00
0.01
0.02
0.03
0.04
*
n.s.
Figure 5: P20 peptide does not have negative effects on HASMC proliferation
Conclusions
• P20 peptide inhibits the formation of stress fibers in HASMCs – Previous studies found a loss of actin in other cell
types
• P20 peptide can inhibit contraction of stimulated pig ASM – Previous studies found P20 peptide induces relaxation
• Together, suggests that actin is depolymerized prior to and during stimulation and prevents contraction
Conclusions
• P20 peptide dramatically increases the pool of G-actin, although only one trial
• Actin depolymerization by P20 peptide is sufficient to inhibit migration
• P20 peptide is not cytotoxic and will not promote hyperplasia of the airway according to cell studies
Further Directions
• More actin assay trials
– Human lungs
• Dose response in cell experiments
• Animal models of asthma
Why is this important?
• Medicine is moving to become more personalized
• Genomic Therapeutics: SNPs such as codon 16
• Targeting the molecular basis of smooth muscle contraction
Acknowledgements
• Thank you to Dr. Brophy, Dr. Komalavilas, Dr. Cheung-Flynn, and Kyle Hocking for supporting me in lab during my time at Vanderbilt
• Thank you to committee members Dr. Broadie and Dr. Gamse and director Dr. Patton for reviewing the project