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