Airway inflammation and reversible bronchoconstriction characterize asthma;however, in severe disease, irreversible airway obstruction and increases in airway smooth muscle (ASM) mass/growth commonly occur. Growth factors require activation of Src, phosphatidylinositol 3-kinase (PI3K) and S6K1 to stimulate ASM growth. Our new data shows that mitogens increase ASM expression of regulators of G-protein signaling (RGS) proteins which inactivate G proteins and co-localize with phospho-p85 (p-p85) PI3K to facilitate S6K1 activation. Importantly, RGS protein knockdown inhibited ASM growth;mitogens inhibited agonist-induced contraction and decreased cytosolic calcium levels [Ca2+]i. These data suggest that specific RGS proteins serve as molecular switches to promote ASM growth while inhibiting excitation-contraction (E-C) coupling. Our central hypothesis states that RGS proteins promote mitogen-induced ASM proliferation in a PI3K-dependent manner and inhibit agonist-induced contraction by altering E-C coupling. Newly developed techniques by the PI using RGS knockdown of human ASM from normals and those with asthma will define how RGS proteins regulate cell growth. The use of precision cut lung slices (PCLS) and calcium imaging will define how RGS proteins regulate ASM contraction. Fluorescence lifetime imaging microscopy (FLIM) will identify RGS protein- protein interactions in biopsies and ASM cells from subjects with asthma.
In Aim 1, levels of RGS protein expression and PI3K activation will be defined in ASM cells and in PCLS. The necessity and sufficiency of RGS proteins to regulate Src, PI3K, S6K1 activation and growth will be defined in ASM transfected with mutant RGS constructs. Using FLIM, alterations in ASM mass, RGS protein expression and p-p85 PI3K in endobronchial biopsies will be assessed.
In Aim 2, the necessity and sufficiency of RGS proteins to regulate [Ca2+]i and contraction will be determined. RGS protein effects on agonist-induced PLC activation, myosin light chain phosphorylation, [Ca2+]i levels and RhoA kinase activation will be clarified in ASM transfected with mutant contracts. Our studies will identify the key mechanisms by which RGS proteins regulate ASM growth and contractility while providing insight into therapeutic targets to abrogate ASM hyperplasia and hyperresponsiveness in asthma.

Public Health Relevance

Although asthma is characterized by airway inflammation and reversible obstruction, in severe asthma, some develop irreversible airflow obstruction that profoundly affects morbidity and mortality. Regulatory G protein Signaling (RGS) molecules promote ASM growth and alter ASM contraction. These molecules may serve as a novel therapeutic target in the treatment of severe asthma and thus prevent aberrant ASM growth while modulating bronchoconstriction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL097796-04
Application #
8316179
Study Section
Special Emphasis Panel (ZHL1-CSR-A (M1))
Program Officer
Banks-Schlegel, Susan P
Project Start
2009-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2012
Total Cost
$490,194
Indirect Cost
$145,460
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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