(3-agonists activate Gs-coupled GPCRs (B2ARs) and attenuate agonist-induced contraction of human airway smooth muscle (HASM) via activation of cAMP/protein kinase A pathways. Evidence suggests, however, that p-agonists can worsen asthma control and contribute to asthma morbidity and mortality. These adverse effects, in part, are attributed to p-agonist tachyphylaxis, mediated by B2AR desensitization, internalization and phosphorylation. Using human precision cut lung slices (hPCLS) and HASM cells, our studies challenge current dogma and show that steroids modulate B2AR tachyphylaxis without increasing B2AR number or density. Steroids decrease B-agonist-induced expression of phosphoinositide 3 kinase (PI3K) that promotes B2AR desensitization and increases protein phosphatase 2A (PP2A) dephosphorylation of the receptor that fosters recycling of the P2AR to the cell surface. Our central hypothesis states that steroids modulate B2AR induced bronchodilation by preventing desensitization and enhancing resensitization ofthe B2AR.
In Aim 1, we will define whether steroids modulate G protein receptor kinase (GRK)-induced B2AR phosphorylation, B-arresting binding and decrease B2AR trafficking/degradation to lysosomes. Using siRNA and lentivirus mutant constructs of B2AR GRKs and B-arrestins, the necessity and sufficiency of these signaling events in mediating steroid effects on B2AR tachyphylaxis will be defined. Using ASM cells derived from fatal asthma subjects, the role ofBP2AR phosphorylation, B-arresting binding and trafficking in the presence of steroids will determine whether steroids modulate asthma-induced alterations in p-agonist tachyphylaxis.
In Aim 2, we will define whether steroids resensitize B2ARs by decreasing PI3K activation and enhancing PP2A dephosphorylation of the B2AR. PI3K isoform expression and activation induced by p-agonists will be determined in the presence and absence of steroids in HASM cells and in hPCLS. The role of PI3K and PP2A in mediating steroid rescue of B2AR tachyphylaxis will be determined using siRNA constructs and lentivirus of mutant PI3K catalytic subunits and PP2A. Identifying mechanisms by which steroids prevent B-agonist-induced tachyphylaxis will provide new therapeutic targets to enhance bronchodilation in asthma.

Public Health Relevance

Beta receptor down regulation by B-agonists limits the clinical efficacy of such therapy. This study will define how steroids reverse B2AR downregulation in order to identify novel therapeutic approaches to treat asthma.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL114471-01A1
Application #
8512852
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2013-07-15
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$478,130
Indirect Cost
$170,063
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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