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A2854 - Non-Canonical Regulation of Beta2 Adrenergic Receptor in Response to RSV Infection in Human Primary Airway Smooth Muscle Cells
Author Block: T. J. Harford1, V. Bokun2, M. K. Gupta3, S. V. Naga Prasad4, F. Rezaee5, G. Piedimonte6; 1Pediatric Research Center, Cleveland Clinic Childrens, Cleveland, OH, United States, 2Patholbiology, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States, 3Molecular Cardiology, Lerner Research Institute, Cleveland, OH, United States, 4Molecular Cardiology, Cleveland Clinic, Cleveland, OH, United States, 5Pediatrics, Center for Pediatric Research, Cleveland, OH, United States, 6Pediatrics, The Cleveland Clinic, Cleveland, OH, United States.
RATIONALE: Airway epithelial cells are the primary target for respiratory syncytial virus (RSV) infection, however studies have shown human airway smooth muscle cells (HASMCs) can also be infected. The primary carepath in treating severe RSV-induced bronchiolitis includes the use of albuterol however, is not recommended in pediatric patients due to lack of effectiveness and potential side effects. A better understanding of the effects of RSV on Beta 2 adrenergic receptor (β2AR) signaling in HASMCs obtained from children is necessary to more effectively treat RSV bronchiolitis in the pediatric population. We therefore hypothesized that RSV infection of hASMCs leads to disruption of the β2AR signaling pathway. METHODS Primary hASMCs isolated from the lungs of pediatric donors were infected with medium, UV inactivated RSV, or RSV strain A2 expressing recombinant red protein (rrRSV) at a multiplicity of infection (MOI) of 1. Changes in protein expression and localization were assessed by Western blot analysis and immunocytochemistry for total and phospho-β2AR. In some experiments, cells were pre-treated with a proteasomal inhibitor (MG-132), an MMP inhibitor (batimastat), or albuterol. Changes in β2AR density in plasma membrane /endosomal fractions were assessed as well as adenylyl cyclase activity. RESULTS: Immunofluorescence analysis showed enhanced phospho-β2AR in RSV infected cells as compared to medium control or UV inactivated virus. Western blot analysis of phosphorylated receptor revealed a cleaved band at approximately 35kDa in RSV-infected cells, in addition to expected bands observed at expected size. Densitometric analysis showed a marked increase of cleaved band in a dose- and time dependent manner post RSV infection. Pretreatment with MG-132, but not batimastat abolished the cleaved band in RSV-infected hASMCs. Plasma membrane associated β2AR was reduced in response to RSV with no respective increase in endosome fraction. Finally, adenylyl cyclase activity increased upon RSV infection. CONCLUSIONS: Results obtained in this study provide evidence as to why children with RSV induced bronchiolitis fail to respond to albuterol. Cleavage of the receptor by a yet to be identified protease results in the β2AR to be incapable of ligand binding. This cleavage disrupts the downstream signaling events required for airway smooth muscle relaxation in response to albuterol. The data presented herein provides critical insights for development of novel treatments for RSV induced bronchiolitis in children who are underserved by current therapies.