A-kinase anchoring proteins als nieuwe targets in de ontwikkeling en progressie van luchtwegvernauw

  • Aanvrager: Dr W.J. Poppinga

EB 2016: Compartmentalized signaling in experimental biology Wilfred J. Poppinga, Rijks Universiteit Groningen 7.1.16.101CO

A-Kinase Ancboring Proteins Coordinate the Contractile Phenotype of Airway Smooth Muscle

Wilfred Jelco Poppinga I ,2, Bing Han I ;2, Carolina R. Elzinga I, Andrew J. Halayko3, Herman Meurs I ;2, Martina Schmidt I ;2. I Molecular Pharmacology, University of Groningen, Groningen, Netherlands, 2Groningen Research Institute for Asthma and COPD, University Medica!Center Groningen, Groningen, Netherlands, 3Physiology and Pathophysiology, and Intemal Medicine, University of Manitoba, Winnipeg, MB, Canada

Smooth muscle contraction is modulated by three pathways: the amount of free [Ca2+]i; Ca2+ sensitivity; and, the regulation of intracellular filaments comprising the contractile machinery. Recently, a new dirneusion has emerged to play a role in airway smooth muscle function, namely
the scaffolding protein family A-kinase anchoring proteins (AKAPs) that bind cAMP effector PKA and other proteins to specific cellular compartments. Here we studied the role of AKAPs in maintaining the contractile phenotype ofthe airway smooth muscle. Treatment with AKAP-PKA interaction inhibitor st-Ht31 (50 f.J.M) for 96 h caused an increase in abundance ofthe contractile proteins a-smooth muscle actin (aSMA) (283% of basal) and calponin (159% of basal) in immortalized hurnan trachea!smooth muscle cells, without an increase in mRNA expression or stability. To understand potential underlying mechanisms for this response, after 72 h incubation with st-Ht31, we treated cells (24 h) with the mRNA transcription inhibitor, actinomycin D (1 f.lg/mL): this decreased abundance of both proteins in absence and presence of st-Ht31 ( 50 aSMA,70% calponin). Nonetheless, st-Ht31 still induced higher protein levels compared to untreated control cultures ( 35% aSMA, 80% calponin). Inhibition of protein synthesis using cycloheximide (5 f.J.g/mL) had no effect on the expression aSMA, however it did increase the expression of calponin under both control and st-Ht31-treatment conditions( 200%), suggesting that aSMA is a rather stabie protein and that calponin degradation requires de novo synthesis of an unknown protein. Lysosomal inhibition using chloroquine (50 f.J.M) did not affect either protein. lnhibition ofthe proteasome with MG-132 (5 f.J.M) reversed st-Ht31-induced increase of aSMA (102% of basal), without affecting the st-Ht31 induced increase of calponin (210% of basal), revealing that st-Ht31-induced increase in aSMA requires proteasomal degradation of an unknown protein. Functionally, incubation of human trachea!smooth muscle strips with st-Ht31 for 96 h increased both the abundance of contractile proteins and the contractile response to methacholine. These data implicate that AKAPs regulate the maintenance of contractile smooth muscle, probably by post-translationally controlling levels of contractile proteins.

Support or Funding Information
This study was supported by the Dutch Lung Foundation (Grant #: 3.2.11.015) and an Ubbo
Emmius grant.