A‐Kinase Anchoring Proteins Coordinate the Contractile Phenotype of Airway Smooth Muscle

Smooth muscle contraction is modulated by three pathways: the amount of free [Ca 2+ ] i ; Ca 2+ sensitivity; and, the regulation of intracellular filaments comprising the contractile machinery. Recently, a new dimension 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 of the airway smooth muscle. Treatment with AKAP‐PKA interaction inhibitor st‐Ht31 (50 μM) for 96 h caused an increase in abundance of the contractile proteins α‐smooth muscle actin (αSMA) (283% of basal) and calponin (159% of basal) in immortalized human tracheal 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 μg/mL): this decreased abundance of both proteins in absence and presence of st‐Ht31 (~50 αSMA, ~70% calponin). Nonetheless, st‐Ht31 still induced higher protein levels compared to untreated control cultures (~35% αSMA, ~80% calponin). Inhibition of protein synthesis using cycloheximide (5 μg/mL) had no effect on the expression αSMA, however it did increase the expression of calponin under both control and st‐Ht31‐treatment conditions (~200%), suggesting that αSMA is a rather stable protein and that calponin degradation requires de novo synthesis of an unknown protein. Lysosomal inhibition using chloroquine (50 μM) did not affect either protein. Inhibition of the proteasome with MG‐132 (5 μM) reversed st‐Ht31‐induced increase of αSMA (102% of basal), without affecting the st‐Ht31 induced increase of calponin (210% of basal), revealing that st‐Ht31‐induced increase in αSMA requires proteasomal degradation of an unknown protein. Functionally, incubation of human tracheal 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.