The Catalytic α Subunit of cAMP‐Dependent Protein Kinase Regulates Formoterol‐Induced Gene Expression in BEAS‐2B Human Bronchial Epithelial Cells

Abstract

RationaleThe β2‐adrenoceptor is a GPCR that classically couples to Gsα, the activation of adenylyl cyclases and the formation of cAMP. Through distinct signalling complexes often containing cAMP‐dependent protein kinase (PKA), cAMP mediates a myriad of functional responses including the regulation of gene expression. We have shown previously that in the BEAS‐2B human airway epithelial cell line, long acting β2‐adrenoceptor agonists (LABAs) induce cAMP response element (CRE) dependent transcription, which we suggest contributes to their therapeutic activity in obstructive lung diseases. In the current study we have extended those findings by identifying the PKA catalytic subunit responsible for mediating gene expression induced by the LABA, formoterol.MethodsMicroarray, RT‐PCR, western blotting and gene silencing were used to identify and implicate PKA catalytic subunits in the regulation of LABA‐induced gene expression. A CRE reporter construct stably transfected into BEAS‐2B cells was used for this purpose together with an assessment of CREB phosphorylation and an analysis of a panel of cAMP‐inducible genes (NR4A3, CRISPLD2, RGS2, NR4A2, CD200 and IL‐6).ResultsMicroarray‐based gene expression profiling indicated that BEAS‐2B cells express mRNA transcripts for PRKACA (Cα), PRKACB (Cβ) and PRKX (Cx) whereas PRKACG (Cg) was absent. These data were confirmed at the protein level by western blotting. siRNAs were optimized to achieve 80 to 90% knockdown of PRKACA, PRKACB and PRKX, which was reproduced at the protein level. Under these conditions, silencing PRKACA was associated with a significant (~50%) abrogation of formoterol‐induced CRE reporter activation, CREB phosphorylation and gene expression. No additional effect was seen when siRNAs that target PRKACB or PRKX were used in combination with those directed towards PRKACA.ConclusionsThese data show that Cα is an essential cAMP signalling component controlling formoterol‐induced gene transcription in BEAS‐2B cells. The incomplete inhibition produced under conditions of 80–90% Cα knockdown implies that this PKA catalytic subunit is present in significant excess of that required to maximally mediate cAMP‐induced gene expression. This is an initial first step at identifying cAMP signalling components (adenylyl cyclases, scaffold proteins, phosphodiesterases and effectors) that control LABA‐induced functional responses in airway structural cells. We propose that identifying these components and their assembly into multiprotein signalling complexes could provide new opportunities for the development of novel therapies for asthma and chronic obstructive pulmonary disease.Support or Funding InformationAlberta Lung Association (ALA)