The Catalytic α Subunit of cAMP‐Dependent Protein Kinase Regulates Formoterol‐Induced Gene Expression in BEAS‐2B Human Bronchial Epithelial Cells
Notice bibliographique
Résumé
Rationale The β 2 ‐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 β 2 ‐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. Methods Microarray, 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 ). Results Microarray‐based gene expression profiling indicated that BEAS‐2B cells express mRNA transcripts for PRKACA (Cα), PRKACB (Cβ) and PRKX (C x ) 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 . Conclusions These 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 Information Alberta Lung Association (ALA)
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,002 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».