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The CREB-Regulated Transcription Coactivators, CRTC2/3, Facilitate the Synergistic Induction of RGS2 by LABAs and Glucocorticoids in Human Bronchial Epithelial Cells
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A3803 - The CREB-Regulated Transcription Coactivators, CRTC2/3, Facilitate the Synergistic Induction of RGS2 by LABAs and Glucocorticoids in Human Bronchial Epithelial Cells
Author Block: M. L. Manson1, Z. Rojnik1, K. Thorn1, C. Karrman Mardh1, R. Newton2; 1Bioscience, Respiratory, Inflammation and Autoimmunity (RIA), IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden, 2Airways Inflammation Research Group, Snyder Institute of Chronic Diseases, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
Rationale: Glucocorticoids (GC) and long-acting β2-agonists (LABAs) form the cornerstone therapy in asthma. The mechanism(s) responsible for their superior efficacy as combination remain incompletely understood, but could involve the synergistic induction of anti-inflammatory genes, like regulator of G-protein signalling 2 (RGS2)1. This study aimed to delineate the molecular interactions underlying this synergy by investigating through which pathways cAMP (LABAs) and glucocorticoids exert their effects on gene transcription. This study focussed thereby specifically on the contribution of transcriptional co-activators of cAMP signalling: EP300, CREBBP, CRTC2 and CRTC3.
Methods: EP300, CREBBP, CRTC2 and CRTC3 were individually or in combination knocked-down by siRNA in BEAS-2B and primary human bronchial epithelial cells (HBECs). GC and cAMP-driven gene induction (mRNA) upon dexamethasone (GC), formoterol (LABA), or the combination (GC+LABA) were examined 48-hours post siRNA transfection and 1-6 hours after the different stimulations. siRNA-interventions were performed in duplicate using different individual siRNA’s and compared against lipofectamine and lamin siRNA transfection controls.
Results: siRNA-targeted interventions resulted in 75-90% knock-down of EP300, CREBBP, CRTC2 and CRTC3 protein levels in BEAS-2B and HBECs. Double knock-down (dkD) of EP300 and CREBBP, co-activators with histone-acetyltransferase activity, reduced global H3K18ac (-80%) and H3K27ac (-20%). However, maximal induction of LABA-driven genes and GC+LABA inducible synergies remained intact or were even amplified in EP300/CREBBP dKD.
In contrast, dKD of CRTC2 and CRTC3 impaired both LABA-driven induction of RGS2 (-65-81%) at 2-hours and RGS2 synergy (-72%) by GC+LABA at 6-hours. Validation of these siRNA findings was confirmed by overexpressing a CRTC2 plasmid, which enhanced the RGS2 synergy and rescued the CRTC2/3 dKD phenotype. Confocal immunofluorescence microscopy studies confirmed the nuclear translocation of CRTC2 upon LABA treatment in BEAS-2B, an effect that was not influenced by GC. Conversely, GC reduced nuclear retention of CRTC2 following LABA stimulation (6-hours), by promoting proteosomal breakdown of CRTC2 (LABA: -23% vs GC+LABA: -75%), thereby revealing a negative feedback loop that limits prolonged signalling through CRTC2.
RGS2 synergy upon GC+LABA co-administration was also prevented in HBECs deficient for both CRTC2 and CRTC3. Finally, an RNA-seq was conducted in CRTC2/3-deficient HBECs to define the global contribution of CRTCs for LABA-driven cAMP-signalling in bronchial epithelial cells.
Conclusions: This study demonstrates for the first time that CRTCs act as key transcriptional regulators in human bronchial epithelial cells. CRTCs were shown to be critical for cAMP-driven transcriptional regulation and necessary for the synergistic induction of RGS2 by glucocorticoids and LABAs.
1 NS Holden, PNAS, 2011
Author Block: M. L. Manson1, Z. Rojnik1, K. Thorn1, C. Karrman Mardh1, R. Newton2; 1Bioscience, Respiratory, Inflammation and Autoimmunity (RIA), IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden, 2Airways Inflammation Research Group, Snyder Institute of Chronic Diseases, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
Rationale: Glucocorticoids (GC) and long-acting β2-agonists (LABAs) form the cornerstone therapy in asthma. The mechanism(s) responsible for their superior efficacy as combination remain incompletely understood, but could involve the synergistic induction of anti-inflammatory genes, like regulator of G-protein signalling 2 (RGS2)1. This study aimed to delineate the molecular interactions underlying this synergy by investigating through which pathways cAMP (LABAs) and glucocorticoids exert their effects on gene transcription. This study focussed thereby specifically on the contribution of transcriptional co-activators of cAMP signalling: EP300, CREBBP, CRTC2 and CRTC3.
Methods: EP300, CREBBP, CRTC2 and CRTC3 were individually or in combination knocked-down by siRNA in BEAS-2B and primary human bronchial epithelial cells (HBECs). GC and cAMP-driven gene induction (mRNA) upon dexamethasone (GC), formoterol (LABA), or the combination (GC+LABA) were examined 48-hours post siRNA transfection and 1-6 hours after the different stimulations. siRNA-interventions were performed in duplicate using different individual siRNA’s and compared against lipofectamine and lamin siRNA transfection controls.
Results: siRNA-targeted interventions resulted in 75-90% knock-down of EP300, CREBBP, CRTC2 and CRTC3 protein levels in BEAS-2B and HBECs. Double knock-down (dkD) of EP300 and CREBBP, co-activators with histone-acetyltransferase activity, reduced global H3K18ac (-80%) and H3K27ac (-20%). However, maximal induction of LABA-driven genes and GC+LABA inducible synergies remained intact or were even amplified in EP300/CREBBP dKD.
In contrast, dKD of CRTC2 and CRTC3 impaired both LABA-driven induction of RGS2 (-65-81%) at 2-hours and RGS2 synergy (-72%) by GC+LABA at 6-hours. Validation of these siRNA findings was confirmed by overexpressing a CRTC2 plasmid, which enhanced the RGS2 synergy and rescued the CRTC2/3 dKD phenotype. Confocal immunofluorescence microscopy studies confirmed the nuclear translocation of CRTC2 upon LABA treatment in BEAS-2B, an effect that was not influenced by GC. Conversely, GC reduced nuclear retention of CRTC2 following LABA stimulation (6-hours), by promoting proteosomal breakdown of CRTC2 (LABA: -23% vs GC+LABA: -75%), thereby revealing a negative feedback loop that limits prolonged signalling through CRTC2.
RGS2 synergy upon GC+LABA co-administration was also prevented in HBECs deficient for both CRTC2 and CRTC3. Finally, an RNA-seq was conducted in CRTC2/3-deficient HBECs to define the global contribution of CRTCs for LABA-driven cAMP-signalling in bronchial epithelial cells.
Conclusions: This study demonstrates for the first time that CRTCs act as key transcriptional regulators in human bronchial epithelial cells. CRTCs were shown to be critical for cAMP-driven transcriptional regulation and necessary for the synergistic induction of RGS2 by glucocorticoids and LABAs.
1 NS Holden, PNAS, 2011
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