Glucocorticoids rapidly stimulate cAMP production in a Gαs‐dependent manner
Authors: Nuñez Francisco J. , Corpuz Maia L. , Fogel David M. , Lyon Katherine T. , Kazarian Austin G. , Johnstone Timothy B. , Ostrom Rennolds S 
Published: April 2020

Glucocorticoids have wide therapeutic applications in inflammation and immune disorders and have traditionally been used to treat diseases such as asthma and COPD. They exert their anti‐inflammatory effects by binding to the cytosolic glucocorticoid receptor (GRα), a nuclear receptor that translocates into the nucleus to regulate gene transcription. Several studies have shown that glucocorticoids can have rapid, non‐genomic actions. For instance, short duration studies on several cell lines revealed that glucocorticoids can rapidly modulate calcium signaling, reactive oxygen species and arachidonic acid release. Moreover, studies show that a membrane GR (mGR) may interact directly with a GPCR coupled to Gαs and/or Gq/11.

Objective

To better understand the underlying mechanism of the non‐genomic effects of glucocorticoids, we tested the hypothesis that glucocorticoids can directly stimulate cAMP production via a Gαs coupled GPCR.

Results

The glucocorticoids prednisone, fluticasone and budesonide stimulate cAMP rapidly in the absence of phosphodiesterase (PDE) inhibitors as measured in real time via the cADDis assay. These effects were seen in human airway smooth muscle (HASM), HEK‐293, HFL‐1, MCF‐7 and A375 cells. Glucocorticoids retained cAMP stimulating activity in HASM cells preincubated with 10 μM IBMX, a broad spectrum PDE inhibitor, implying that they do not increase cAMP via inhibition of PDEs. Cortisol‐albumin conjugate, a cell membrane impermeable glucocorticoid, stimulated cAMP suggesting that an outward facing membrane‐bound receptor mediates glucocorticoid non‐genomic signaling. GRα knockdown had no effect on glucocorticoid‐stimulation of cAMP, implying a GRα‐independent mechanism. We tested whether glucocorticoid‐stimulated cAMP depends upon the G protein Gαs by transfecting HASM with siRNA for GNAS. Immunoblot analysis confirmed a reduction in expression of both long and short forms of Gαs in the knockdown condition. Gαs knockdown lead to diminished cAMP production by fluticasone, budesonide and formoterol but not by forskolin, indicating that Gαs is an essential part of the non‐genomic signaling mechanism of glucocorticoids. The G protein estrogen receptor (GPER) is a G‐protein coupled receptor (GPCR) that couples to Gαs and binds to specific steroid ligands and is expressed in various cell types including HASM, HFL‐1 and HEK‐293. In the presence of either G15 or G36, GPER‐specific antagonists, both budesonide‐ and fluticasone‐stimulated cAMP was unaltered, suggesting that GPER does not mediate cAMP signaling by glucocorticoids.

Conclusion

Taken together these results reveal that glucocorticoids induce rapid, non‐genomic signaling via the Gαs‐cAMP pathway. The identity of the cell surface receptor for glucocorticoids is unknown, but given the rapid, GRα‐independent, Gαs‐dependent nature of the response we hypothesize that a GPCR or family of GPCRs is involved.

Support or Funding Information

This work was supported by NIH grant GM107094