Origin and functional activity of the membrane-bound glucocorticoid receptor
Article first published online: 29 NOV 2011
Copyright © 2011 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 63, Issue 12, pages 3779–3788, December 2011
How to Cite
Strehl, C., Gaber, T., Löwenberg, M., Hommes, D. W., Verhaar, A. P., Schellmann, S., Hahne, M., Fangradt, M., Wagegg, M., Hoff, P., Scheffold, A., Spies, C. M., Burmester, G.-R. and Buttgereit, F. (2011), Origin and functional activity of the membrane-bound glucocorticoid receptor. Arthritis & Rheumatism, 63: 3779–3788. doi: 10.1002/art.30637
- Issue published online: 29 NOV 2011
- Article first published online: 29 NOV 2011
- Accepted manuscript online: 26 AUG 2011 01:20PM EST
- Manuscript Accepted: 16 AUG 2011
- Manuscript Received: 9 MAR 2011
- DFG. Grant Number: Bu 1015/7-1
- Deutsche Sparkassenstiftung Medizin
- DFG Graduate School GSC 203 Project at the Berlin-Brandenburg School for Regenerative Therapies
Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR.
We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference–mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)–bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR–expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting.
Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA.
Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR–mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.