Origin and functional activity of the membrane-bound glucocorticoid receptor

Abstract

Objective. 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.Methods. 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 highsensitivity 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.Results. 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.Conclusion. 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 GRmediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.Glucocorticoids (GCs) are the most commonly used drugs in the treatment of a wide range of rheumatic diseases. These substances exert their immunosuppressive, antiinflammatory, and antiallergic effects on primary and secondary immune cells, tissues, and organs (1). Unfortunately, GCs have the potential to exert unwanted effects on metabolism, bone tissue, muscles, eyes, and skin, especially when the dose and duration of treatment are increased (2-5). These disadvantages drive the need for an improved risk/benefit ratio for GCs, for instance by understanding the mechanisms of GC action in greater detail. The therapeutic effects of GCs are primarily mediated by cytosolic GC receptor ␣ (cGR␣), the classic genomic mechanism of GC action Dr. Buttgereit's work was supported by grants from the DFG (Bu 1015/7-1) and the Deutsche Sparkassenstiftung Medizin. Mr. Hahne's work was supported by the DFG Graduate School GSC 203 Project at the Berlin-Brandenburg School for Regenerative Therapies.

Membrane Glucocorticoid Receptor Activation Induces Proteomic Changes Aligning with Classical Glucocorticoid Effects

“…This strongly suggests that the mGR and the cGR have a high sequence homology (at least within these first 400 residues) and most probably originate from the same gene. Accordingly, a mild decrease in mGR levels was previously observed when the classical GR transcript was stably silenced with siRNAs (21). We also confirmed that the Ligand Binding Domain is exposed on the cell surface and is accessible to interact with the membrane-impermeable Cort-BSA conjugate.…”

“…It is now accepted that the mGR is a product of the NR3C1 gene, as is the classical cytosolic GR. First, antibodies raised and directed against the cGR epitopes are able to specifically detect a membranebound form (19,20) and additionally, a recent report demonstrated that stable silencing of the classical GR gene is able to down-regulate mGR expression (21). However the over-expression of the classical GR transcript did not lead to an increased level of mGR (22), suggesting that the membrane isoform is not simply an unmodified GR localized on the cell surface.…”

“…However, bovine serum albumin conjugated GCs (GC-BSA) have been successfully used to discriminate the specific activities of the mGR from those of the cytosolic GR (21). Here, we present the first proteomic study of the effects of the mGR selectively activated by BSA-conjugated cortisol in a human lymphoma cell line.…”

Recent Topics in Steroid and Asthma: Beyond the 'Classic' Concept of Action

“…The non-genomic effects of GC in asthma are becoming clearer. A physical and functional interaction between the GR and the T-cell receptor (TCR) complex [9] and membrane-bound GR induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) due to DEX/bovine serum albumin (BSA) treatment [10] has been reported. Low concentrations of DEX rapidly regulate intracellular pH, Ca 2+ and cAMP-dependent protein kinase activity, and inhibit Cl -secretion in bronchial epithelial cells via nongenomic mechanisms [11].…”

The Clinical Role of Glucocorticoids in the Management of Rheumatoid Arthritis

“…Interestingly in patients with RA who have an activated immune system, the percentage memebrane GCR expressing monocytes is increased, in keeping with the in-vitro observations. These membrane expressed receptors are thought to be variants of the classical cytoplasmic GCRs (Löwenberg et al, 2007) and have recently been shown to also interact with the MAP kinase pathway (Strehl et al, 2011) Moreover, the engagement of these receptors is thought to inhibit T cell signalling by acting through downstream TCR associated signalling proteins lymphocytespecific tyrosine kinase (LCK) and FYN oncogene (Lowenberg et al, 2006). It is possible that memberane glucocorticoid receptors will prove to have therapeutic implications.…”