title: 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 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Charité University Hospital and German Rheumatism Research Centre, Berlin, Germany. strehl@drfz.de
authors: Cindy
authors: C
authors: Strehl
authors: Timo
authors: T
authors: Gaber
authors: Mark
authors: M
authors: Löwenberg
authors: Daniel W
authors: DW
authors: Hommes
authors: Auke P
authors: AP
authors: Verhaar
authors: Saskia
authors: S
authors: Schellmann
authors: Martin
authors: M
authors: Hahne
authors: Monique
authors: M
authors: Fangradt
authors: Markus
authors: M
authors: Wagegg
authors: Paula
authors: P
authors: Hoff
authors: Alexander
authors: A
authors: Scheffold
authors: Cornelia M
authors: CM
authors: Spies
authors: Gerd-Rüdiger
authors: GR
authors: Burmester
authors: Frank
authors: F
authors: Buttgereit
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid; D012710:Serum Albumin, Bovine; D003907:Dexamethasone; D048051:p38 Mitogen-Activated Protein Kinases