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

  • Sara Vernocchi
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg

    Department of Immunology, Research Institute of Psychobiology, University of Trier, D-54290 Trier, Germany
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  • Nadia Battello
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg
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  • Stephanie Schmitz
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg
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  • Dominique Revets
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg
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  • Anja M. Billing
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg
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  • Jonathan D. Turner
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg

    Department of Immunology, Research Institute of Psychobiology, University of Trier, D-54290 Trier, Germany
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  • Claude P. Muller
    Correspondence
    To whom correspondence should be addressed: Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg. Tel.: +352 490604 220; Fax: +352 490686;
    Affiliations
    From the Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Grand-Duchy of Luxembourg

    Department of Immunology, Research Institute of Psychobiology, University of Trier, D-54290 Trier, Germany
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  • Author Footnotes
    * This work was supported by a grant from the Fonds National de la Recherche, Luxembourg (AFR grants PHD-08-053).
    This article contains supplemental Figs. S1 to S5 and Tables S1 to S3.
Open AccessPublished:January 22, 2013DOI:https://doi.org/10.1074/mcp.M112.022947
      Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly “priming” classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.
      Classically, glucocorticoids (GCs)
      The abbreviations used are:
      GC
      Glucocorticoid
      aa
      amino acid
      Ab
      antibody
      AF1
      transactivation domain
      ATP
      Adenosine triphosphate
      Cav-1
      caveolin-1
      cGR
      cytosolic glucocorticoid receptor
      Co-A
      coenzyme-A
      Cort
      cortisol
      Cort-BSA
      BSA-conjugated cortisol
      CoxVb
      Cytochrome C oxidase, subunit Vb
      FBS
      fetal bovine serum
      GC-BSA
      BSA-conjugated Glucocorticoid
      GILZ
      GC-induced leucine zipper
      GR
      glucocorticoid receptor
      HADH
      3-hydroxyacyl-CoA dehydrogenase type-2
      IPA
      Ingenuity pathway analysis
      mAb
      Monoclonal antibody
      MAT II
      Methionine adenosyltransferase 2
      mGR
      membrane glucocorticoid receptor
      PBS
      Phosphate buffered saline
      PLA
      Proximity ligation assay
      PMT
      Photomultiplier Tubes
      siRNA
      Short interfering RNA.
      1The abbreviations used are:GC
      Glucocorticoid
      aa
      amino acid
      Ab
      antibody
      AF1
      transactivation domain
      ATP
      Adenosine triphosphate
      Cav-1
      caveolin-1
      cGR
      cytosolic glucocorticoid receptor
      Co-A
      coenzyme-A
      Cort
      cortisol
      Cort-BSA
      BSA-conjugated cortisol
      CoxVb
      Cytochrome C oxidase, subunit Vb
      FBS
      fetal bovine serum
      GC-BSA
      BSA-conjugated Glucocorticoid
      GILZ
      GC-induced leucine zipper
      GR
      glucocorticoid receptor
      HADH
      3-hydroxyacyl-CoA dehydrogenase type-2
      IPA
      Ingenuity pathway analysis
      mAb
      Monoclonal antibody
      MAT II
      Methionine adenosyltransferase 2
      mGR
      membrane glucocorticoid receptor
      PBS
      Phosphate buffered saline
      PLA
      Proximity ligation assay
      PMT
      Photomultiplier Tubes
      siRNA
      Short interfering RNA.
      exert their immunomodulatory effect by activating the cytosolic glucocorticoid receptor (cGR), which translocates to the nucleus and regulates gene expression (
      • Zanchi N.E.
      • Filho M.A.
      • Felitti V.
      • Nicastro H.
      • Lorenzeti F.M.
      • Lancha Jr., A.H.
      Glucocorticoids: extensive physiological actions modulated through multiple mechanisms of gene regulation.
      ). However, there is increasing evidence of GCs effects on a large number of tissues and organs, which are independent of transcriptional changes and occur rapidly, within minutes or seconds of exposure to GCs (
      • Buttgereit F.
      • Scheffold A.
      Rapid glucocorticoid effects on immune cells.
      ,
      • Boldizsar F.
      • Talaber G.
      • Szabo M.
      • Bartis D.
      • Palinkas L.
      • Nemeth P.
      • Berki T.
      Emerging pathways of non-genomic glucocorticoid (GC) signalling in T cells.
      ,
      • Haller J.
      • Mikics E.
      • Makara G.B.
      The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings.
      ). One of the mechanisms proposed for these rapid nongenomic GC-effects is the activation of a membrane-bound GR (mGR).
      The existence of a glucocorticoid receptor (GR) in plasma membrane was first reported in a mouse lymphoma cell line (S-49) and it was proposed to be functionally associated with glucocorticoid-induced cell death (
      • Gametchu B.
      Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis.
      ). Subsequently, a corticosterone binding protein was identified in synapses of amphibian brain, with characteristics similar to G-protein coupled receptors (
      • Orchinik M.
      • Murray T.F.
      • Franklin P.H.
      • Moore F.L.
      Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes.
      ,
      • Orchinik M.
      • Murray T.F.
      • Moore F.L.
      A corticosteroid receptor in neuronal membranes.
      ,
      • Evans S.J.
      • Moore F.L.
      • Murray T.F.
      Solubilization and pharmacological characterization of a glucocorticoid membrane receptor from an amphibian brain.
      ,
      • Evans S.J.
      • Murray T.F.
      • Moore F.L.
      Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain.
      ). The existence of such a receptor was also reported in a mouse pituitary cell line (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ,
      • Maier C.
      • Rünzler D.
      • Schindelar J.
      • Grabner G.
      • Waldhausl W.
      • Kohler G.
      • Luger A.
      G-protein-coupled glucocorticoid receptors on the pituitary cell membrane.
      ), suggesting that a second gene is involved in the expression of this GC-binding proteins at least in the central nervous system. However, in rats a GR immunoreactive protein was detected on the plasma membrane of liver cells (
      • Grote H.
      • Ioannou I.
      • Voigt J.
      • Sekeris C.E.
      Localization of the glucocorticoid receptor in rat liver cells: evidence for plasma membrane bound receptor.
      ), of hippocampal and hypothalamic neurons (
      • Liposits Z.
      • Bohn M.C.
      Association of glucocorticoid receptor immunoreactivity with cell membrane and transport vesicles in hippocampal and hypothalamic neurons of the rat.
      ), and of neuronal and glial cells in the lateral amygdala. These data support the hypothesis that the mGR originate from the NR3C1 gene, as the cytosolic receptor (
      • Johnson L.R.
      • Farb C.
      • Morrison J.H.
      • McEwen B.S.
      • LeDoux J.E.
      Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala.
      ).
      The origin and the function of this GR isoform were further investigated in the S-49 mouse T-lymphoma cell line (
      • Gametchu B.
      • Watson C.S.
      • Shih C.C.
      • Dashew B.
      Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells.
      ,
      • Chen F.
      • Watson C.S.
      • Gametchu B.
      Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells.
      ,
      • Chen F.
      • Watson C.S.
      • Gametchu B.
      Multiple glucocorticoid receptor transcripts in membrane glucocorticoid receptor-enriched S-49 mouse lymphoma cells.
      ,
      • Powell C.E.
      • Watson C.S.
      • Gametchu B.
      Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49++ lymphoma cells: biochemical characterization and interaction with Hsp 70 and Hsp 90.
      ,
      • Gametchu B.
      • Watson C.S.
      • Pasko D.
      Size and steroid-binding characterization of membrane-associated glucocorticoid receptor in S-49 lymphoma cells.
      ,
      • Gametchu B.
      • Watson C.S.
      Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines.
      ). The presence of the mGR appeared to be linked to the expression of exon 1A-containing GR transcripts and the production of a high molecular weight (150 kDa) GR immuno-reactive protein. The mammalian mGR was proposed to be a variant of the classical cytosolic GR. 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 membrane-bound form (
      • Gametchu B.
      • Watson C.S.
      • Wu S.
      Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.
      ,
      • Berki T.
      • Kumánovics G.
      • Kumánovics A.
      • Falus A.
      • Ujhelyi E.
      • Németh P.
      Production and flow cytometric application of a monoclonal anti-glucocorticoid receptor antibody.
      ) and additionally, a recent report demonstrated that stable silencing of the classical GR gene is able to down-regulate mGR expression (
      • 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.
      • Buttgereit F.
      Origin and functional activity of the membrane-bound glucocorticoid receptor.
      ). However the over-expression of the classical GR transcript did not lead to an increased level of mGR (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ), suggesting that the membrane isoform is not simply an unmodified GR localized on the cell surface.
      The number of mGR molecules per cell is particularly low. In CCRF-CEM cells, a human T-cell lymphoblast-like cell line the detection was possible only after enrichment of mGR+ cells using immunopanning methods (
      • Gametchu B.
      • Watson C.S.
      • Wu S.
      Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.
      ,
      • Gametchu B.
      • Chen F.
      • Sackey F.
      • Powell C.
      • Watson C.S.
      Plasma membrane-resident glucocorticoid receptors in rodent lymphoma and human leukemia models.
      ,
      • Sackey F.N.
      • Watson C.S.
      • Gametchu B.
      Cell cycle regulation of membrane glucocorticoid receptor in CCRF-CEM human ALL cells: correlation to apoptosis.
      ). To date liposome-based fluorescence amplification techniques have been used (
      • Scheffold A.
      • Assenmacher M.
      • Reiners-Schramm L.
      • Lauster R.
      • Radbruch A.
      High-sensitivity immunofluorescence for detection of the pro- and anti-inflammatory cytokines gamma interferon and interleukin-10 on the surface of cytokine-secreting cells.
      ), allowing the detection of as few as 50 receptor molecules per cell. By applying this method, Bartholome et al. confirmed the presence of the mGR on CCRF-CEM cells and demonstrated that the mGR is physiologically present in monocytes and B-cells from healthy donors, while circulating T-lymphocytes were consistently negative (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ).
      The proportion of mGR positive monocytes was proposed to be linked to the activity of the immune system. The frequency of CD14+/mGR+ cells was increased in patients with systemic lupus erythematosus (SLE) (
      • Spies C.M.
      • Schaumann D.H.
      • Berki T.
      • Mayer K.
      • Jakstadt M.
      • Huscher D.
      • Wunder C.
      • Burmester G.R.
      • Radbruch A.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway.
      ). It positively correlated with parameters of disease activity in patients with rheumatoid arthritis (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ) and was slightly induced after vaccination (
      • Spies C.M.
      • Bartholome B.
      • Berki T.
      • Burmester G.R.
      • Radbruch A.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) on monocytes are up-regulated after vaccination.
      ). In addition the number of mGR positive monocytes dramatically increased on lipopolysaccharides (LPS) stimulation (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ), whereas decreasing in a dose-dependent manner on GC treatment in SLE patients (
      • Spies C.M.
      • Schaumann D.H.
      • Berki T.
      • Mayer K.
      • Jakstadt M.
      • Huscher D.
      • Wunder C.
      • Burmester G.R.
      • Radbruch A.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway.
      ). On the other hand, the proportion of mGR positive B-cells was stable and not affected by any of these factors. The regulation of the mGR expression by lipopolysaccharides and GCs was dependent on a functional secretory pathway (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ) and required both transcription and translation (
      • Spies C.M.
      • Schaumann D.H.
      • Berki T.
      • Mayer K.
      • Jakstadt M.
      • Huscher D.
      • Wunder C.
      • Burmester G.R.
      • Radbruch A.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway.
      ).
      To date a clear mechanism by which the GR is targeted to the membrane has not been defined. However, the GR has been found in association with Cav-1 in membrane lipid rafts (
      • Jain S.
      • Li Y.
      • Kumar A.
      • Sehgal P.B.
      Transcriptional signaling from membrane raft-associated glucocorticoid receptor.
      ,
      • Matthews L.
      • Berry A.
      • Ohanian V.
      • Ohanian J.
      • Garside H.
      • Ray D.
      Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the antiproliferative program.
      ,
      • Chidlow Jr., J.H.
      • Sessa W.C.
      Caveolae, caveolins, and cavins: complex control of cellular signalling and inflammation.
      ) and Cav-1 was shown to facilitate the membrane translocation of the estrogen receptor (ER) (
      • Razandi M.
      • Oh P.
      • Pedram A.
      • Schnitzer J.
      • Levin E.R.
      ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions.
      ), suggesting a similar role for the mGR. Caveolae are important for signal transduction of many receptors (
      • Pike L.J.
      Lipid rafts: bringing order to chaos.
      ) and the localization of the GR in these lipid rafts suggests that the membrane-bound form exerts its function through a mechanism distinct from its cytosolic counterpart. For example, GC stimulation of a lung cell line led to the activation of protein kinase B (PKB) in a Caveolin-dependent mechanism (
      • Matthews L.
      • Berry A.
      • Ohanian V.
      • Ohanian J.
      • Garside H.
      • Ray D.
      Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the antiproliferative program.
      ). Similarly the presence of Cav-1 and G-protein was required for membrane estrogen receptor (
      • Watson C.S.
      • Jeng Y.J.
      • Hu G.
      • Wozniak A.
      • Bulayeva N.
      • Guptarak J.
      Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-alpha interactions with G protein-alphai and caveolin I.
      ) and membrane androgen receptor activation (
      • Liu J.
      • Youn H.
      • Yang J.
      • Du N.
      • Liu J.
      • Liu H.
      • Li B.
      G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells.
      ), suggesting that Cav-1 and probably the associated lipid rafts, may play a role in steroid membrane signaling.
      Previous studies have reported several signal transduction pathways activated on short GC-stimulation (
      • Haller J.
      • Mikics E.
      • Makara G.B.
      The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings.
      ,
      • Cato A.C.
      • Nestl A.
      • Mink S.
      Rapid actions of steroid receptors in cellular signaling pathways.
      ), but only a few of the rapid CG-effects have been shown to be selectively triggered by the mGR. 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 (
      • 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.
      • Buttgereit F.
      Origin and functional activity of the membrane-bound glucocorticoid receptor.
      ). 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. The dissection of mGR specific functions identified effects on the proteome that aligned with classical GC-activities. In CCRF-CEM cells, Cort-BSA activated RhoA signaling and no other signal transduction pathway was identified. We visualized the mGR and its association with Cav-1 using the highly sensitive in situ Proximity Ligation Assay (PLA) (
      • Fredriksson S.
      • Gullberg M.
      • Jarvius J.
      • Olsson C.
      • Pietras K.
      • Gústafsdottir S.M.
      • Ostman A.
      • Landegren U.
      Protein detection using proximity-dependent DNA ligation assays.
      ). This membrane protein seems to modulate Cort-BSA effects, but is not necessary for activity.

      DISCUSSION

      Glucocorticoids exert their therapeutic effects by activating the cytosolic GR leading to classical genomic effects. In addition there is a fast nongenomic pathway that is at least partially mediated by a membrane bound GR. Here, we report the first study on proteomic effects induced by selective activation of the mGR only. The lymphoblast-like T-cell line CCRF-CEM was chosen as it was the first human cell line shown to have a functional mGR (
      • Gametchu B.
      • Watson C.S.
      • Wu S.
      Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.
      ), responsible for the well-known pharmacological effect of GCs, GC-induced apoptosis (
      • Gametchu B.
      • Watson C.S.
      Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines.
      ,
      • Gametchu B.
      • Chen F.
      • Sackey F.
      • Powell C.
      • Watson C.S.
      Plasma membrane-resident glucocorticoid receptors in rodent lymphoma and human leukemia models.
      ,
      • Sackey F.N.
      • Watson C.S.
      • Gametchu B.
      Cell cycle regulation of membrane glucocorticoid receptor in CCRF-CEM human ALL cells: correlation to apoptosis.
      ). This cell line was also used to validate the liposome-based mGR detection technique by Bartholome et al. (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ) and it is the cell line in which the mGR has been mostly studied. For their lymphocytolytic activity, GCs are widely used in the treatment of acute lymphoblastic leukemia (
      • Gametchu B.
      • Watson C.S.
      Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines.
      ). For their anti-inflammatory and immuno-suppressive effects they are often used in the treatment of autoimmune and inflammatory diseases (
      • Löwenberg M.
      • Stahn C.
      • Hommes D.W.
      • Buttgereit F.
      Novel insights into mechanisms of glucocorticoid action and the development of new glucocorticoid receptor ligands.
      ). Proteomic analysis identified 128 proteins that were differentially regulated in CCRF-CEM cells on specific activation of the mGR using BSA-conjugated cortisol. These activities were confirmed to be unique to the mGR as there was no activation of the cGR target gene GILZ. In line with the above known pharmacological effects of GCs, we observed that the top networks activated on mGR activation were mainly involved in cell death of this lymphoma cell line via apoptosis. MAT II was one of the proteins identified in our proteomic data set and subsequently validated as a mGR target protein. This protein catalyzes the biosynthesis of S-adenosylmethionine, which is the principal methyl donor in the cell and is required for T-lymphocyte activation and proliferation (
      • Zeng Z.
      • Yang H.
      • Huang Z.Z.
      • Chen C.
      • Wang J.
      • Lu S.C.
      The role of c-Myb in the up-regulation of methionine adenosyltransferase 2A expression in activated Jurkat cells.
      ). In Jurkat cells, GCs were previously shown to inhibit MAT II expression thus blocking T-cell proliferation, inflammation, and initiation of an immune response (
      • Zeng Z.
      • Yang H.
      • Huang Z.Z.
      • Chen C.
      • Wang J.
      • Lu S.C.
      The role of c-Myb in the up-regulation of methionine adenosyltransferase 2A expression in activated Jurkat cells.
      ). Our observation of a rapid decrease of this protein in the cytosolic fraction on mGR activation of Jurkat cells suggests that these effects are already initiated by the early activation of the mGR.
      Also some other anti-inflammatory effects seem to be triggered by mGR activation. We showed that Cort-BSA reduced Prostaglandin E synthase 3 (p23) in CCRF-CEM cells. This protein is part of the multiprotein complex binding the inactive GR and is required for GR nuclear translocation and target gene trans-activation and trans-repression (
      • Lovgren A.K.
      • Kovarova M.
      • Koller B.H.
      cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis.
      ). This protein also catalyzes the production of prostaglanding E2 (PGE2) as an immediate response to pro-inflammatory stimuli and GCs were previously shown to exert their anti-inflammatory function, partially by inhibiting the up-regulation of other prostaglanding E2 synthases (
      • Hara S.
      • Kamei D.
      • Sasaki Y.
      • Tanemoto A.
      • Nakatani Y.
      • Murakami M.
      Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models.
      ). Our results suggest that the mGR plays an early role in this GC activity.
      Furthermore, we found several metabolic pathways to be associated with Cort-BSA stimulation, including the glycolysis/gluconeogenesis and amino-acyl tRNA biosynthesis pathways. It is well established that GCs are important for the maintenance of metabolic homeostasis. They mediate the switch from amino acid and protein anabolism to catabolism by down-regulating enzymes, such as aminoacyl-tRNA synthetases (
      • Revollo J.R.
      • Cidlowski J.A.
      Mechanisms generating diversity in glucocorticoid receptor signaling.
      ). CGs also stimulate hepatic gluconeogenesis; inhibit glucose uptake by peripheral tissues (
      • Munck A.
      • Guyre P.M.
      • Holbrook N.J.
      Physiological functions of glucocorticoids in stress and their relation to pharmacological actions.
      ); liberate energy substrates as glucose, amino acids, glycerol, and fatty acids; and increase lipolysis by up-regulating lipase in adipocytes (
      • Peckett A.J.
      • Wright D.C.
      • Riddell M.C.
      The effects of glucocorticoids on adipose tissue lipid metabolism.
      ). Our IPA results strongly suggest that these effects are at least partially the result of the rapid activation of mGR. In our data set, HADH was found to be inhibited rapidly after Cort-BSA stimulation in all cell lines investigated. This protein is a metabolic enzyme involved in the β-oxidation of fatty acids and their transformation into acetyl Co-A fueling the citric acid cycle. HADH was also shown to directly interact with the glutamate dehydrogenase (GDH), an enzyme oxidizing and deaminating glutamate to α-ketoglutarate. HADH inhibits glutamate dehydrogenase activity controlling insulin release in pancreatic cells and glycemia (
      • Li C.
      • Chen P.
      • Palladino A.
      • Narayan S.
      • Russell L.K.
      • Sayed S.
      • Xiong G.
      • Chen J.
      • Stokes D.
      • Butt Y.M.
      • Jones P.M.
      • Collins H.W.
      • Cohen N.A.
      • Cohen A.S.
      • Nissim I.
      • Smith T.J.
      • Strauss A.W.
      • Matschinsky F.M.
      • Bennett M.J.
      • Stanley C.A.
      Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.
      ).
      On mGR activation we also observed a rapid translocation of COX Vb from the cytosol to the nuclear fraction, which may also contain mitochondria. This Cort-BSA target protein is an important regulatory subunit of the cytochrome C oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain (
      • Bender E.
      • Kadenbach B.
      The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation.
      ). In CEM cells the recruitment of COX Vb to the mitochondria increases oxydative phosphorylation and mitochondrial respiration encouraging adenosine triphosphate (ATP) production (
      • Chen Z.X.
      • Pervaiz S.
      Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2.
      ). Thus the mGR seems to be involved in multiple ways in the effects of GC on the fatty acid, glucose, and citric acid pathways as well as the respiratory chain, to rapidly provide energy under stress. Although we have only started to dissect mGR specific activities and to discriminate them from the cytosolic GR, our data seem to suggest that the mGR supports a number of GC-effects by rapid “priming” of the cell for the subsequent genomic actions (
      • Chen H.C.
      • Farese R.V.
      Steroid hormones: Interactions with membrane-bound receptors.
      ).
      The mechanism by which mGR activation leads to these proteomic changes requires further investigation. CoxVb was previously reported as a protein kinase A target (
      • Bender E.
      • Kadenbach B.
      The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation.
      ) and prohibitin appears to play a role in several processes such as proliferation, apoptosis, maintenance of mitochondria function and morphology, gene transcription and signal transduction pathways, especially phosphatidylinositol 3-kinase/PKB and Ras/ Mitogen activated protein kinase /extracellular-signal-regulated kinase signaling (
      • Mishra S.
      • Ande S.R.
      • Nyomba B.L.
      The role of prohibitin in cell signaling.
      ,
      • Theiss A.L.
      • Sitaraman S.V.
      The role and therapeutic potential of prohibitin in disease.
      ). Modulation of prohibitin by Cort-BSA was not validated by Western blot in CCRF-CEM cells, but in Jurkat and MCF-7 cells mGR activation lead to a significant change in prohibitin levels. Therefore, we studied 84 downstream target genes of 18 established signaling cascades, such as mitogenic, survival, cAMP response element-binding protein, and PKC pathways. In these experiments, CCRF-CEM cells were stimulated with Cort-BSA for 6 h to allow transcriptional changes to occur, however, for all genes differential expression (measured by qPCR) was below a twofold change and not statistically significant. In conclusion, in CCRF-CEM cells no shifts characteristic for phosphorylation were observed in our 2D-DIGE experiments, none of the above mentioned pathways emerged from Ingenuity analysis and target genes of the most important signal transduction pathways were not transcriptionally up-regulated. We cannot totally exclude that effects on the proteome may be mediated by rapidly phosphorylated kinases or other regulatory proteins, that are functionally very important, but are not necessarily detected by 2D-DIGE because of their low abundance. Nevertheless if this was the case it did not translate into transcriptional activation of target genes of major signaling pathways. Recently p38 Mitogen activated protein kinase was shown to be phosphorylated in human monocytes on mGR activation by dexamethasone-BSA (
      • Berki T.
      • Kumánovics G.
      • Kumánovics A.
      • Falus A.
      • Ujhelyi E.
      • Németh P.
      Production and flow cytometric application of a monoclonal anti-glucocorticoid receptor antibody.
      ), suggesting cell- or GC-specific mGR effects.
      In CCRF-CEM T-lymphoma cells the mGR appears to exert its function at least partially through the modulation of RhoA activity. In our proteomic data set, this signaling mechanism emerged as a significantly enriched canonical pathway from the Ingenuity analysis. RhoA belongs to a family of guanosine triphosphatases, which are essential for adhesion, migration, morphological polarization, and activation of T-cells (
      • Rougerie P.
      • Delon J.
      Rho GTPases: masters of T lymphocyte migration and activation.
      ). Accordingly, in the cytoplasmic fraction we found that the top functions included adhesion of endothelial cell lines and susceptibility to infection of lymphoma cell lines, which is part of the category “Cell morphology.” These Rho guanosine triphosphatases are highly conserved and ubiquitously expressed and their modulatory mechanism differs considerably from other molecular regulators such as kinases. They exert their function only when associated to the membrane and on activation they induce a conformational change in their effector molecules requiring protein-protein interactions (
      • Rougerie P.
      • Delon J.
      Rho GTPases: masters of T lymphocyte migration and activation.
      ). Although major signaling pathways activating transcriptional events were not detectable, the short Cort-BSA stimulation used suggests that the effects observed are most probably caused by protein translocation, post-translational modifications, or protein-protein interactions.
      In all stimulations we used a physiologically relevant dose of cortisol reflecting the concentration observed in human sera after moderate psychosocial stress. Similar GC levels in the in vivo study by Datson et al. resulted in an equally mild gene response in brain cells (
      • Datson N.A.
      • Speksnijder N.
      • Mayer J.L.
      • Steenbergen P.J.
      • Korobko O.
      • Goeman J.
      • de Kloet E.R.
      • Joëls M.
      • Lucassen P.J.
      The transcriptional response to chronic stress and glucocorticoid receptor blockade in the hippocampal dentate gyrus.
      ). Higher concentrations of Cort-BSA may have elicited stronger GC effects, but their physiological relevance could be questionable. Interestingly, our proteomic experiments are in direct agreement with another recent 2D-DIGE study from our laboratory (
      • Billing A.M.
      • Revets D.
      • Hoffmann C.
      • Turner J.D.
      • Vernocchi S.
      • Muller C.P.
      Proteomic profiling of rapid non-genomic and concomitant genomic effects of acute restraint stress on rat thymocytes.
      ). After acute stress in vivo similar fold changes were observed in an overlapping set of proteins in rat thymocytes. For example, after 15 min of restraint stress rat thymocytes showed a −2.23-fold change of SET protein in the cytosolic fraction whereas in the present study we observed a −2.6-fold change for SET. Similarly other proteins were found commonly regulated in the two data sets: Peroxiredoxin II (PRDX2; −1.74 in our study and −1.45 in stressed rats), the Heterogeneous nuclear ribonucleoprotein C (HNRPC; +1.16 in our study and −2.11 in stressed rats) and the ATP synthase subunit beta (ATPB; −1.12 in our study and −1.45 in stressed rats). This comparison between the two studies further underlines the physiological significance of our findings.
      Furthermore, we confirmed the existence of the mGR at very low levels on the cell surface, by using the very sensitive in situ PLA method, which in principle generates a signal for each target molecule in a cell (
      • Fredriksson S.
      • Gullberg M.
      • Jarvius J.
      • Olsson C.
      • Pietras K.
      • Gústafsdottir S.M.
      • Ostman A.
      • Landegren U.
      Protein detection using proximity-dependent DNA ligation assays.
      ). Considering the high sensitivity of this PLA technique our 5% of CCRF-CEM cells having more than 50 molecules of mGR agree well with the 11% positivity found by flow cytometry using a liposome-based immunofluorescence amplification technique (
      • Bartholome B.
      • Spies C.M.
      • Gaber T.
      • Schuchmann S.
      • Berki T.
      • Kunkel D.
      • Bienert M.
      • Radbruch A.
      • Burmester G.R.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis.
      ). Three additional cell lines, including Jurkat cells, which were previously reported to be essentially mGR negative (
      • Spies C.M.
      • Schaumann D.H.
      • Berki T.
      • Mayer K.
      • Jakstadt M.
      • Huscher D.
      • Wunder C.
      • Burmester G.R.
      • Radbruch A.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway.
      ), were positive for mGR expression by in situ PLA. MCF-7 cells had a considerably higher numbers of detectable receptors, whereas Jurkat and CCRF-CEM had the lowest. Even the low numbers of signals in these cell lines were consistently and significantly reduced to background levels when the antibodies were blocked with their cognate peptides, confirming the specificity of the mGR detection. Interestingly three antibodies directed against different epitopes within the first 400 residues of the classical GR gave the same results in three of the four cell lines tested. 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 (
      • 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.
      • Buttgereit F.
      Origin and functional activity of the membrane-bound glucocorticoid receptor.
      ). 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. This is also compatible with our finding that the GR antagonist RU486 binds to the mGR and blocks its activity. We speculate that the residues between aa 420 and 527 may be involved in the association with the membrane or other membrane anchoring proteins. Specific splicing variants may be responsible for the membrane localization on this receptor. In any case no signal peptide or transmembrane domain has so far been identified in the classical GR protein sequence.
      We extended the validation of the five Cort-BSA modulated proteins to the other mGR-positive cell lines. Although prostaglandin E synthase 3 was a Cort-BSA target in CCRF-CEM, this protein was not expressed in Jurkat, U2-OS, and MCF-7 cells. CoxVb, HADH, prohibitin, and MAT II were all modulated by Cort-BSA in at least one other cell line in the same manner as in CCRF-CEM cells. HADH was found to be down-regulated in all cell types, whereas the other validated Cort-BSA modulated proteins showed more heterogeneity. For example, MCF-7 showed an up-regulation of CoxVb in the nuclear fraction comparable to CCRF-CEM cells, whereas in U2-OS mGR activation down-regulated CoxVb with a different kinetics. Overall, these results indicate that these proteins are genuine mGR specific targets, albeit with some differences in kinetics.
      The mechanism by which the GR is targeted to the membrane remains elusive. Our observation that Cav1-mGR dimers were detectable with the M20 GR antibody, and not with the 5E4 mAb covering the AF1 domain could be explained by a previous suggestion that the GR interacts with Cav-1 through this transactivation domain (
      • Matthews L.
      • Berry A.
      • Ohanian V.
      • Ohanian J.
      • Garside H.
      • Ray D.
      Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the antiproliferative program.
      ). Cav-1 is a structural protein of caveolae that previously has been shown to facilitate the translocation of the estrogen receptor to the cell membrane (
      • Razandi M.
      • Oh P.
      • Pedram A.
      • Schnitzer J.
      • Levin E.R.
      ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions.
      ). For the mGR, it would appear that Cav-1 is not necessary for membrane localization, as previously suggested (
      • Spies C.M.
      • Schaumann D.H.
      • Berki T.
      • Mayer K.
      • Jakstadt M.
      • Huscher D.
      • Wunder C.
      • Burmester G.R.
      • Radbruch A.
      • Lauster R.
      • Scheffold A.
      • Buttgereit F.
      Membrane glucocorticoid receptors are down regulated by glucocorticoids in patients with systemic lupus erythematosus and use a caveolin-1-independent expression pathway.
      ), because both CCRF-CEM and Jurkat cells do not express Cav-1. However, irrespective of the Cav-1 expression, the changes in the Cort-BSA modulated proteins were visible in all the cell lines investigated. To assess the role played by Cav-1 in rapid GCs effects we silenced Cav-1 in U2-OS cells, which had the highest number of mGR/Cav-1 dimers. After a 70% reduction in Cav-1 protein levels, HADH was no longer down-regulated by Cort-BSA and CoxVb showed an opposite regulation, with a marked and significant up-regulation in the nuclear fraction already after 5 min exposure. In absence of Cav-1, Cort-BSA induces an up-regulation of CoxVb as observed in CCRF-CEM cells and knocked-down U2-OS cells, whereas it is down-regulated when Cav-1 is present and associated with the mGR. However, in all cell lines tested Cort-BSA induced a down-regulation of HADH independent of Cav-1. Membrane-GR stimulation after Cav-1 silencing lead to a mild up-regulation of HADH, which was not observed in CCRF-CEM cells that do not express Cav-1. Thus, Cav-1 seems to act as a modulator of mGR activity and the outcome of mGR activation may ultimately depend on the relative numbers of mGR associated with Cav-1 or other members of the caveolin family, such as Cav-2 α, also expressed in leukemia T cells (
      • Tsuji Y.
      • Hatanaka M.
      • Maeda T.
      • Seya T.
      • Takenaka H.
      • Shimizu A.
      Differential-expression and tyrosine-phosphorylation profiles of caveolin isoforms in human T cell leukemia cell lines.
      ,
      • Tsuji Y.
      • Nakagawa T.
      • Hatanaka M.
      • Takeuchi T.
      • Matsumoto E.
      • Takenaka H.
      • Shimizu A.
      Quantification of caveolin isoforms using quantitative real-time RT-PCR, and analysis of promoter CpG methylation of caveolin-1alpha in human T cell leukemia cell lines.
      ).
      We conclude that similar to the cytosolic GR, at least the first 400 amino acids of the mGR are the product of the NR3C1 gene. The expression of this receptor varies considerably among cell lines and our results suggest that even cells considered so far as mGR negative express this receptor. The activation of the mGR induces proteomic changes, which were largely validated in four cell lines despite some differences in kinetics and regulation. The IPA provides strong evidences that the mGR is involved in pro-apoptotic, immune-modulatory, and metabolic pathways, which are also modulated by GC and the cytosolic GR, suggesting that mGR activation triggers rapid early priming events that pave the way for the slower genomic GC activities. In CCRF-CEM cells, mGR activation was strongly related to cell death and seems to be largely responsible for the GC induced therapeutic lymphocytolysis. No clear genomic effect that would suggest transcriptional activation of the important kinases and their signaling pathways was identified on Cort-BSA stimulation and RhoA emerged as the most prominent signaling pathway. Finally, Cav-1 was not required for membrane localization of the GR and it was not necessary for activity, however if it dimerizes with the mGR it modulates Cort-BSA induced proteomic effects.

      Acknowledgments

      We thank Professor Timea Berki from the University of Pécs, Hungary for supplying the anti-GR antibody (clone 5E4) used in the study. We are grateful to Hartmut Schächinger for his initiatives within the Trier-Leiden International Research Training Group (IRTG GRK 1389/1) and the Graduate School of Psychobiology.

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