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and the glucocorticoid receptor in vivo and in nontransformed human cells
1
* Departement Forschung, University of Basel, 4031 Basel, Switzerland;
Department of Pharmacology and Institute of Respiratory Medicine, University Sydney, Camperdown NSW-2050, Australia;
Department of Respiratory Commercial Strategy, GlaxoSmithKline, Research & Development, Stockley Park, Middlesex UB11 1BU, UK; and
Department of Internal Medicine IV, University Hospital Vienna, 1090 Vienna, Austria
1Correspondence: Department of Internal Medicine IV, AKH, University Hospital Vienna, Währinger Gürtel 18–22, 1090 Vienna, Austria. E-mail: lutz-henning.block{at}akh-wien.ac.at
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In in vitro experiments, this interaction turned out to be responsible for the inhibitory action of glucocorticoids on lymphocytic and mesenchymal cell proliferation. Our results suggest that complex formation of the GR with C/EBP accounts for a novel pathway of glucocorticoid action.—Rüdiger, J. J., Roth, M., Bihl, M. P., Cornelius, B. C., Johnson, M., Ziesche, R., Block, L.-H. Interaction of C/EBP and the glucocorticoid receptor in vivo and in nontransformed human cells.
Key Words: glucocorticoids • CCAAT/enhancer binding protein alpha • cell proliferation • protein complex
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, cell proliferation/differentiation (2)
, and cell metabolism (3)
. Glucocorticoids act through binding to its cytosolic glucocorticoid receptor (GR), serving as transcription factor. Once activated by a glucocorticoid, GR homodimerize and translocate into the nucleus, where they subsequently bind to specific glucocorticoid responsive elements on gene promoters (4)
. However, this mechanism does not explain all actions of glucocorticoids. Several studies demonstrated a direct protein–protein interaction of the GR with other transcription factors such as AP-1 (5
, 6)
, NF-
B (7
, 7
8
9)
, Stat3 (10)
, Stat5 (11)
, and Stat6 (12)
, thereby inhibiting their transcriptional activity.
Regulation of the antiproliferative effects of glucocorticoids has been hypothesized to depend on the coexistence of the GR and the CCAAT/enhancer binding protein alpha (C/EBP) (13
, 14)
. Both studies suggested that the antiproliferative action of glucocorticoids was mediated through the GR leading to de novo synthesis and activation of C/EBP (13
, 14)
. Subsequently, C/EBP causes cell cycle arrest through activation of p21(waf1/cip1). However, de novo synthesis of C/EBP alone was insufficient to achieve p21(waf1/cip1) activation and inhibit cell proliferation (13
, 14)
. Therefore, the authors postulated an additional direct mechanism for C/EBP activation by the GR, but no proof was provided (14)
. Analyzing glucocorticoid resistance, Ramos et al. identified a rat hepatoma cell line bearing a GR mutation at the DNA binding domain as well as a deficiency for C/EBP activation by glucocorticoids (15)
. Substitutive transfection with the wild-type gene of either of these factors alone was unable to restore the antiproliferative effects of glucocorticoids. The authors postulated that the DNA binding abilities of the GR are essential for the activation C/EBP and subsequent p21(waf1/cip1) induction (15)
. However, the molecular mechanism of the GR-C/EBP transmitted gene induction of p21(waf1/cip1) is not conclusive. Previously, we and others described a glucocorticoid-dependent gene activation p21(Waf1/Cip1) occurring within 1–3 h after addition of the drug in fibroblasts (16
17
18)
. Similarly, Beurton et al. reported a glucocorticoid-induced activation of C/EBP DNA binding in rat hepatoma cells within 2 h (19)
. Such a fast activation of C/EBP and/or p21(waf1/cip1) by glucocorticoids is unlikely to involve previous gene transcription, translation, and activation of C/EBP.
Here we describe that glucocorticoid-induced activation of C/EBP, which is due to the formation of a GR–C/EBP complex, is activating the C/EBP-dependent p21(waf1/cip1) gene. In addition to the classical binding of the GR to glucocorticoid responsive elements (GRE), glucocorticoid-mediated GR–C/EBP complex formation may serve as an additional important pathway of glucocorticoid action in mesenchymal cells and lymphocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cells were cultured in RPMI 1640, 5% FCS, 20 mM HEPES or DMEM, 10% FCS, MEM vitamins, and 20 mM HEPES. All cell culture media and additives were purchased from Facola/Seromed (Basel, Switzerland). Treatment and experiments were performed between passages 2 and 5. Before the preparation of nuclear and cytosolic extracts, cells were subcultured in Petri dishes and kept for 24 to 48 h in serum-rich medium until they reached 60–80% confluency. Prior to stimulation, cells were serum deprived for 24 h with 0.1% FCS. Low serum medium was exchanged every 12 h. To exclude de novo synthesis as responsible for GR-C/EBP interaction, cells were pretreated with cycloheximide (1 µM) for 3 h before incubation with dexamethasone.
Peripheral blood leukocytes (PBLs) were isolated from healthy volunteers (n=3) using Ficoll gradient centrifugation (Histopaque 1077®, Sigma, St Louis, MO). For cell proliferation assays, PBLs were incubated in cell culture dishes overnight to remove macrophages (21)
.
Nuclear and cytosolic extracts
Nuclear and cytosolic extracts were prepared from 60% confluent cells or PBLs were resuspended in 80 µl of low-salt buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10% glycerol, complete protease inhibitor (Roche Diagnostics, Mannheim, Germany) and incubated for 10 min on ice. After centrifugation for 1 min at 13,000 g at room temperature, supernatants were taken as a cytosolic fraction. The remaining pellet was dissolved in 50 µl high-salt buffer (420 mM NaCl, 20 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, complete protease inhibitor); samples were kept on ice for 30 min, followed by a centrifugation step at 13,000 g for 10 min at room temperature. Supernatants were taken as nuclear fractions (18)
. Protein concentration was measured according to Bradford’s method (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assay (EMSA)
Binding of C/EBP–GR complex to C/EBP binding sites was characterized in EMSA by determining shifting of either promoter fragments of the p21(Waf1/Cip1) gene or a C/EBP consensus sequence. Both p21(Waf1/Cip1) promoter fragments contained a C/EBP binding site. The 144 base pair (bp) fragment, ranging from 3266 to 3409 bp according to the sequence #U24170 (p21–144), was labeled by random priming (Highprime, Roche Diagnostics). The 22 bp p21(Waf1/Cip1) promoter fragment (p21–22; ATC CAT CCT CTG CAA TTT TTT A), its mutated fragment (p21mut; CCA TCC TCT GCG GCC GCT TAA AAG CAA AAC TGC), and the C/EBP consensus sequence (TGC AGA TTG CGC AAT CTG CA; Santa Cruz Biotechnology, CA) were end-labeled using T4 kinase (Roche Diagnostics). For the binding assay, 60 fmol of the labeled DNA probes were incubated in a final volume of 10 µl for 30 min at room temperature with 1 µg of cytosolic or nuclear protein extracts under binding conditions (10 mM Tris, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM MgCl2, 8% glycerol, 1 µg poly-[dI-dC]). After incubation, formation of DNA/protein complexes was determined on a nondenaturing 4% polyacrylamide gel electrophoresis, which was dried and exposed to X-ray film. Specificity of the observed DNA/protein complexes was demonstrated by incubating extracts of dexamethasone-treated cells with either competing unlabeled C/EBP consensus sequence or in the presence of polyclonal antibodies against C/EBP, GR, and Oct1 (all supplied by Santa Cruz Biotechnology, Santa Cruz, CA), respectively.
Western blot analysis
To determine expression of C/EBP protein, 5 µg total protein was size-fractionated in a 4–15% SDS-PAGE. After electrophoresis, proteins were transferred on PVDF membrane (Millipore Corp., Bedford, MA) by semidry transfer. Protein transfer was controlled by Ponceau’s staining. Membranes were blocked for 1 h (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% skimmed milk). Incubation with antibodies specific to either C/EBP or GR was performed at 4°C overnight, followed by incubation with a second horseradish peroxidase-linked antibody for 1 h. Protein abundance was detected by an enhanced chemiluminescence (ECL) method (Pierce, Rockford, IL).
Polymerase chain reaction
Expression of C/EBP was determined by reverse transcription-polymerase chain reaction (RT-PCR). RNeasy® Mini kit was used for total RNA extraction according to the manufacturer’s instructions (Qiagen, Basel, Switzerland). First-strand cDNA synthesis was performed from 1 µg of total RNA in a volume of 25 µl using 200 units of M-MLV Reverse Transcriptase RNase H Minus, Point Mutant (Promega, Madison, WI). 600 ng of cDNA was amplified using ß-actin primers: 5' GTA CGT TGC TAT CCA GGC TGT GC; 3' TCA GGC AGC TCG TAG CTC TTC TC, generating a 336 bp fragment; C/EBP primers: 5' AAA GGG GTG GAA ACA TAG GG; 3' CCA CGA CCT AGC TTT CTG GT revealing a 227 bp fragment. All primer pairs were synthesized by MWG-Biotech GmbH (Ebersberg, Germany). PCR conditions were denaturation of double-stranded DNA: 98°C for 15 s; primer annealing step: 58°C for 22 s; and elongation step: 72°C for 40 s. Amplification profile involved different cycle numbers: ß-actin bands were analyzed after 37 cycles, C/EBP after 49 cycles. PCR reaction was size fractionated by electrophoresis on 2% agarose gel (NuSieve GTG, FMC BioProducts, Rockland, ME) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and visualized by ethidium bromide stain under UV light. The housekeeping gene ß-actin served as control of equal conditions for amplification.
Luciferase reporter gene assay
p21(Waf1/Cip1) promoter/luciferase construct (WWP-Luc) was a kind gift from B. Vogelstein (Johns Hopkins University, Baltimore, MD) and contained a 2.4 kb promoter fragment of the p21(Waf1/Cip1) gene (22
, 23)
. Luciferase assay was performed in 60% confluent cells, which were transfected for 1 h in the presence of serum-free DMEM medium containing 1.25 ng/µl of the WWP-Luc construct and Tfx50 (Promega) in a Tfx50/DNA ratio of 3:1. Growth medium was added and cells were incubated for 24 to 36 h (18)
. After an additional 6 h of dexamethasone treatment, cells were lysed and luciferase activity was determined for 10 s in a LUMAC Biocounter M1500P (Landgraaf, Netherlands).
Cell proliferation
FCS-induced mesenchymal cell proliferation was determined by counting the incorporation of [3H]-thymidine, as originally described by Chesterman et al. (24)
. [3H]-Thymidine incorporation was determined 36 h after fibroblast stimulation with FCS with or without dexamethasone; 60% confluent cells were serum deprived for 24 h, followed by serum stimulation for 24 h in the presence or absence of dexamethasone. To specify whether the antiproliferative action of dexamethasone depends at least in part on C/EBP expression, cells were pretreated for 24 h with single-stranded DNA antisense oligonucleotides or random single-stranded DNA oligonucleotides reapplied every 12 h with 0.5 µM. For [3H]-thymidine incorporation, 1 µCi [3H]-thymidine (Amersham, Little Chalfont, UK) per milliliter was added to culture medium for 24 h. DNA was extracted after cell fixation by methanol (4°C, 10 min), washing with 0.1 N trichloric acetic acid, and lysis in 0.3 N NaOH. Incorporated [3H]-thymidine was determined by liquid scintillation counting.
For PBLs, we cultured freshly prepared cells in a 96-well plate with 50,000 cells per well in RPMI 1640, 5% autologous serum, 5 µg/ml concanavalin A, and 20 mM HEPES in the presence of either random single-stranded DNA oligonucleotide or C/EBP antisense (2 µM) for 36 h. PBLs were treated with dexamethasone for 24 h. Partial adhesion of the cells due to 36 h of treatment made the thymidine incorporation method inappropriate for detection of cell proliferation for technical reasons; we therefore instead used a colorimetric assay for cell proliferation activity (celltiter96-AQ®, Promega). According to the instructions of the manufacturer, we mixed the PMS solution with MTS solution (1:20) immediately before adding 20 µl of that mixture to each well. After incubation for 2 h, absorbance at 490 nm was measured using an ELISA plate reader (Molecular Device Corp., Sunnyvale, CA).
Antisense treatment of human primary VSMC and fibroblasts
In proliferation assays, a 23 nucleotide (nt) C/EBP phosphorothioated, DNA single-strand (5'-GAA GGC GGC GCT GCT GGG CGC GT-3', 220–242 nt according the C/EBP sequence with the accession number Y11525; ref 25
) served as antisense specific against C/EBP mRNA. Binding characteristics and specificity of this oligonucleotide were preevaluated by RNA folding prediction, Blast2-search, and by multiple sequence alignment among C/EBP isoforms (26)
. Specificity of C/EBP antisense actions was verified by the use of a random phosphorothioated oligonucleotide sequence (AGC TCG GAT GCA TGG AGG AG-3'). For determination of the time-dependent distribution of C/EBP antisense, the molecule was labeled with fluorescein at the 3', followed by light microscopy using excitation at 488 nm. Antisense/sense were added 24 h before stimulation of cells. Cell culture medium containing half of the initial oligonucleotide concentration was exchanged every 12 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nuclear extracts from primary VSMC and fibroblasts were incubated with dexamethasone (10-7 M) over a period of 3 h. The extracts were incubated with a 32P-labeled 144 bp fragment of the p21(Waf1/Cip1) promoter (p21–144) responsible for GR-induced p21(Waf1/Cip1) promoter activation (13
, 14)
. This fragment contains a known C/EBP binding site but lacks a GRE (13
, 14)
. In untreated cells, we detected a faint signal for DNA binding proteins, whereas treatment with dexamethasone resulted in a specific protein binding to the p21–144 oligonucleotide as early as 1 h (Fig. 1
a). The size of the DNA–protein complex increased during the period investigated.
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To further characterize the DNA sequence responsible for the dexamethasone-induced mobility shift, we performed stepwise deletion analysis of the p21–144 promoter fragment. The dexamethasone-induced DNA mobility shift was detectable, even though the p21(Waf1/Cip1) promoter region was reduced to 22 nt (p21–22) that earlier had been described to maintain the binding of C/EBP to the p21(Waf1/Cip1) promoter (13
, 14)
. As depicted in Fig. 1b
, this p21–22 fragment was sufficient for the dexamethasone-inducible DNA mobility shift with a kinetic similar to that obtained with the full-length promoter (Fig. 1a
). To prove the specificity of the DNA/protein interaction, we used a mutated C/EBP binding site (GCAATTTTT to GCGGCCGC; p21mut) with the same nuclear protein extracts. Using p21mut did not show an induction of DNA mobility shift in the presence of dexamethasone (Fig. 1c
). In contrast to the p21–144 oligonucleotide, the size of GC-induced complex binding to the p21–22 oligonucleotide did not increase, suggesting that additional transcription factors may interact with the 144 bp promoter region. However, these transcription factors are not necessary for the dexamethasone-induced protein–DNA binding. To confirm that dexamethasone in general induces C/EBP binding to its cognate DNA sequences, we performed EMSA using a commercially available C/EBP consensus sequence. As shown in Fig. 2
a, a mobility shift similar to that observed with both p21(Waf1/Cip1) promoter oligonucleotides was seen. Pretreatment with cycloheximide showed similar results.
|
The formation of the GR–C/EBP complex in vivo was demonstrated in three healthy probands who inhaled a single therapeutical dose of glucocorticoids (800 µg budesonide, n=3; 500 µg fluticasone, n=1). Probands’ peripheral blood leukocytes were isolated at 0, 1, and 3 h after inhalation and activation of the GR–C/EBP complex was measured in the nuclei using EMSA as described above. As shown in Fig. 2b
, inhaled glucocorticoids caused rapid binding of the GR to its cognate DNA binding site. This binding was paralleled by an increase in C/EBP binding to its corresponding DNA sequence (Fig. 2c
).
Dexamethasone-induced C/EBP–GR complex formation
Transcription factors responsible for binding the C/EBP consensus sequence were further investigated by supershifts. Fibroblasts treated with dexamethasone (10-7 M, 3 h) were preincubated with antibodies (Ab) specific against either the GR or the C/EBP protein (Fig. 2d, GR
Ab and C/EBP Ab, respectively). Both antibodies led to a decrease in signal and caused the appearance of a supershift (Fig. 2d
). These findings suggest GC-induced complex formation of the dexamethasone-activated GR with C/EBP.
De-novo synthesis of C/EBP is not essential for GC-dependent p21(Waf1/Cip1) activation
As hypothesized earlier, C/EBP de novo synthesis essentially precedes GC-dependent p21(Waf1/Cip1) gene activation in mouse hepatoma cell lines (13
14
15)
. To determine whether this mechanism is also effective in human nontransformed cells, we performed Western blot experiments with cells pretreated with cycloheximide. Treatment with dexamethasone resulted in an increased C/EBP protein content in the nuclear fraction as early as 1 h (Fig. 3
). Consequently, the corresponding cytosolic fractions showed a time-dependent decrease in C/EBP abundance. There was no indication for any change in total C/EBP protein content at any time point investigated (Fig. 3)
.
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p21(Waf1/Cip1) promoter activity depends on C/EBP activation
To ascertain whether the complex formation with subsequent nuclear shift is capable of promoter activation, we used a p21(Waf1/Cip1) promoter-driven luciferase reporter gene assay. In transiently transfected primary human VSMC and fibroblasts, dexamethasone induced luciferase activity within 6 h in a dose-dependent manner, reaching a plateau at 10-8 M (Fig. 4
). Inasmuch as GC-mediated activation of this GRE-less promoter depends on C/EBP coexpression (13
14
15)
, our data support the hypothesis of a C/EBP–GR complex formation in human primary VSMC and fibroblasts.
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Inhibition of cell proliferation by the C/EBP–GR complex
Functional response to dexamethasone treatment was examined by measuring the proliferation of human primary fibroblasts. Addition of C/EBP antisense was used to evaluate the involvement of C/EBP in the antiproliferative action of glucocorticoids. To determine the intracellular localization of the antisense after addition into the media, the antisense molecule was labeled with fluorescein and its trafficking was monitored through light and fluorescence microscopy (Fig. 5
a–f). After addition to the media at 2 µM, the single-stranded DNA was enriched at the cell membrane (1 h; Fig. 5c, d
); 6 h after treatment, the antisense was distributed homogeneously within the cytosol (Fig. 5e, f
).
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In the proliferation assay, dexamethasone counteracted FCS-induced cell proliferation in a dose-dependent manner, starting at 10-10 M (Fig. 6
a). At a concentration of 10-7 M, dexamethasone almost abolished the effects evoked by FCS stimulation. DNA antisense against C/EBP neutralized the glucocorticoid-induced inhibition of cell proliferation unless dexamethasone concentrations higher than 10-9 M were applied. Even at a concentration of 10-7 M, dose-response curves of dexamethasone treatment with and without C/EBP antisense still did not meet (Fig. 6a
). The crucial role for C/EBP alone in the control of cell proliferation was supported by the observation that C/EBP antisense treatment markedly increased thymidine incorporation (data not shown). Specificity of DNA antisense effects was verified by adding a random single-stranded DNA oligonucleotide, which neither increased cell proliferation nor changed the potency of the glucocorticoid on cell proliferation (data not shown).
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Similar to our tests in fibroblasts and VSMC, we sought to determine the functionality of the glucocorticoid-induced GR-C/EBP interaction in PBLs using a colorimetric proliferation assay (MTS). To approximate the in vivo situation, we isolated PBLs before and after inhalation of budesonide (800 µg). The proliferation assay was performed using autologous serum instead of FCS. To enhance cell proliferation, PBLs were stimulated with concanavalin A and cell proliferation was determined after 24 h. The colorimetric proliferation test revealed similar effects of the inhaled glucocorticoid in PBLs as observed in fibroblasts or VSMCs. If C/EBP amounts were depleted by 12 h preincubation with C/EBP antisense, glucocorticoids even at 10-7 M did not change MTS reduction to formazan of cultured PBLs (Fig. 6b
).
Function of C/EBP antisense itself was confirmed by Western blot experiments (mesenchymal cells) or PCR analysis (PBLs) presenting a 30–50% decrease of C/EBP protein or mRNA preincubated with C/EBP antisense (Fig. 6c, d
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that mediates glucocorticoid actions through stimulation of promoters containing C/EBP recognition sites.
Using specific antibodies for supershift assay, we demonstrated that the dexamethasone-dependent mobility shift with the C/EBP consensus fragment depends on a complex formed by the GR and C/EBP (Fig. 2d
). The Western blot experiments further ruled out that de novo synthesis was responsible for the rapid cellular response on glucocorticoid treatment (13
14
15)
. Moreover, they are supported by additional observations in cultured hepatoma cell lines, where glucocorticoids rapidly activate C/EBP DNA binding (19)
. This cellular response to dexamethasone is functional, as shown by the rapid translation of p21(Waf1/Cip1) promoter-driven genes (14
, 18)
.
It remains unclear, though, which protein region of the GR is responsible for binding and activating the C/EBP protein. Based on two earlier reports on the function of the second GR zinc finger, we assume this region is critical for C/EBP–GR complex formation (15
, 27)
. Ramos and colleagues found a mutation of the GR at the second zinc finger region, which is essential for DNA binding causing resistance to GC-suppressed cell proliferation via C/EBP (15)
. A mouse model for a functional mutation in the second zinc finger exhibited some symptoms similar to those of C/EBP knockout mice (27
, 28)
. This suggests a close link between the GR and C/EBP. We hypothesize that the presence of C/EBP at the time of GR activation affects the net result of GC activity. Whereas activation of the GR in the absence of C/EBP should lead mainly to modulation of GRE-controlled genes alone, in the presence of C/EBP, GC effects include antiproliferative action and C/EBP-dependent gene regulation.
Since it is known that C/EBP dimerizes with other members of the C/EBP family, the GR may also heterodimerize with other members of this transcription factor family such as C/EBPß, C/EBP
, or C/EBP
. These transcription factors also serve as regulators of the immune response, as indicated by their synonyms NF-IL-6, NF-IL6ß, and Ig/EBP, respectively (29
30
31)
.
Our work proves that cross-talk between the activated GR and C/EBP is not only important for antiproliferative actions of glucocorticoids in rat hepatoma cell lines, but also in mesenchymal cells and peripheral white blood cells. Such interactions may be significant for understanding the action of glucocorticoids in the treatment of a variety pathophysiological circumstances such as hyperproliferation of fibroblasts in lung fibrosis (32)
, bronchial smooth muscle cell proliferation in asthma (33
34
35)
, or subsets of T-lymphocyte proliferation, e.g., in sarcoidosis (36
, 37)
and asthma (1
, 38
39
40)
. Moreover, we have shown in PBLs that GCs indeed activate C/EBP in vivo, underscoring the relevance of the novel pathway suggested by us.
We conclude that the direct interaction of C/EBP with the GR by complex formation may provoke important physiological actions of GCs.
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ACKNOWLEDGMENTS |
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Received for publication April 3, 2001.
Revision received October 2, 2001.
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B activity through induction of I-B synthesis. Science 270,286-290B and the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 91,752-756 transcription factor in the glucocorticoid stimulation of p21(waf1/cip1) gene promoter activity in growth-arrested rat hepatoma cells. J. Biol. Chem. 273,2008-2014). Biochem. Biophys. Res. Commun. 215,106-113[CrossRef][Medline]
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