Correspondence to: Alberto Muñoz, Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain. Tel:+34 91-5854640 Fax:+34 91-5854587 E-mail:amunoz{at}iib.uam.es.
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Abstract |
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The immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFB) transcription factors. Inhibition of the c-Jun NH2-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor
(TNF-
)induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH2-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-
induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-
induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution.
Key Words:
dexamethasone, activating protein-1, tumor necrosis factor , c-Jun NH2-terminal kinase, nuclear translocation
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Introduction |
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Glucocorticoid hormones have many important regulatory roles in the organism. Their activities are exerted through the control of genes via three mechanisms: direct transcriptional regulation, indirect transcriptional regulation through interference with other transcription factors, and posttranscriptional effects (B) transcription factors. There is evidence that the immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of AP-1 and NF
B (
AP-1 is a group of dimeric factors constituted by members of the c-Jun and c-Fos families of protooncogenic products ( (TNF-
) and interleukin-1, and ultraviolet radiation. c-Jun, the main component of AP-1, is activated by NH2-terminal phosphorylation on serines 63 and 73 (Ser63/73) by members of the c-Jun NH2-terminal Kinase (JNK) family (
To gain insight into the mechanism of AP-1 interference by hormone-activated nuclear receptors, we have addressed the following questions. Does the synthetic glucocorticoid dexamethasone (Dex) inhibit JNK phosphorylation/activation in intact cells? Can Dex affect nuclear translocation of JNK? And, what is the mechanism of hormone-activated GR and what is its primary target?
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Materials and Methods |
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Cell Culture and Transfections
HeLa and Cos7 cells were grown in DME supplemented with 10% FCS. Media, tissue culture reagents, and FCS were purchased from GIBCO BRL. Cells were serum starved by changing the culture medium to DME supplemented with 0.5% FCS 16 h before treatment. Cos-7 cells were transfected with 3 µg of plasmid encoding HA-JNK (pCDNA3-JNK1) and 0.4 µg of those encoding either GRwt (pSB-hGR) or GRdim (pSB-hGR(A458T)), or with the empty vector (pRSh-R-; donated by Dr. A.C.B. Cato, Karlsruhe, Germany). Treatments: HeLa or Cos-7 cells (2448 h after transfection) were pretreated with Dex (1 µM) or vehicle (ethanol) for 45 min. This period was chosen because of previous studies showing that it is sufficient for maximum inhibition of JNK activation by TNF- (
(10 ng/ml) or its vehicle (ethanol) was added to the cells. Thus, TNF-
+ Dex cells were incubated with Dex for 45 min plus the indicated period of TNF-
stimulation. Dex-alone control cells were incubated with hormone during the pretreatment and throughout for the period of stimulation.
Immunocytochemistry
HeLa and Cos7 cells were rinsed twice in PBS, fixed with 3.7% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 15 min, and were then treated with 0.1 M glycine in PBS for 15 min. The nonspecific sites were blocked by incubation with PBS containing 1% BSA or goat serum for 30 min at room temperature. Cells were then washed in PBS containing 0.05% Tween-20 for 5 min and incubated with the primary antibodies diluted in PBS for 1 h at room temperature or overnight at 4°C.
The following primary antibodies were used: rabbit polyclonal or mouse monoclonal antic-Jun phosphorylated on serine-63 (New England Biolabs, Inc., 9261S; or Santa Cruz Biotechnology, Inc., sc-822), rabbit polyclonal antic-Jun (Oncogene Research, PC06), mouse monoclonal against human JNK1 (BD PharMingen, 15701A), mouse monoclonal anti-JNK1 phosphorylated on threonine-183 and tyrosine-185 (Santa Cruz Biotechnology, Inc., sc-6254), and rabbit polyclonal anti-GR (Santa Cruz Biotechnology, Inc., sc-1003). The cells were incubated for 45 min with one of the following secondary antibodies: Texas red (TxR)-conjugated goat antirabbit (Jackson ImmunoResearch or Vector), FITC-conjugated goat antirabbit (Jackson ImmunoResearch), and TxR-conjugated goat antimouse (Jackson ImmunoResearch). To amplify the phospho-JNK and JNK1 staining, anti-mouse Ig-digoxigenin, F(ab')2-fragment, followed by antidigoxigenin-rhodamine (Boehringer), or biotinylated anti-mouse, followed by streptavidin-rhodamine (Jackson ImmunoResearch), secondary antibodies were used. Double immunofluorescence with anti-GR and antiphospho-JNK or anti-JNK1 was performed on samples of Cos7 cells transfected with wild-type GR (GRwt) or GRdim.
Confocal Microscopy and Quantification
Confocal microscopy was performed with an MRC-1024 laser scanning microscope (BioRad), equipped with an Axiovert 100 invert microscope (ZEISS), and using excitation wavelengths of 488 nm (for FITC) and 543 nm (for TxR). To measure fluorescence intensity in cell cultures treated with TNF- or preincubated with Dex before TNF-
, 30 cells without prior selection of each culture were analyzed by laser spectroscopy performed with the confocal microscope and using a Plan-Apochromat 63x/1.4 oil immersion objective. The selection of rectangular regions of interest in the nucleus, excluding nucleoli, the adjustment of the confocal settings (iris, gain, black level), the same for each cell series, and the quantification of fluorescence intensities on a grey scale (0255) was performed following the procedure of
) for the JNK signal was calculated as described (
Subcellular Fractionation
To prepare whole-cell extracts, the monolayers were washed twice in PBS and the cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1.5 mM MgCl2, 10 mM NaF, 10% glycerol, 4 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 50 mM Hepes, pH 7.4, plus PPIM [25 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin]) for 15 min on ice followed by centrifugation at 13,000 rpm for 10 min at 4°C. For subcellular fractionation, cells were lysed in nuclear precipitation buffer (NPB: 10 mM Tris HCl, pH 7.4, 2 mM MgCl2, 140 mM NaCl, plus PPIM) supplemented with 0.1% Triton X-100 by incubating on ice for 10 min. The lysate was layered onto 50% wt/vol sucrose/NPB and centrifuged at 13,000 rpm for 10 min. Supernatants were taken as cytosolic fraction. Pellets (nuclei) were washed with NPB and extracted with Dignam C buffer (20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, plus PPIM) to obtain the nuclear fraction.
Immune Complex Kinase Assay
JNK was immunoprecipitated from each subcellular fraction using 0.4 µg of an anti-JNK antibody (sc-474 from Santa Cruz Biotechnology, Inc.). JNK activity in the immunocomplexes was assayed by incubation with 1 µg glutathione Stransferasec-Jun1-79 (GST-c-Jun) as substrate in the presence of 20 µM cold ATP and 1 µCi of [32P]ATP as described (
Western Blotting
JNK, phosphorylated JNK, and MEK-1 were detected in subcellular extracts by immunoblotting using the specific antibodies sc-474 (Santa Cruz Biotechnology, Inc.), V7931 (Promega), and sc-219 (Santa Cruz Biotechnology, Inc.), respectively. Histone H10 was detected using an mAb donated by Prof. A. Alonso (Deutches Krebsforschung Zentrum, Heidelberg). Western blots were performed and developed using the ECL detection system (Amersham Pharmacia Biotech) using either HRP-conjugated anti-rabbit (for anti-JNK, antiphospho-JNK, and anti-MEK-1) or HRP-conjugated anti-mouse (for antihistone H10) antibodies (ICN).
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Results |
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Glucocorticoids Inhibit both Nuclear Increase in NH2-terminal Phosphorylated and Total c-Jun, and in Phosphorylated/Activated JNK in HeLa Cells
To study whether glucocorticoids inhibit phosphorylation of c-Jun NH2-terminal domain in intact cells in vivo, we analyzed by confocal laser scanning microscopy the immunofluorescence of HeLa cells incubated with an antibody against c-Jun protein phosphorylated on serine-63 (hereafter referred to as phospho-c-Jun) upon treatment with TNF-. No signal over background was found in cells treated with vehicle or Dex alone (Fig 1 A, a and b). Upon TNF-
treatment, an intense punctate staining against a more diffuse signal appeared throughout the nucleus (except the nucleoli), whereas the cytoplasm remained negative (Fig 1 A, c). The increase in phospho-c-Jun induced by 30-min treatment with TNF-
was efficiently inhibited in Dex-treated cells (Fig 1 A, c and d). Dex reduced the level of nuclear phospho-c-Jun induced by TNF-
in HeLa cells by
60% (Fig 1 B). Unstimulated vehicle- and Dex-treated cells showed a similar low nuclear staining when an antitotal c-Jun antibody was used (Fig 1 C, a and b). Upon TNF-
treatment, a strong nucleoplasmic immunolabeling was observed (Fig 1 C, c). In agreement with previous data (
(Fig 1 C, c and d). Computer-assisted quantification revealed a 40% inhibition in TNF-
+ Dex cells with respect to cells treated with TNF-
alone (Fig 1 D).
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c-Jun phosphorylation is a consequence of the TNF-induced dual phosphorylation and subsequent activation of JNK and its concomitant translocation into the cell nucleus (
(Fig 2A and Fig B). This result was confirmed by immunoblotting analysis (Fig 2 C). To check whether this effect of Dex correlated with a decrease in enzymatic activity in both cellular compartments, we measured JNK activity in cytosolic and nuclear fractions. In agreement with immunofluorescence and immunoblotting data, Dex pretreatment led to significant reductions in JNK activity in both cytosolic and nuclear fractions (Fig 2 D), which were consistent with the lower levels of nuclear phospho-JNK (Fig 2A and Fig B). In turn, these data are consistent with the reduction of phospho-c-Jun in Dex-treated cells when stimulated with TNF-
(Fig 1 A). Verification that nuclear proteins did not leak into the cytosolic fractions during the fractionation process was obtained through subsequent hybridization of Western membranes to detect histone H10, which localizes exclusively in the nucleus. Conversely, MEK-1 was detected only in cytosolic fractions (Fig 2 D).
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Glucocorticoids Do Not Inhibit the TNF-induced Nuclear Accumulation of JNK
We used antibodies against total JNK to test the possibility that the inhibitory effect of Dex on the nuclear levels of phospho-JNK and JNK activity could be the consequence of a reduction in the nuclear content of this enzyme. In unstimulated HeLa cells, JNK immunoreactivity was diffusely distributed in the cytosol and nucleus (Fig 3 A, a). TNF- induced the nuclear accumulation of the enzyme, which was unchanged by Dex treatment (Fig 3 A, b and c). Using two distinct antibodies (anti-JNK1/JNK3 sc-474 and anti-JNK1 15701A), the same increase (five- to sixfold) in fluorescence was found in the nucleus of TNF-
treated cells, irrespective of Dex pretreatment (Fig 3 A, right), whereas cytoplasmic fluorescence did not change significantly upon any treatment (not shown). Measurement of JNK translocation by immunoblotting of subcellular fractions revealed that JNK was predominantly cytosolic in unstimulated HeLa cells, and confirmed that TNF-
induced a similar increase in nuclear JNK content, irrespective of pretreatment with Dex (Fig 3 B). A large amount of JNK remained in the cytosol even in TNF-
treated cells. The lack of significant differences in cytosolic JNK can be explained by the fact that JNK is abundant in the cytosol (
30-fold higher than in the nucleus in unstimulated cells) and therefore only a small fraction of cytosolic JNK is mobilized to the nucleus. The apparent discrepancy between the levels of extranuclear JNK content observed by immunoblotting and immunofluorescence may be explained by the fact that the image obtained by confocal microscopy corresponds to a cellular slice, whereas immunoblotting of subcellular fractions allows a more precise estimation of JNK content in each fraction as takes into account their total size. It should be kept in mind that a minor degree of protein leakage during cellular fractionation and/or differences in fluorescence signal due to variations in epitope accessibility and microenvironment cannot be ruled out. Dex alone also caused a slight, but reproducible, accumulation of JNK in the nucleus (Fig 3 C). This effect was progressive and led to a 22.5-fold increase in nuclear JNK content (Fig 3 D). To rule out the possibility that Dex could affect basal JNK activity by itself, we performed JNK assays in cytosolic and nuclear fractions of HeLa cells upon hormone addition. As expected, Dex did not induce JNK activity, but inhibited the activation of this enzyme by TNF-
(Fig 3 E). The inhibition was simultaneous in cytosol and nucleus, with kinetics compatible with that of nuclear entry of hormone-bound GR (
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Mechanism of JNK Inhibition by Hormone-activated GR
We found no evidence of interaction between activated, hormone-bound GR and JNK when we coimmunoprecipitated these two proteins from HeLa cell extracts (not shown). In view of recent reports ( addition to control or Dex-treated HeLa cells (not shown). In addition, we examined whether Dex treatment affected the amount of Hsp70 that is bound to JNK. However, the highly unspecific binding of this protein (even to agarose beads) precluded this analysis.
Next, we examined whether a mutant form of GR (GRdim;
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To verify that GRdim has the same effects on JNK activity and nuclear accumulation as GRwt, we analyzed the effects of Dex in GR-deficient Cos-7 cells that were transfected with either of these two genes. As seen in Fig 5 A, Dex treatment of GRwt- or GRdim-transfected Cos-7 caused the same inhibition of the TNF-induced JNK phosphorylation as observed in HeLa cells. Measurement of fluorescence intensity showed a 73 and 74% inhibition of nuclear phospho-JNK in GRwt- and GRdim-transfected cells, respectively. Likewise, in both types of transfected cells, Dex inhibited the increase in phospho-c-Jun and total c-Jun (not shown). Also as in HeLa cells, the accumulation of total JNK in the nucleus was not inhibited, but even slightly increased, by the hormone in both GRwt- and GRdim-transfected Cos-7 cells (Fig 5 B, b). In agreement with this, Dex did not change the nuclear translocation of JNK induced by TNF-
(Fig 5 B, c and d).
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Discussion |
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Our results show that Dex inhibits JNK phosphorylation and activity in intact cells, and that this effect is not due to inhibition of the nuclear translocation of the enzyme induced by TNF-. Rather, Dex by itself can promote the nuclear entry of inactive JNK in noninduced cells. These results were observed in two cell types, HeLa and Cos-7, and extend previous observations that JNK activity was reduced in extracts from cells treated with glucocorticoids, retinoids, thyroid hormone, or estrogens (
Our results differ from those reported for extracellular-regulated kinase (ERK), which suggested that kinase phosphorylation was necessary to induce homodimerization and subsequent nuclear translocation (
The effects of Dex have at least two possible explanations: direct or indirect inhibition of JNK activation in the cytoplasm in response to stress, and the activation of putative phosphatase(s). The negative result of the coimmunoprecipitation approach do not rule out a direct interaction between JNK and activated GR. Likewise attempts to coimmunoprecipitate JNK and its substrate c-Jun have failed, probably due to the existence of transient and/or weak interaction (
Known JNK targets are mainly nuclear, such as c-Jun, ATF-2, Elk-1, and p53 (
In two-hybrid experiments in yeast and in vitro, domain) of c-Jun binding a putative
inhibitor that could block its activation by JNK has been predicted (
domain in resting cells led to the hypothesis that JNK itself could be a form of this
inhibitor (
inhibitor, thus preventing AP-1 activation. JNK activation is believed to contribute to inflammatory responses (
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Footnotes |
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María Victoria González and Benilde Jiménez contributed equally to this work.
1 Abbreviations used in this paper: AP-1, activating protein-1; Dex, dexamethasone; GR, glucocorticoid receptor; GRdim, GR mutant deficient in dimerization; GRwt, GR wild-type; Hsp70, heat shock protein 70; JNK, c-Jun NH2-terminal kinase; TNF-, tumor necrosis factor-
.
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Acknowledgements |
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We thank M. Karin, P. Crespo, A. Alonso, and A.C.B. Cato for plasmids or antibodies, J. Bernal for his critical reading of the manuscript, and M. González and T. Martínez for technical help.
M.V. González and J.M. González-Sancho were supported by postdoctoral fellowships from the Comunidad Autónoma de Madrid. This work was supported by grants from the Plan Nacional de Investigación y Desarrollo (SAF98-0060, 1FD97-0281-CO2-01), Comisión Interministerial de Ciencia y Tecnología, and Plan General de Conocimiento (PM96-0035), Ministerio de Educación y Cultura of Spain.
Submitted: 7 October 1999
Revised: 7 July 2000
Accepted: 7 July 2000
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References |
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