Role of 14-3-3 as a Positive Regulator of the Glucocorticoid Receptor Transcriptional Activation
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内分泌学杂志 2005年第7期
School of Life Sciences and Biotechnology, Korea University (Y.S.K., S.-W.J., H.J.S., J.K.), Seoul 136-701; Graduate School of Biotechnology, Kyung Hee University (S.-W.J.), Yongin 449-701; Department of Biomedical Laboratory Science, College of Health Science, Yonsei University (H.J.S.), Wonju 222-701; Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine (H.J.L., D.S.N.), Seoul 138-736; and Department of Clinical Laboratory Science, School of Medicine, Eulji University (I.S.K.), Daejun 301-832, South Korea
Address all correspondence and requests for reprints to: Dr. Jesang Ko, School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, South Korea. E-mail: jesangko@korea.ac.kr.
Abstract
The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates the effects of glucocorticoids. It is known that 14-3-3 family proteins interact with GR and regulate its transcriptional activity. They also bind to several molecules and influence many cellular events by altering their subcellular localization and/or acting as a chaperone. Recently, it has been proposed that ligand-activated degradation of GR occurs via the ubiquitin-proteasomal degradation pathway and that inhibition of proteasomal activity induces up-regulation of GR and enhances the transcriptional activity of GR. To examine the function of 14-3-3 in the glucocorticoid-dependent signal pathway, we studied the regulatory role of 14-3-3 in ligand-induced GR transcriptional activation. 14-3-3 Enhanced the transcriptional activity of GR, and the levels of GR were higher in cells transfected with the 14-3-3 expression vector in response to glucocorticoid. The GR level increased in both cytosol and nucleus, and endogenous GR was also elevated by 14-3-3 in HeLa cells. 14-3-3 Inhibited ligand-induced down-regulation of GR. Proteasomal inhibition did not induce any synergistic effect on the 14-3-3-induced increase in GR in response to glucocorticoid, and inhibition of translation did not block elevation of GR by 14-3-3, indicating that 14-3-3 induces stabilization of GR. These results suggest that 14-3-3 functions as a positive regulator in the glucocorticoid signal pathway by blocking the degradation of GR and inducing an elevation of GR, thus enhancing the transcriptional activity of GR.
Introduction
THE GLUCOCORTICOID RECEPTOR (GR), a member of the nuclear receptor superfamily, is a ligand-dependent transcription factor that mediates the effects of glucocorticoids in diverse cellular processes, including homeostasis, cell growth, development, stress response, and inflammation (1, 2, 3). GR contains three functional motifs: an N-terminal transactivation domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (4). GR regulates the transcription of target genes either by binding to specific glucocorticoid response elements (GREs) within the target genes or by interacting with other transcription factors, such as nuclear factor-B and activating protein-1 (5, 6, 7). In the inactive state, GR forms a complex with other proteins, including the 90-kDa heat shock protein, the 70-kDa heat shock protein, and FK506 binding protein 52, and is sequestered in the cytoplasm (8). GR dissociates from 90-kDa heat shock protein upon ligand binding, resulting in unmasking of the GR nuclear localization signal, and enters the nucleus, where it acts as a transcription factor. Coactivators or corepressors of transcription, such as receptor-interacting protein 140 (RIP140), cAMP response element (CREB)-binding protein, and thyroid hormone receptor-associated protein 220 (9, 10, 11), and signaling molecules, such as nuclear factor-B and signal transducer and activator of transcription-3 and -5 (12, 13), interact with GR and modulate the transcriptional activity of GR. Recently, it has been reported that 14-3-3 family proteins interact with GR and regulate its transcriptional activity (14, 15, 16). 14-3-3 Is known to bind to GR and enhances its transcriptional activity (14). 14-3-3 Also interacts with and retains corepressor RIP140 in the cytoplasm, leading to an increase in GR transcriptional activity (17). In contrast, 14-3-3 binds to GR and favors the cytoplasmic subcellular localization of GR, thus acting as a negative regulator of GR-mediated signal transduction (18).
The 14-3-3 protein, a member of a highly conserved family of acidic and dimeric proteins, is known to be involved in numerous cellular events, including regulation of the cell cycle, cell growth and differentiation, and apoptosis (19, 20, 21). 14-3-3 Family proteins interact with many signaling molecules, such as MAPK kinase kinase, Raf-1, Wee1, Cdc25, cyclin B1, protein kinase C, IGF-I receptor, insulin receptor substrate 1, Bad, and Bcl (22, 23, 24, 25, 26), and regulate several signal transduction pathways (27, 28, 29). 14-3-3 Family proteins influence diverse biological activities by regulating the subcellular localization of target proteins (30, 31, 32, 33, 34) and protecting target proteins from proteolysis by acting as chaperones (35, 36). Also, 14-3-3 proteins help two molecules to interact or to interrupt the association between two molecules by functioning as molecular scaffolds (37).
Glucocorticoid induces down-regulation of GR; thus, long-term treatment with glucocorticoid reduces hormone responsiveness (38, 39). GR is regulated at both the mRNA and protein levels in response to its ligand (40, 41, 42, 43, 44, 45, 46). Studies have shown that glucocorticoid treatment decreases the GR mRNA level in different tissues and leads to a reduction of the GR protein level via the ubiquitin-proteasome-dependent pathway (40, 41, 42, 43, 44, 45, 46). Recently, it has been reported that proteasomal inhibition increases accumulation of GR and enhances GR-mediated transactivation (47). Inhibition of proteasomal activity also induces increased association of GR with the nuclear matrix and reduces the mobility of GR within the nucleus (48).
Because the GR level is reduced via proteasomal degradation, and 14-3-3 family proteins protect target proteins from proteolysis by acting as molecular chaperones, 14-3-3 probably increases the GR protein level by inhibiting proteasomal degradation, resulting in an enhanced transcriptional activity of GR. In this study we aimed to elucidate the regulatory mechanism of GR transcriptional activation due to 14-3-3. Our results indicate that 14-3-3 induces up-regulation of GR in response to glucocorticoid resulting from an inhibition of ligand-induced down-regulation of GR. Down-regulation of GR is inhibited via 14-3-3-induced GR stabilization. We propose a novel mechanism by which 14-3-3 enhances the transcriptional activity of GR involved in the GR-mediated signal pathway.
Materials and Methods
Materials
DMEM, MEM, sodium pyruvate, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). The luciferase assay kit was obtained from Promega Corp. (Madison, WI). Dexamethasone, N-acetyl-Leu-Leu-Norleu-al (ALLN), and cycloheximide were products of Sigma-Aldrich Corp. (St. Louis, MO). Polyclonal anti-GR antibody, monoclonal anti-14-3-3 antibody, polyclonal retinoid X receptor antibody, and polyclonal anti-ERK2 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Lactacystin and MG-132 were obtained from Calbiochem (San Diego, CA).
Cell culture and transient transfection
COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 C under a 5% CO2 atmosphere. HeLa cells were incubated in MEM supplemented with 1 mM sodium pyruvate, 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were seeded into six-well plates at 3 x 105 cells/well, then transiently transfected with appropriate expression vectors. After 24 h, cells were grown in the same medium supplemented with 0.5% FBS for 24 h. Serum-starved cells were treated with dexamethasone for the indicated times.
Reporter assay
COS-7 cells were transiently transfected with 2 μg of the reporter construct containing three GREs upstream of the luciferase reporter gene, 0.5 μg pSV-?-galactosidase reporter construct, 0.5 μg GR expression vector, and 4 μg mock vector or the HA-14-3-3 expression vector using Lipofectamine-2000 reagents. After 24 h, cells were grown in the same medium supplemented with 0.5% FBS for 24 h. Serum-starved cells were treated with 1 μM dexamethasone for 24 h and harvested. Luciferase assays were performed using the luciferase assay system (Promega Corp.). Cells were extracted with 100 μl/well reporter lysis buffer, and the supernatants were collected and assayed using a Luminoskan Ascent (Thermo Labsystems Oy, Helsinki, Finland). For ?-galactosidase assay, pSV-?-galactosidase was cotransfected with the luciferase reporter gene. Cell extracts were assayed for ?-galactosidase activity using the ?-galactosidase enzyme assay system (Promega Corp.) and were analyzed by a PerkinElmer LS50 spectrofluorometer at 350-nm excitation and 450-nm emission. The ratio of luciferase to ?-galactosidase activity was determined in triplicate samples. All data are presented as the mean ± SE of at least three independent experiments.
Western blot analysis
Cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate]. Cell lysates or fractionated protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose filters. The blots were incubated with anti-GR antibody for 1 h. After washing three times with Tris-buffered saline containing 0.1% Tween 20, the blots were incubated with antirabbit antibody conjugated with horseradish peroxidase for 1 h, washed three times with Tris-buffered saline containing 0.1% Tween 20, then developed with the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). The same blot was stripped and reprobed with anti-14-3-3 antibody to confirm the expression of 14-3-3 and with anti-ERK2 antibody for use as an internal control.
Cell fractionation
After transfection, cells were harvested and washed with ice-cold PBS. Cells were then resuspended in cytosol extraction buffer (10 mM HEPES, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mM dithiothreitol, 0.1 mM sodium orthovanadate, and protease inhibitors) and incubated on ice for 10 min. The nuclei were collected by centrifugation at 2,000 x g for 5 min at 4 C. The supernatant was collected as a cytosolic fraction. Collected nuclei were washed with ice-cold PBS, resuspended in nuclear extraction buffer (20 mM HEPES, 25% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mM sodium orthovanadate, and protease inhibitors), incubated on ice for 30 min, and centrifuged at 10,000 x g for 20 min at 4 C. The supernatant was collected as a nuclear fraction. Protein concentrations of each sample were determined.
Results
14-3-3 enhances dexamethasone-induced transcriptional activation of GR
We first determined whether 14-3-3 interacts with GR to increase its transcriptional activity. COS-7 cells were transiently transfected with the pLuc-GRE reporter vector, the CMV-hGR expression vector, and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector, then incubated in the absence or presence of dexamethasone. Although 14-3-3 slightly enhanced transcriptional activity in ligand-free cells, overexpression of 14-3-3 induced an increase in ligand-dependent transactivation by approximately 2-fold (Fig. 1). These data indicate that 14-3-3 is a positive regulator of GR transcriptional activity.
FIG. 1. 14-3-3 Enhances GR transcriptional activity. COS-7 cells were transiently transfected with 2 μg pLuc-GRE reporter construct, 0.5 μg pCMV-hGR expression vector, 1 μg pSV40-?-galactosidase construct, and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were treated with or without 1 μM dexamethasone (Dex) for 24 h and harvested for luciferase assay. Luciferase activity was normalized by ?-galactosidase activity, and transfections for luciferase assay were performed in triplicate. Data are expressed as the mean ± SD and are presented as the relative fold induction (luciferase activity in the absence of dexamethasone and 14-3-3 is set at 1). Expression of 14-3-3 was confirmed by Western blot analysis using the same samples for luciferase assay, and ERK2 was used as an internal control.
14-3-3 Increases the GR protein level in dexamethasone-stimulated cells
Because ligand-induced transactivation of GR is dependent on the amount of receptors (49, 50), we determined whether 14-3-3 plays a role in regulation of the GR protein level. COS-7 cells were cotransfected with the CMV-hGR expression vector and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector in the absence or presence of dexamethasone. The GR protein level was then detected by Western blotting. Although a slight increase in the level of GR was observed in ligand-untreated cells, the level of the GR protein was dramatically increased approximately 7-fold by overexpression of 14-3-3 in glucocorticoid-treated cells (Fig. 2, A and B). However, 14-3-3 had no effect on the level of the nuclear receptor retinoid X receptor, which was used as a control (Fig. 2A). 14-3-3 Increased the GR protein level in a dose-dependent manner in dexamethasone-treated cells (Fig. 2, C and D). These results indicate that 14-3-3 induces up-regulation of the GR protein in response to glucocorticoid.
FIG. 2. The GR protein is increased by 14-3-3. A, COS-7 cells were cotransfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were incubated in the absence or presence of 1 μM dexamethasone (Dex) for 24 h. Cell lysates were prepared and resolved by 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Up-regulation of the GR protein was detected by Western blotting. B, Densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA). Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the absence of dexamethasone and 14-3-3 is set at 100%). The data shown represent three independent experiments. C, COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and the indicated amounts of the pCMV-HA-14-3-3 expression vector. The total amounts of DNA (4.5 μg) were kept constant using the pCMV empty vector. Cells were treated with or without 1 μM dexamethasone for 24 h, and whole cell extracts were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gels. An 14-3-3-dependent increase in GR protein was detected by immunoblotting using polyclonal anti-GR antibody. D, Densitometric analysis was performed using Quantity One software. The data shown represent three independent experiments. Results are expressed as the percentage of GR, where the 100% value represents the amount of GR in the absence of dexamethasone. E, COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were stimulated with or without 1 μM dexamethasone for 24 h. Cells were harvested and fractionated into cytosol and nucleus, then samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. RXR, Retinoid X receptor.
To elucidate the subcellular distribution of the increased amounts of GR, transfected cells were incubated in the absence or presence of dexamethasone and separated into cytoplasmic and nuclear fractions. As shown in Fig. 2E, in ligand-activated cells, the GR protein level increased in both cytoplasmic and nuclear fractions, indicating that the increase in the level of GR in the nuclear fraction is probably responsible for enhanced GR transcriptional activation.
14-3-3 Increases endogenous GR in dexamethasone-treated HeLa cells
To determine whether 14-3-3 is also an effective regulator of the endogenous GR, HeLa cells harboring endogenous GR were transfected with either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector; treated, or not, with dexamethasone; then subjected to Western blot analysis. The endogenous GR level was increased by 14-3-3 in glucocorticoid-treated HeLa cells (Fig. 3), indicating that 14-3-3 also regulates the level of endogenous GR.
FIG. 3. 14-3-3 Increases endogenous GR. A, HeLa cells were transiently transfected with 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were stimulated with or without 1 μM dexamethasone (Dex) for 24 h. Cell lysates were resolved by 12% sodium dodecyl sulfate-polyacrylamide gels and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. B, Densitometric analysis was performed using Quantity One software. Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the absence of dexamethasone and 14-3-3 is set at 100%). The data shown represent three independent experiments.
14-3-3 Inhibits down-regulation of GR in response to dexamethasone
GR is down-regulated in response to glucocorticoid. Because the GR level was increased more by 14-3-3 in glucocorticoid-treated cells than in untreated cells, 14-3-3 probably inhibits glucocorticoid-induced down-regulation of GR. To test this hypothesis, we examined the effect of 14-3-3 on glucocorticoid concentration-dependent down-regulation of GR. As shown in Fig. 4, A and B, dexamethasone induced a decrease in the GR protein level in a concentration-dependent manner in untransfected cells. In contrast, down-regulation of GR in response to dexamethasone was significantly inhibited in 14-3-3-overexpressed cells. We also examined the effect of 14-3-3 on time-dependent down-regulation of GR in response to glucocorticoid. In untransfected cells, GR was down-regulated in response to glucocorticoid in a time-dependent manner (Fig. 4, C and D). However, 14-3-3 significantly reduced the time-dependent decrease in the GR level. These results indicate that 14-3-3 inhibits ligand-activated down-regulation of GR, resulting in an increase in the GR protein level in glucocorticoid-treated cells.
FIG. 4. Ligand-induced down-regulation of GR is inhibited by 14-3-3. COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. A, Cells were treated with the indicated concentrations of dexamethasone (Dex) for 24 h. Whole cell lysates were prepared and resolved by 12% sodium dodecyl sulfate-polyacrylamide gels, then transferred to nitrocellulose membrane. The amount of the GR protein was detected by Western blotting using anti-GR antibody. B, Densitometry was determined using the results of three independent experiments. Data are expressed as the percentage of GR, where the 100% value is the amount of GR in the absence of dexamethasone. C, The transfected cells were incubated in the presence of 1 μM dexamethasone for the indicated times. Whole cell lysates were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. D, Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the presence of dexamethasone for 0 h is set at 100%). The data shown represent three independent experiments.
14-3-3 Inhibits dexamethasone-induced down-regulation of GR via an enhancement of its stability
The reduction in the GR level in response to glucocorticoid is regulated by both translational and posttranslational processes (40). The amount of GR mRNA is decreased and/or proteasome-dependent degradation of GR is induced in response to dexamethasone (42, 47). To elucidate at which step 14-3-3 acts to regulate the GR protein level, COS-7 cells were transiently cotransfected with the pCMV-hGR expression vector and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector. The proteasome inhibitor ALLN inhibited down-regulation of GR in response to dexamethasone in cells transfected with vector alone (Fig. 5, A and B). However, in 14-3-3-overexpressing cells, ALLN treatment alone prevented the dexamethasone-induced increase in GR levels, but the GR levels were not further elevated when 14-3-3 was also overexpressed in ALLN-treated cells (Fig. 5A). The same results were obtained from experiments using the proteasomal inhibitors MG-132 and lactacystin (Fig 5, C and D). Cycloheximide, an inhibitor of protein translation, did not inhibit 14-3-3-induced elevation of GR in response to dexamethasone (Fig. 5, E and F). These results indicate that 14-3-3 is probably involved in stabilization of GR via inhibition of proteasome-dependent degradation.
FIG. 5. 14-3-3 Protects GR from proteasomal degradation. COS-7 cells were transiently cotransfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. A and C, Cells were preincubated in the absence or presence of 20 μM ALLN, 10 μM MG-132, and 5 μM lactacystin for 1 h and treated with or without 1 μM dexamethasone (Dex) for 16 h as indicated. Cell extracts were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane. The protein level of GR was determined using anti-GR antibody. B and D, Densitometry was determined using Quantity One software. Data are expressed as the percentage of GR, where the 100% value represents the amount of GR in the absence of dexamethasone, ALLN, and 14-3-3 (B) or in the presence of dexamethasone and 14-3-3 (D). E, The transfected cells were treated with or without 10 μg/ml cycloheximide (CHX) for 1 h and incubated in the presence or absence of 1 μM dexamethasone for 24 h. Whole cell lysates were prepared and resolved by 12% sodium dodecyl sulfate-polyacrylamide gels, then transferred to nitrocellulose membrane. A change in the GR level was detected by Western blotting using antibodies specific for GR, 14-3-3, and ERK2. F, Densitometric analysis was performed using Quantity One software. Data are presented as the percentage of GR (the protein level of GR in the absence of 14-3-3, dexamethasone, and CHX is set at 100%). The data shown represent three independent experiments.
Discussion
There have been advances in our understanding of the roles of the 14-3-3 proteins in the glucocorticoid-dependent signal pathway since it was first reported that 14-3-3 family proteins bind to GR and regulate its transcriptional activity (14). However, the exact mechanism by which 14-3-3 proteins function in GR-mediated signaling is not fully understood. In this study we elucidated the regulatory mechanism by which 14-3-3 enhances the ligand-induced transcriptional activation of GR and demonstrated that 1) 14-3-3 induces stabilization of the GR protein; 2) 14-3-3 reduces ligand-activated down-regulation of GR; 3) 14-3-3 increases the GR protein level; and 4) 14-3-3 enhances the transcriptional activity of GR in response to glucocorticoid.
14-3-3 Family proteins are known to have chaperone activity. They are involved in many cellular events by protecting partner proteins from proteolysis. It has been reported that 14-3-3 interacts with and relocalizes the A20 zinc finger protein from the insoluble to the soluble fraction, suggesting a chaperone function (35). 14-3-3 Binds to the nicotinic acetylcholine receptor 4 subunit and induces stabilization of the subunit (36). Our results show that 14-3-3 inhibits down-regulation of GR in response to glucocorticoid. Inhibition of proteasome did not induce any synergistic effect on the 14-3-3-induced increase in the GR level in response to glucocorticoid, and inhibition of translation did not block up-regulation of GR by 14-3-3. This indicates that 14-3-3 regulates the protein level of GR at the posttranslational stage. 14-3-3 Probably binds to GR and increases its stabilization via inhibition of proteasome-dependent degradation; consequently, 14-3-3 acts as a chaperone in the GR-dependent signal pathway.
It has been proposed that GR interacts with 14-3-3 without ligand stimulation (16). Our results show that 14-3-3 induces a slight increase in the basal transcriptional activity and the protein level of GR in dexamethasone-untreated cells. These data indicate that 14-3-3 protects GR from proteolysis in the resting state, resulting in an enhanced basal transcriptional activity of GR. Therefore, it is speculated that under physiological conditions or in the resting state, 14-3-3 exists as a GR-binding protein and regulates the turnover rate of GR. 14-3-3 Influences the steady-state protein level of GR and affects the basal transcriptional activity of GR in the resting state. However, it cannot be ruled out that 14-3-3 has an effect on other molecules involved in the ubiquitin-proteasome pathway and indirectly affects the protein level of GR. It may also be possible that 14-3-3 stimulates a GR-responsive gene in a hormone-independent fashion. Collectively, 14-3-3 seems not only to inhibit down-regulation of GR in response to glucocorticoid, but also to regulate the turnover rate of GR in the resting state.
Several lines of evidence suggest that 14-3-3 family proteins act as regulators of GR transcriptional activity. 14-3-3 Interacts with and sequesters GR in the cytoplasm, resulting in a decrease in GR transcriptional activation (18). 14-3-3 Has been proposed to bind to and export RIP140, a corepressor of the nuclear receptor, out of the nucleus, leading to enhancement of GR transcriptional activity (17). We found that 14-3-3 induces up-regulation of GR itself. The sensitivity of cells to glucocorticoid is dependent on the amount of GR in the cells (49, 50), and long-term treatment with glucocorticoid induces down-regulation of GR, resulting in a reduction of hormone responsiveness (38, 39). Therefore, a 14-3-3-induced increase in GR probably causes an increase in GR transcriptional activation. It is possible that 14-3-3 acts as a positive regulator of glucocorticoid-induced transcriptional activation of GR via dual mechanisms. 14-3-3 May inhibit the functions of the corepressor RIP140 and also increase the level of GR, leading to a synergistic enhancement of the transcriptional activation of GR. A recent report indicates that inhibition of proteasomal activity induces increased association of GR with the nuclear matrix and reduces the mobility of GR within the nucleus (48). We found that 14-3-3 induces an increase in the GR protein level in the nuclear fraction. It is possible that equilibrium exists between the cytosol and the nucleus, and it is worth examining whether 14-3-3 is also involved in GR subnuclear trafficking.
GR is up-regulated by certain stimuli, such as glucocorticoid and IL-2 (51, 52). Glucocorticoid is known to increase GR expression in human leukemic T cells, followed by apoptosis, and IL-2 up-regulates the amount of GR in human peripheral blood mononuclear cells and in the osteosarcoma cell line. The expression and binding activity of GR are also up-regulated in septic muscle (53). An increase in GR in response to glucocorticoid in T cells may result from up-regulation of GR mRNA (54), but the molecular mechanism of GR up-regulation by other stimuli was not fully understood. We have shown that 14-3-3 up-regulates GR via stabilization of the receptor, leading to an increase in GR transcriptional activation. Additional studies should identify stimuli that selectively induce up-regulation of 14-3-3. These stimuli can be used in drug development for alleviating reduced hormone responsiveness due to long-term treatment with glucocorticoid.
In the present study we have proposed a novel mechanism by which 14-3-3 enhances the transcriptional activation of GR. We have demonstrated that 14-3-3 inhibits ligand-induced degradation of GR, thus inducing elevation of GR levels, leading to increased transcriptional activity of GR. Although additional studies are required to determine whether other mechanisms exist by which 14-3-3 regulates GR transcriptional activity, we suggest, for the first time, that 14-3-3 regulates GR transcriptional activation by acting as a chaperone.
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Address all correspondence and requests for reprints to: Dr. Jesang Ko, School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, South Korea. E-mail: jesangko@korea.ac.kr.
Abstract
The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates the effects of glucocorticoids. It is known that 14-3-3 family proteins interact with GR and regulate its transcriptional activity. They also bind to several molecules and influence many cellular events by altering their subcellular localization and/or acting as a chaperone. Recently, it has been proposed that ligand-activated degradation of GR occurs via the ubiquitin-proteasomal degradation pathway and that inhibition of proteasomal activity induces up-regulation of GR and enhances the transcriptional activity of GR. To examine the function of 14-3-3 in the glucocorticoid-dependent signal pathway, we studied the regulatory role of 14-3-3 in ligand-induced GR transcriptional activation. 14-3-3 Enhanced the transcriptional activity of GR, and the levels of GR were higher in cells transfected with the 14-3-3 expression vector in response to glucocorticoid. The GR level increased in both cytosol and nucleus, and endogenous GR was also elevated by 14-3-3 in HeLa cells. 14-3-3 Inhibited ligand-induced down-regulation of GR. Proteasomal inhibition did not induce any synergistic effect on the 14-3-3-induced increase in GR in response to glucocorticoid, and inhibition of translation did not block elevation of GR by 14-3-3, indicating that 14-3-3 induces stabilization of GR. These results suggest that 14-3-3 functions as a positive regulator in the glucocorticoid signal pathway by blocking the degradation of GR and inducing an elevation of GR, thus enhancing the transcriptional activity of GR.
Introduction
THE GLUCOCORTICOID RECEPTOR (GR), a member of the nuclear receptor superfamily, is a ligand-dependent transcription factor that mediates the effects of glucocorticoids in diverse cellular processes, including homeostasis, cell growth, development, stress response, and inflammation (1, 2, 3). GR contains three functional motifs: an N-terminal transactivation domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (4). GR regulates the transcription of target genes either by binding to specific glucocorticoid response elements (GREs) within the target genes or by interacting with other transcription factors, such as nuclear factor-B and activating protein-1 (5, 6, 7). In the inactive state, GR forms a complex with other proteins, including the 90-kDa heat shock protein, the 70-kDa heat shock protein, and FK506 binding protein 52, and is sequestered in the cytoplasm (8). GR dissociates from 90-kDa heat shock protein upon ligand binding, resulting in unmasking of the GR nuclear localization signal, and enters the nucleus, where it acts as a transcription factor. Coactivators or corepressors of transcription, such as receptor-interacting protein 140 (RIP140), cAMP response element (CREB)-binding protein, and thyroid hormone receptor-associated protein 220 (9, 10, 11), and signaling molecules, such as nuclear factor-B and signal transducer and activator of transcription-3 and -5 (12, 13), interact with GR and modulate the transcriptional activity of GR. Recently, it has been reported that 14-3-3 family proteins interact with GR and regulate its transcriptional activity (14, 15, 16). 14-3-3 Is known to bind to GR and enhances its transcriptional activity (14). 14-3-3 Also interacts with and retains corepressor RIP140 in the cytoplasm, leading to an increase in GR transcriptional activity (17). In contrast, 14-3-3 binds to GR and favors the cytoplasmic subcellular localization of GR, thus acting as a negative regulator of GR-mediated signal transduction (18).
The 14-3-3 protein, a member of a highly conserved family of acidic and dimeric proteins, is known to be involved in numerous cellular events, including regulation of the cell cycle, cell growth and differentiation, and apoptosis (19, 20, 21). 14-3-3 Family proteins interact with many signaling molecules, such as MAPK kinase kinase, Raf-1, Wee1, Cdc25, cyclin B1, protein kinase C, IGF-I receptor, insulin receptor substrate 1, Bad, and Bcl (22, 23, 24, 25, 26), and regulate several signal transduction pathways (27, 28, 29). 14-3-3 Family proteins influence diverse biological activities by regulating the subcellular localization of target proteins (30, 31, 32, 33, 34) and protecting target proteins from proteolysis by acting as chaperones (35, 36). Also, 14-3-3 proteins help two molecules to interact or to interrupt the association between two molecules by functioning as molecular scaffolds (37).
Glucocorticoid induces down-regulation of GR; thus, long-term treatment with glucocorticoid reduces hormone responsiveness (38, 39). GR is regulated at both the mRNA and protein levels in response to its ligand (40, 41, 42, 43, 44, 45, 46). Studies have shown that glucocorticoid treatment decreases the GR mRNA level in different tissues and leads to a reduction of the GR protein level via the ubiquitin-proteasome-dependent pathway (40, 41, 42, 43, 44, 45, 46). Recently, it has been reported that proteasomal inhibition increases accumulation of GR and enhances GR-mediated transactivation (47). Inhibition of proteasomal activity also induces increased association of GR with the nuclear matrix and reduces the mobility of GR within the nucleus (48).
Because the GR level is reduced via proteasomal degradation, and 14-3-3 family proteins protect target proteins from proteolysis by acting as molecular chaperones, 14-3-3 probably increases the GR protein level by inhibiting proteasomal degradation, resulting in an enhanced transcriptional activity of GR. In this study we aimed to elucidate the regulatory mechanism of GR transcriptional activation due to 14-3-3. Our results indicate that 14-3-3 induces up-regulation of GR in response to glucocorticoid resulting from an inhibition of ligand-induced down-regulation of GR. Down-regulation of GR is inhibited via 14-3-3-induced GR stabilization. We propose a novel mechanism by which 14-3-3 enhances the transcriptional activity of GR involved in the GR-mediated signal pathway.
Materials and Methods
Materials
DMEM, MEM, sodium pyruvate, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). The luciferase assay kit was obtained from Promega Corp. (Madison, WI). Dexamethasone, N-acetyl-Leu-Leu-Norleu-al (ALLN), and cycloheximide were products of Sigma-Aldrich Corp. (St. Louis, MO). Polyclonal anti-GR antibody, monoclonal anti-14-3-3 antibody, polyclonal retinoid X receptor antibody, and polyclonal anti-ERK2 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Lactacystin and MG-132 were obtained from Calbiochem (San Diego, CA).
Cell culture and transient transfection
COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 C under a 5% CO2 atmosphere. HeLa cells were incubated in MEM supplemented with 1 mM sodium pyruvate, 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were seeded into six-well plates at 3 x 105 cells/well, then transiently transfected with appropriate expression vectors. After 24 h, cells were grown in the same medium supplemented with 0.5% FBS for 24 h. Serum-starved cells were treated with dexamethasone for the indicated times.
Reporter assay
COS-7 cells were transiently transfected with 2 μg of the reporter construct containing three GREs upstream of the luciferase reporter gene, 0.5 μg pSV-?-galactosidase reporter construct, 0.5 μg GR expression vector, and 4 μg mock vector or the HA-14-3-3 expression vector using Lipofectamine-2000 reagents. After 24 h, cells were grown in the same medium supplemented with 0.5% FBS for 24 h. Serum-starved cells were treated with 1 μM dexamethasone for 24 h and harvested. Luciferase assays were performed using the luciferase assay system (Promega Corp.). Cells were extracted with 100 μl/well reporter lysis buffer, and the supernatants were collected and assayed using a Luminoskan Ascent (Thermo Labsystems Oy, Helsinki, Finland). For ?-galactosidase assay, pSV-?-galactosidase was cotransfected with the luciferase reporter gene. Cell extracts were assayed for ?-galactosidase activity using the ?-galactosidase enzyme assay system (Promega Corp.) and were analyzed by a PerkinElmer LS50 spectrofluorometer at 350-nm excitation and 450-nm emission. The ratio of luciferase to ?-galactosidase activity was determined in triplicate samples. All data are presented as the mean ± SE of at least three independent experiments.
Western blot analysis
Cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate]. Cell lysates or fractionated protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose filters. The blots were incubated with anti-GR antibody for 1 h. After washing three times with Tris-buffered saline containing 0.1% Tween 20, the blots were incubated with antirabbit antibody conjugated with horseradish peroxidase for 1 h, washed three times with Tris-buffered saline containing 0.1% Tween 20, then developed with the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). The same blot was stripped and reprobed with anti-14-3-3 antibody to confirm the expression of 14-3-3 and with anti-ERK2 antibody for use as an internal control.
Cell fractionation
After transfection, cells were harvested and washed with ice-cold PBS. Cells were then resuspended in cytosol extraction buffer (10 mM HEPES, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mM dithiothreitol, 0.1 mM sodium orthovanadate, and protease inhibitors) and incubated on ice for 10 min. The nuclei were collected by centrifugation at 2,000 x g for 5 min at 4 C. The supernatant was collected as a cytosolic fraction. Collected nuclei were washed with ice-cold PBS, resuspended in nuclear extraction buffer (20 mM HEPES, 25% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mM sodium orthovanadate, and protease inhibitors), incubated on ice for 30 min, and centrifuged at 10,000 x g for 20 min at 4 C. The supernatant was collected as a nuclear fraction. Protein concentrations of each sample were determined.
Results
14-3-3 enhances dexamethasone-induced transcriptional activation of GR
We first determined whether 14-3-3 interacts with GR to increase its transcriptional activity. COS-7 cells were transiently transfected with the pLuc-GRE reporter vector, the CMV-hGR expression vector, and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector, then incubated in the absence or presence of dexamethasone. Although 14-3-3 slightly enhanced transcriptional activity in ligand-free cells, overexpression of 14-3-3 induced an increase in ligand-dependent transactivation by approximately 2-fold (Fig. 1). These data indicate that 14-3-3 is a positive regulator of GR transcriptional activity.
FIG. 1. 14-3-3 Enhances GR transcriptional activity. COS-7 cells were transiently transfected with 2 μg pLuc-GRE reporter construct, 0.5 μg pCMV-hGR expression vector, 1 μg pSV40-?-galactosidase construct, and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were treated with or without 1 μM dexamethasone (Dex) for 24 h and harvested for luciferase assay. Luciferase activity was normalized by ?-galactosidase activity, and transfections for luciferase assay were performed in triplicate. Data are expressed as the mean ± SD and are presented as the relative fold induction (luciferase activity in the absence of dexamethasone and 14-3-3 is set at 1). Expression of 14-3-3 was confirmed by Western blot analysis using the same samples for luciferase assay, and ERK2 was used as an internal control.
14-3-3 Increases the GR protein level in dexamethasone-stimulated cells
Because ligand-induced transactivation of GR is dependent on the amount of receptors (49, 50), we determined whether 14-3-3 plays a role in regulation of the GR protein level. COS-7 cells were cotransfected with the CMV-hGR expression vector and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector in the absence or presence of dexamethasone. The GR protein level was then detected by Western blotting. Although a slight increase in the level of GR was observed in ligand-untreated cells, the level of the GR protein was dramatically increased approximately 7-fold by overexpression of 14-3-3 in glucocorticoid-treated cells (Fig. 2, A and B). However, 14-3-3 had no effect on the level of the nuclear receptor retinoid X receptor, which was used as a control (Fig. 2A). 14-3-3 Increased the GR protein level in a dose-dependent manner in dexamethasone-treated cells (Fig. 2, C and D). These results indicate that 14-3-3 induces up-regulation of the GR protein in response to glucocorticoid.
FIG. 2. The GR protein is increased by 14-3-3. A, COS-7 cells were cotransfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were incubated in the absence or presence of 1 μM dexamethasone (Dex) for 24 h. Cell lysates were prepared and resolved by 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Up-regulation of the GR protein was detected by Western blotting. B, Densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA). Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the absence of dexamethasone and 14-3-3 is set at 100%). The data shown represent three independent experiments. C, COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and the indicated amounts of the pCMV-HA-14-3-3 expression vector. The total amounts of DNA (4.5 μg) were kept constant using the pCMV empty vector. Cells were treated with or without 1 μM dexamethasone for 24 h, and whole cell extracts were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gels. An 14-3-3-dependent increase in GR protein was detected by immunoblotting using polyclonal anti-GR antibody. D, Densitometric analysis was performed using Quantity One software. The data shown represent three independent experiments. Results are expressed as the percentage of GR, where the 100% value represents the amount of GR in the absence of dexamethasone. E, COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were stimulated with or without 1 μM dexamethasone for 24 h. Cells were harvested and fractionated into cytosol and nucleus, then samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. RXR, Retinoid X receptor.
To elucidate the subcellular distribution of the increased amounts of GR, transfected cells were incubated in the absence or presence of dexamethasone and separated into cytoplasmic and nuclear fractions. As shown in Fig. 2E, in ligand-activated cells, the GR protein level increased in both cytoplasmic and nuclear fractions, indicating that the increase in the level of GR in the nuclear fraction is probably responsible for enhanced GR transcriptional activation.
14-3-3 Increases endogenous GR in dexamethasone-treated HeLa cells
To determine whether 14-3-3 is also an effective regulator of the endogenous GR, HeLa cells harboring endogenous GR were transfected with either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector; treated, or not, with dexamethasone; then subjected to Western blot analysis. The endogenous GR level was increased by 14-3-3 in glucocorticoid-treated HeLa cells (Fig. 3), indicating that 14-3-3 also regulates the level of endogenous GR.
FIG. 3. 14-3-3 Increases endogenous GR. A, HeLa cells were transiently transfected with 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. Cells were stimulated with or without 1 μM dexamethasone (Dex) for 24 h. Cell lysates were resolved by 12% sodium dodecyl sulfate-polyacrylamide gels and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. B, Densitometric analysis was performed using Quantity One software. Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the absence of dexamethasone and 14-3-3 is set at 100%). The data shown represent three independent experiments.
14-3-3 Inhibits down-regulation of GR in response to dexamethasone
GR is down-regulated in response to glucocorticoid. Because the GR level was increased more by 14-3-3 in glucocorticoid-treated cells than in untreated cells, 14-3-3 probably inhibits glucocorticoid-induced down-regulation of GR. To test this hypothesis, we examined the effect of 14-3-3 on glucocorticoid concentration-dependent down-regulation of GR. As shown in Fig. 4, A and B, dexamethasone induced a decrease in the GR protein level in a concentration-dependent manner in untransfected cells. In contrast, down-regulation of GR in response to dexamethasone was significantly inhibited in 14-3-3-overexpressed cells. We also examined the effect of 14-3-3 on time-dependent down-regulation of GR in response to glucocorticoid. In untransfected cells, GR was down-regulated in response to glucocorticoid in a time-dependent manner (Fig. 4, C and D). However, 14-3-3 significantly reduced the time-dependent decrease in the GR level. These results indicate that 14-3-3 inhibits ligand-activated down-regulation of GR, resulting in an increase in the GR protein level in glucocorticoid-treated cells.
FIG. 4. Ligand-induced down-regulation of GR is inhibited by 14-3-3. COS-7 cells were transfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. A, Cells were treated with the indicated concentrations of dexamethasone (Dex) for 24 h. Whole cell lysates were prepared and resolved by 12% sodium dodecyl sulfate-polyacrylamide gels, then transferred to nitrocellulose membrane. The amount of the GR protein was detected by Western blotting using anti-GR antibody. B, Densitometry was determined using the results of three independent experiments. Data are expressed as the percentage of GR, where the 100% value is the amount of GR in the absence of dexamethasone. C, The transfected cells were incubated in the presence of 1 μM dexamethasone for the indicated times. Whole cell lysates were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and subjected to immunoblotting using antibodies specific for GR, 14-3-3, and ERK2. D, Data are expressed as the mean ± SD and are presented as the percentage of GR (the protein level of GR in the presence of dexamethasone for 0 h is set at 100%). The data shown represent three independent experiments.
14-3-3 Inhibits dexamethasone-induced down-regulation of GR via an enhancement of its stability
The reduction in the GR level in response to glucocorticoid is regulated by both translational and posttranslational processes (40). The amount of GR mRNA is decreased and/or proteasome-dependent degradation of GR is induced in response to dexamethasone (42, 47). To elucidate at which step 14-3-3 acts to regulate the GR protein level, COS-7 cells were transiently cotransfected with the pCMV-hGR expression vector and either the pCMV empty vector or the pCMV-HA-14-3-3 expression vector. The proteasome inhibitor ALLN inhibited down-regulation of GR in response to dexamethasone in cells transfected with vector alone (Fig. 5, A and B). However, in 14-3-3-overexpressing cells, ALLN treatment alone prevented the dexamethasone-induced increase in GR levels, but the GR levels were not further elevated when 14-3-3 was also overexpressed in ALLN-treated cells (Fig. 5A). The same results were obtained from experiments using the proteasomal inhibitors MG-132 and lactacystin (Fig 5, C and D). Cycloheximide, an inhibitor of protein translation, did not inhibit 14-3-3-induced elevation of GR in response to dexamethasone (Fig. 5, E and F). These results indicate that 14-3-3 is probably involved in stabilization of GR via inhibition of proteasome-dependent degradation.
FIG. 5. 14-3-3 Protects GR from proteasomal degradation. COS-7 cells were transiently cotransfected with 0.5 μg pCMV-hGR expression vector and 4 μg pCMV empty vector or pCMV-HA-14-3-3 expression vector. A and C, Cells were preincubated in the absence or presence of 20 μM ALLN, 10 μM MG-132, and 5 μM lactacystin for 1 h and treated with or without 1 μM dexamethasone (Dex) for 16 h as indicated. Cell extracts were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane. The protein level of GR was determined using anti-GR antibody. B and D, Densitometry was determined using Quantity One software. Data are expressed as the percentage of GR, where the 100% value represents the amount of GR in the absence of dexamethasone, ALLN, and 14-3-3 (B) or in the presence of dexamethasone and 14-3-3 (D). E, The transfected cells were treated with or without 10 μg/ml cycloheximide (CHX) for 1 h and incubated in the presence or absence of 1 μM dexamethasone for 24 h. Whole cell lysates were prepared and resolved by 12% sodium dodecyl sulfate-polyacrylamide gels, then transferred to nitrocellulose membrane. A change in the GR level was detected by Western blotting using antibodies specific for GR, 14-3-3, and ERK2. F, Densitometric analysis was performed using Quantity One software. Data are presented as the percentage of GR (the protein level of GR in the absence of 14-3-3, dexamethasone, and CHX is set at 100%). The data shown represent three independent experiments.
Discussion
There have been advances in our understanding of the roles of the 14-3-3 proteins in the glucocorticoid-dependent signal pathway since it was first reported that 14-3-3 family proteins bind to GR and regulate its transcriptional activity (14). However, the exact mechanism by which 14-3-3 proteins function in GR-mediated signaling is not fully understood. In this study we elucidated the regulatory mechanism by which 14-3-3 enhances the ligand-induced transcriptional activation of GR and demonstrated that 1) 14-3-3 induces stabilization of the GR protein; 2) 14-3-3 reduces ligand-activated down-regulation of GR; 3) 14-3-3 increases the GR protein level; and 4) 14-3-3 enhances the transcriptional activity of GR in response to glucocorticoid.
14-3-3 Family proteins are known to have chaperone activity. They are involved in many cellular events by protecting partner proteins from proteolysis. It has been reported that 14-3-3 interacts with and relocalizes the A20 zinc finger protein from the insoluble to the soluble fraction, suggesting a chaperone function (35). 14-3-3 Binds to the nicotinic acetylcholine receptor 4 subunit and induces stabilization of the subunit (36). Our results show that 14-3-3 inhibits down-regulation of GR in response to glucocorticoid. Inhibition of proteasome did not induce any synergistic effect on the 14-3-3-induced increase in the GR level in response to glucocorticoid, and inhibition of translation did not block up-regulation of GR by 14-3-3. This indicates that 14-3-3 regulates the protein level of GR at the posttranslational stage. 14-3-3 Probably binds to GR and increases its stabilization via inhibition of proteasome-dependent degradation; consequently, 14-3-3 acts as a chaperone in the GR-dependent signal pathway.
It has been proposed that GR interacts with 14-3-3 without ligand stimulation (16). Our results show that 14-3-3 induces a slight increase in the basal transcriptional activity and the protein level of GR in dexamethasone-untreated cells. These data indicate that 14-3-3 protects GR from proteolysis in the resting state, resulting in an enhanced basal transcriptional activity of GR. Therefore, it is speculated that under physiological conditions or in the resting state, 14-3-3 exists as a GR-binding protein and regulates the turnover rate of GR. 14-3-3 Influences the steady-state protein level of GR and affects the basal transcriptional activity of GR in the resting state. However, it cannot be ruled out that 14-3-3 has an effect on other molecules involved in the ubiquitin-proteasome pathway and indirectly affects the protein level of GR. It may also be possible that 14-3-3 stimulates a GR-responsive gene in a hormone-independent fashion. Collectively, 14-3-3 seems not only to inhibit down-regulation of GR in response to glucocorticoid, but also to regulate the turnover rate of GR in the resting state.
Several lines of evidence suggest that 14-3-3 family proteins act as regulators of GR transcriptional activity. 14-3-3 Interacts with and sequesters GR in the cytoplasm, resulting in a decrease in GR transcriptional activation (18). 14-3-3 Has been proposed to bind to and export RIP140, a corepressor of the nuclear receptor, out of the nucleus, leading to enhancement of GR transcriptional activity (17). We found that 14-3-3 induces up-regulation of GR itself. The sensitivity of cells to glucocorticoid is dependent on the amount of GR in the cells (49, 50), and long-term treatment with glucocorticoid induces down-regulation of GR, resulting in a reduction of hormone responsiveness (38, 39). Therefore, a 14-3-3-induced increase in GR probably causes an increase in GR transcriptional activation. It is possible that 14-3-3 acts as a positive regulator of glucocorticoid-induced transcriptional activation of GR via dual mechanisms. 14-3-3 May inhibit the functions of the corepressor RIP140 and also increase the level of GR, leading to a synergistic enhancement of the transcriptional activation of GR. A recent report indicates that inhibition of proteasomal activity induces increased association of GR with the nuclear matrix and reduces the mobility of GR within the nucleus (48). We found that 14-3-3 induces an increase in the GR protein level in the nuclear fraction. It is possible that equilibrium exists between the cytosol and the nucleus, and it is worth examining whether 14-3-3 is also involved in GR subnuclear trafficking.
GR is up-regulated by certain stimuli, such as glucocorticoid and IL-2 (51, 52). Glucocorticoid is known to increase GR expression in human leukemic T cells, followed by apoptosis, and IL-2 up-regulates the amount of GR in human peripheral blood mononuclear cells and in the osteosarcoma cell line. The expression and binding activity of GR are also up-regulated in septic muscle (53). An increase in GR in response to glucocorticoid in T cells may result from up-regulation of GR mRNA (54), but the molecular mechanism of GR up-regulation by other stimuli was not fully understood. We have shown that 14-3-3 up-regulates GR via stabilization of the receptor, leading to an increase in GR transcriptional activation. Additional studies should identify stimuli that selectively induce up-regulation of 14-3-3. These stimuli can be used in drug development for alleviating reduced hormone responsiveness due to long-term treatment with glucocorticoid.
In the present study we have proposed a novel mechanism by which 14-3-3 enhances the transcriptional activation of GR. We have demonstrated that 14-3-3 inhibits ligand-induced degradation of GR, thus inducing elevation of GR levels, leading to increased transcriptional activity of GR. Although additional studies are required to determine whether other mechanisms exist by which 14-3-3 regulates GR transcriptional activity, we suggest, for the first time, that 14-3-3 regulates GR transcriptional activation by acting as a chaperone.
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