Departments of Medical Nutrition (L.-G.B., A.P., I.R., S.O.) and Biosciences (J.L., K.D.-W.), Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden; and KaroBio AB (L.-G.B., S.N.), Novum, SE-141 57 Huddinge, Sweden
Address all correspondence and requests for reprints to: Professor Sam Okret, Department of Medical Nutrition, Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden. E-mail: Sam.Okret{at}mednut.ki.se.
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ABSTRACT |
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INTRODUCTION |
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GCs act by binding to the intracellular GC receptor (GR), which then stimulates (transactivates) or inhibits (transrepresses) transcription of specific target genes (8, 9). With regard to GC inhibition of cell proliferation, GR may either enhance the expression of genes that exert an antiproliferative effect or alternatively repress expression of genes, which stimulate cell proliferation. Stimulation of gene expression by the GR can mechanistically occur by two distinct ways. In the first case, the ligand-activated GR interacts directly with specific DNA sequences, designated glucocorticoid response elements (GREs), present in the target genes. In the second case, GR-mediated stimulation of gene expression takes place by a GR interaction with other DNA-bound transcription factors without itself contacting DNA (Refs. 8, 9, 10 and references therein). This GR interaction with other DNA-bound transcription factors is usually referred to as tethering. Tethering seems to be the most common mechanism by which GR represses gene expression. In this case, GR binds to and interferes with the transcriptional activity of other DNA-bound transcription factors, as exemplified by GR-mediated inhibition of nuclear factor-B (NF-
B) and activator protein 1 (AP-1) activity (8). In some cases, repression of gene expression seems to involve a direct GR interaction with so-called negative GREs present in target genes (8, 9). Irrespective of the mechanism of GR target gene regulation, binding to GREs or negative GREs, or repression of NF-
B or AP-1 activity, the DNA-binding domain (DBD) of the GR seems to be involved (11, 12, 13, 14).
NF-B, a transcription factor complex, is most commonly a heterodimer between p65 (RelA) and p50 (NF-
B1). It regulates many cellular functions such as inflammation, immune response, apoptosis, oncogenesis, and differentiation (15, 16). It has also been implicated in regulation of cell growth (17). NF-
B is activated by a number of stimuli that include cytokines, mitogens, phorbol esters, radiation, cellular stress, and bacterial or viral products. In nonstimulated cells, NF-
B is sequestered in the cytoplasm in an inactive form through its association with one of several NF-
B-inhibitory molecules (I
Bs) including I
B
. Activation of NF-
B involves a stimuli-initiated degradation of I
B
through a signal cascade that causes phosphorylation at serines 32 and 36 in the I
B
protein. This subsequently results in the release of NF-
B, its nuclear translocation, binding to NF-
B response elements in target genes, and initiation of transcription. Mutation of serines 32 and 36 results in a stable (superrepressive) form of I
B
that very efficiently prevents NF-
B activation (18).
AP-1 is a dimeric transcription factor complex consisting of proteins that include the Jun and Fos subgroups of transcription factors. Jun and Fos proteins may also dimerize with other transcription factors such as members of the ATF (activating transcription factor) or Maf families (19, 20). AP-1 is activated by a plethora of physiological stimuli and environmental cellular stressors and regulates a wide range of cellular processes including cell proliferation, apoptosis, transformation, and differentiation. Many signals that activate NF-B will activate AP-1. Furthermore, several target genes including both inflammatory cytokines and cell cycle regulators contain binding sites for both NF-
B and AP-1 (Refs. 8 and 21 and references therein).
Based on the knowledge that AP-1, as well as NF-B, are involved in the regulation of cell proliferation and that the activity of these transcription factors are repressed by activated GR, it has been hypothesized that GR-AP-1 and/or NF-
B cross-talk is a mechanism involved in the antiproliferative effects exerted by GCs. However, so far, this has not been experimentally demonstrated.
In this report, we tested this hypothesis using GR DBD mutants that separate GR-mediated repression of NF-B and AP-1 signaling, respectively. We used GR mutants in which subdomains of the GR DBD had been exchanged with the corresponding region of the thyroid hormone receptor ß (TRß). The basis for this approach was that previous results have indicated that different GR DBD subdomains are involved in NF-
B and AP-1 repression, respectively (11, 12). Furthermore, the mutants are unable to transactivate via GREs, excluding genes thought to be activated through this mechanism from being involved in the GC-mediated antiproliferative effect. Using two GR DBD mutants, one able to exert an antiproliferative effect and the other not stably introduced into human embryonic kidney (HEK)293 cells, we found a better correlation between the antiproliferative response and NF-
B repression than to AP-1 repression. Gene expression profiling identified the I
B
gene as a primary candidate target gene involved in the GC-mediated inhibition of cell proliferation. This was in line with the observation that the NF-
B signaling pathway is an important target for the antiproliferative effect by GCs. This was verified by selective expression of the I
B
gene.
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RESULTS |
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Analysis of the ability of the GR DBD mutants in the presence of dexamethasone (Dex) to repress NF-B activity, after transfection with the 3xNF-
B-luc reporter gene, demonstrated that repression of NF-
B activity in HEK293 cells relied solely on the presence of the C-terminal zinc finger of GR (Fig. 2C
). Replacement of the C-terminal zinc finger of the GR by the corresponding zinc-finger from the TRß (GggtG) totally abolished the ability of the receptor to repress NF-
B activity. In fact, this variant displayed a weak stimulation of NF-
B activity. Consistent with a critical role for the second zinc finger in mediating GR repression of NF-
B activity, most of the repressive activity remained when the C-terminal zinc finger alone was from the GR (i.e. GttgG). If the complete GR DBD was replaced by the corresponding region from TRß (GgggG
GtttG), the resulting variant displayed less than 10% of the NF-
B repression of the wild-type protein.
To examine the ability of the different GR DBD mutants to repress AP-1 activity, the GR DBD mutant-containing Flp-In HEK293 cells were transfected with the matrix metalloproteinase-1 (MMP1) luciferase reporter gene (MMP1-luc). It has previously been shown that repression of the MMP-1 (collagenase-1) gene by GCs takes place by cross-talk between the GR and AP-1 at the AP-1 site present in the MMP-1 promoter [72 to 65 bp from the transcription start site (22)] through a tethering mechanism. Whereas the wtGR (GgggG) repressed the MMP1-luc activity by 90%, the GtttG, GttgG, and the GggtG mutants retained the ability to repress the activity by 4060% (Fig. 2D). Only the GgttG receptor had completely lost its ability to repress the MMP-1 reporter gene. The GttgG and GtttG mutants were also able to repress AP-1 activity from a reporter gene controlled by five synthetic AP-1 response elements, similar to the wtGR (Fig. 2E
). Furthermore, this demonstrated the NF-
B and AP-1 discriminatory property of GtttG. Most important, the above results from the GR DBD mutants demonstrate that NF-
B and AP-1 repression can be separated as demonstrated by the effect of the GtttG and GggtG receptors on NF-
B and AP-1 activity, respectively.
Correlation between NF-B-, But Not AP-1 Repression, and the Antiproliferative Response to GCs in HEK293 Cells
The Flp-In HEK293 cells stably transfected with the GR DBD mutants were analyzed for the effect on proliferation after treatment with 100 nM Dex. As can be seen in Fig. 3A, Dex treatment of cells containing the wtGR (GgggG) resulted in an antiproliferative response. No effect on proliferation by Dex was seen in the nontransfected, GtttG, GggtG, or GgttG cells. However, maintaining the GR C-terminal zinc finger alone was sufficient to preserve most of the antiproliferative response after Dex administration, as demonstrated by the GttgG cells (Fig. 3A
). Flow cytometric analysis of the cell cycle of GttgG HEK293 Flp-In cells showed that the inhibition of cell proliferation was associated with a reduction in the percentage of cells in the S/G2/M phase from 42.6 ± 0.5% to 33.3 ± 0.6% (n = 3, P < 0.001) (Fig. 3B
) with a corresponding increase of cells in the G1 phase, similar to what was observed in the wtGR cells (data not shown). No indication of apoptosis was observed after Dex treatment (data not shown). No significant effect of Dex on the distribution of the percentage of cells in the S/G2/M phase in GtttG cells was observed (45.0 ± 1.0% in control vs. 42.8 ± 0.7% in Dex-treated cells, n = 3) (Fig. 3B
). These results show that the antiproliferative effect is dependent on the GR C-terminal zinc finger. As the GttgG receptor is unable to transactivate a GRE-dependent reporter gene, the antiproliferative effect exerted by GCs in HEK293 cells is independent of direct GR interaction with a GRE. It was observed that GtttG- and GggtG-containing cells showed no Dex-mediated inhibition of cell proliferation and were unable to repress NF-
B but still repressed AP-1 activity, whereas the GttgG cells demonstrated a Dex-mediated antiproliferative response and inhibited both AP-1 and NF-
B activity (see above). Thus, a correlation between NF-
B repression, rather than AP-1 repression, and antiproliferative response is demonstrated.
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Statistical analysis identified 18 genes that were significantly up-regulated at least 1.7-fold and three that were repressed by at least 30% in GttgG cells after Dex administration (Table 1). In GtttG cells, six genes were induced at least 1.7-fold, and one gene was repressed by at least 30%. One gene regulated in GttgG cells [matrix metalloproteinase 15 (membrane-inserted)] was also regulated in GtttG cells and thus does not correlate to the antiproliferative response. This suggests that at least one of the remaining 20 genes regulated in GttgG but not in GtttG cells is involved in the GC-mediated antiproliferative response observed in HEK293 cells. One of the genes strongly regulated in the GttgG cells, but not in the GtttG cells, was I
B
(Table 1
). This was particularly interesting, because the previous transfection experiments (see above) suggested a correlation between NF-
B repression and the antiproliferative response after Dex administration.
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Selective Expression of IB
in HEK293 Cells Inhibits Proliferation
To assess the effect of IB
expression on HEK293 cell cycling, cells were transiently transfected with an expression vector for the superrepressive form of I
B
(I
B
-DN) together with an expression vector for green fluorescence protein (GFP). The cell cycle of transfected cells (GFP positive) was analyzed by flow cytometry (Fig. 5A
). The results demonstrated that the percentage of cells in S/G2/M phase was significantly reduced from 44.0 ± 2.9% to 36.0 ± 1.4% (n = 6, P < 0.001) in I
B
-DN transfected cells as compared with the cells transfected with the control vector (Fig. 5B
). An analogous reduction of cells in the S/G2/M phase was obtained if cells were transfected with the wild type I
B
(data not shown). No indication of apoptosis was observed. Notably, I
B
expression had an effect on the cell cycle pattern similar to what was observed after Dex treatment of GttgG cells (see Fig. 3B
).
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DISCUSSION |
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In this report, we describe the novel observation that GC repression of HEK293 cell proliferation involves GR-mediated inhibition of NF-B activity. Several results suggested that inhibition of NF-
B (and not AP-1) activity is responsible for the antiproliferative effects of GCs on HEK293 cells. Using GR mutants unable to transactivate through GREs and with different efficacy to repress AP-1 and NF-
B activity, a good correlation between the antiproliferative response and the ability of GCs to suppress NF-
B activity was observed. The antiproliferative domain of the GR was localized to the C-terminal zinc finger, which was the same region shown to be required for NF-
B repression. Our results also indicated that GR-AP-1 cross-reactivity involves the linker region of the GR DBD, because maintaining the GR linker region (GgttG
GggtG) sustained approximately 50% of the AP-1 repressing activity as compared with the wtGR. AP-1 repressing activity is also present in the N-terminal zinc finger of TRß DBD, because replacing the GR N-terminal zinc finger with the N-terminal zinc finger of TRß (GgttG
GtttG) restored 5060% of the AP-1 repressing activity of wtGR. However, definitive proof of important GR DBDs involved in AP-1 repression will require testing additional mutants. Furthermore, our results demonstrated that it is possible to separate GR repression of AP-1 and NF-
B, indicating that repression of these two signaling pathways operates through separate receptor domains and mechanisms. This is in line with a report by Yamamoto and associates (28) who demonstrated that coactivators are involved in the repression of AP-1 activity; in contrast, a recent report excluded the involvement of coactivators in the repression of NF-
B (29). Our results in HEK293 cells are also in line with reports from Jurkat, CEM-C7 T cells, and U2OS cells in which it has been demonstrated that repression rather than activation of genes via GR binding to GREs is important for the antiproliferative response (1, 4, 30). In our case, this conclusion was based on the results obtained using the GttgG cells, which lacked the ability to transactivate target genes via GREs but maintained the transrepressive and antiproliferative effects. However, the antiproliferative mechanism of GR may differ in different cell lines, because in S49 or Saos2 cells, a GR with sustained ability to transactivate via GREs is required for the antiproliferative effect (1, 31). We have also shown that GC induction of p57Kip2 is involved in the antiproliferative response of HeLa cells, and this requires GR binding to a GRE present in the p57Kip2 promoter (32).
Further evidence for the involvement of NF-B repression in the GC-mediated antiproliferative response was obtained from our gene expression profiling studies in HEK293 cells containing two GR mutants (GttgG and GtttG) that lack the ability to transactivate GRE-controlled target genes, of which only one can repress NF-
B activity (GttgG), whereas both can repress AP-1 activity. Only the GttgG receptor that was able to repress NF-
B activity executed an antiproliferative response. These studies identified increased I
B
expression after GC administration in the GttgG-containing HEK293 cells, but not in the GtttG-containing HEK293 cells, in line with NF-
B repression being involved in the GC-induced antiproliferative response. Northern and Western blotting confirmed the I
B
regulation identified by the microarray analysis. Further evidence for a role of NF-
B repression in the antiproliferative response was derived from our studies using ectopically expressed I
B
. Transient transfection of HEK293 cells with the I
B
resulted in an increased percentage of cells in the G1 phase, similar to what was seen in wtGR or GttgG-containing HEK293 cells after Dex treatment. A G1 accumulation is in accordance with what is seen in most other cell types in which GCs result in inhibition of cell proliferation (4, 5, 6, 24). Selective expression of I
B
-DN using the tetracycline-inducible system in HEK293 cells also resulted in inhibited cell proliferation. However, the growth-inhibitory effect in these cells was not as pronounced as in GR-containing (wtGR or GttgG) HEK293 cells after GC administration. This could be explained by the fact that GR not only induced I
B
expression in these latter cells, but also directly inhibited NF-
B activity by tethering, as compared with the selective increase in I
B
expression in Dox-treated HEK293-rtTA I
B
-DN cells. An alternative explanation is that the I
B
cooperates with other Dex-induced gene products in the GttgG cells, e.g. the Nijmegen breakage syndrome gene (Table 1
), that play a role in the control of cell proliferation (33). An additional possibility is that GCs, via the GR, affect phosphorylation events that will result in an altered activity but not in the abundance of proteins involved in cell cycle control, because GCs have been shown to influence, for example, ERK activity (34).
By comparing two GR mutants that are transactivation deficient and in which only one executes an antiproliferative response, we could restrict the number of candidate target genes involved in the antiproliferative response to 20 genes. The expression profiling data do not exclude the involvement of additional genes in the antiproliferative response, because the arrays used in the profiling studies cover only about 8,400 of the approximately 38,000 genes present in the human genome. Furthermore, genes regulated less than the applied filter for selecting regulated genes may be involved. In addition, the microarray does not record regulation at the posttranslational level.
The B-cell CLL/lymphoma 2 gene (Bcl-2), that can act as a negative regulator of cell proliferation (35) does not seem to be involved in the antiproliferative response in HEK293 cells, because its induction in GtttG cells did not result in inhibition of cell growth.
Interestingly, IB
gene expression was induced by the GttgG mutant despite its inability to transactivate target genes controlled by GRE. Furthermore, because I
B
mRNA induction was observed in the presence of the protein synthesis inhibitor CHX, the effect must be directly mediated by the GR mutant without the involvement of newly synthesized intermediary factors. A direct effect for GR induction of I
B
gene expression is in line with what previously has been described for the wtGR (26, 27). Furthermore, the GttgG mutant did not transactivate from either a palindromic or direct repeat (spaced by 4 bp) thyroid response element (Bladh, our unpublished data). This suggests that the GttgG receptor mutant induces I
B
expression by a mechanism independent of DNA-binding, e.g. through a tethering mechanism. Indeed, it has been demonstrated that tethering of the GR to other transcription factors results not only in transrepression but in some cases led to enhanced transcriptional activation of target genes (10, 36, 37). Also GR DBD mutants unable to transactivate from a GRE are able, in some cases, to enhance transcriptional activation through the tethering mechanism (10). This is in agreement with recent data showing that the dimer interface of the GR DBD is dispensable for activation of some genes (38). A tethering mechanism for GR activation of the I
B
gene is also indirectly supported by the fact that a GRE has not been identified in the I
B
promoter. The target protein for GR interaction with the I
B
gene remains to be established.
Although the involvement of NF-B in the control of normal cell proliferation is less clear as compared with AP-1, a few reports suggest the involvement of NF-
B in the control of cell proliferation. NF-
B activation has been shown to be required for cell cycling in several cell types, such as regenerating liver cells, fibroblasts, and breast cancer cells (for a review see Ref. 17). Furthermore, inhibition of NF-
B caused impairment of cell cycle progression in glioma cells and retarded G1/S transition in HeLa cells (39, 40). This also demonstrated a small constitutive NF-
B activity present in these proliferating nonstimulated cells. It has also been found that the level of NF-
B activation in Jurkat cells was linked to signals that control cell cycle progression (41). As shown in Fig. 6
, a small constitutive NF-
B activity seems to be present in nonstimulated HEK293 cells, as selective I
B
expression reduced basal NF-
B activity (Fig. 6
). In line with this, we detected a weak DNA-binding NF-
B activity in proliferating nonstimulated HEK293 cells that could be competed specifically by excess unlabeled NF-
B binding site in gel shift experiments (data not shown).
All together, this suggests, as demonstrated in this report, that GC-mediated repression of NF-B activity is an important mechanism contributing to the antiproliferative effect of GCs. How general this mechanism is remains to be established. However, it opens up the possibility that the identification of GR ligands that will induce the GR into a conformation that discriminates between AP-1 and NF-
B repression may generate ligands with improved therapeutic profiles.
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MATERIALS AND METHODS |
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Expression Vectors and Reporter Genes
The plasmids containing the GR mutants with chimeric DBDs have been described previously (12). Briefly, the DBDs of GR and thyroid receptor ß (TRß) were separated into three parts (the N-terminal zinc finger, the linker region between the two fingers, and the C-terminal zinc finger) and termed ggg or ttt, respectively; the first, second, and third lowercase letter represent the N-terminal zinc finger, linker region, and C-terminal zinc finger region of GR (g) or TRß (t), respectively. The wild- type (wt) GR (GgggG) is equivalent to GRnx and GtttG to GTG (Ref. 12 and references therein). The GR cDNAs (KpnI-DraI fragments) containing the complete coding sequence were subcloned into the KpnI-EcoRV sites of the pcDNA5/FRT expression vector (Invitrogen) to generate stably expressing Flp-In HEK293 cell lines (www.invitrogen.com). Flp-In cells, which contain a single integrated Flp recombination target site, allow stable integration of cDNAs at a specific genomic site and, subsequently, similar expression in individual cell clones.
The luciferase reporter plasmids 3xNFB, 2xGREtk-Luc, and 5xAP1-ALP (alkaline phosphatase) have previously been described (12, 42). The MMP-1 (517/+63 collagenase-1)-luc reporter plasmid was kindly provided by M. Göttlicher (Munich, Germany).
The superrepressive form of IB
(I
B
-DN, I
B
with mutations at serines 32 and 36 to a glycine and alanine, respectively) has been described elsewhere (43) and was subcloned into pcDNA3 (Invitrogen) to generate cytomegalovirus (CMV)-I
B
-DN. The pTRE-I
B
-DN-FLAG plasmid is the I
B
-DN containing a C-terminal FLAG epitope and was subcloned into the tetracycline-inducible vector pTRE (CLONTECH/BD Biosciences, Palo Alto, CA).
Cells, Cell Culturing Conditions, and Establishment of Stably Transfected Cell Lines
Flp-In HEK293 (Invitrogen) or HEK293 Tet-On cells (CLONTECH) were grown at 37 C in 5% CO2 in a 1:1 mixture of high-glucose DMEM and HAM F12 medium containing 10% fetal bovine serum, and penicillin and streptomycin at 10 IU/ml and 100 µg/ml, respectively, and 2 mM L-glutamine. In the case of nontransfected Flp-In HEK293 cells, the medium also contained 100 µg/ml Zeocin. HEK293 Tet-On cells were grown in the presence of 500 µg/ml geniticin. Establishing Flp-In HEK293 cells stably expressing wtGR or GR mutants was performed according to the instructions of the manufacturer (Invitrogen), using the calcium phosphate precipitation method. Individual clones were selected and maintained in the presence of 100 µg/ml hygromycin B (no Zeocin). Individual clones established in the HEK293 Flp-In cells expressed similar amounts of GR. Pools consisting of five to 10 individual clones were used for the subsequent experiments.
To establish the HEK293-rtTA IB
-DN cell lines, HEK293 Tet-On cells were cotransfected as described above with 10 µg of the pTRE-I
B
-DN-FLAG plasmid and 1 µg of the pKSV-Hygromycin selection plasmid. Selection was performed in the presence of 100 µg/ml hygromycin B, and individual clones were isolated.
Transient Transfections of HEK293 Cells
Lipofectin reagent was used in all transfections according to the manufacturers instructions. Briefly, 50,000 cells were seeded in 24-well plates 20 h before transfection. Reporter plasmid (200 ng) was used per well together with 2 ng of the CMV-ALP plasmid as an internal control to correct for differences in transfection efficiency. Cells were treated 24 h post transfection for 20 h, after which the cell medium was collected. Preparation of cell extract and measurement of alkaline phosphatase and luciferase activity were performed as previously described (12, 42). Quadruplicate wells were analyzed for each experimental condition and experiments were repeated three to five times.
In the case of cell cycle analysis after IB
transfection, 10-cm plates were transfected with 5 µg pEGFP-C2 (CLONTECH) and 15 µg CMV-I
B
-DN or pcDNA3 (Invitrogen) expression vector per plate using the Lipofectin reagent. Medium was replaced 22 h after transfection, and incubation was continued for an additional 48 h before analysis.
Western Immunoblotting
Whole-cell extracts were prepared from nontreated cells or cells treated for various time periods with 100 nM Dex or 2 µg/ml Dox as indicated in the different experiments, and Western immunoblotting was performed as previously described (32). For detection of GR, the E-20 antibody (sc-1003) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) was used. IB
-DN in the HEK293-rtTA I
B
-DN cells was specifically detected by an anti-FLAG-M2 antibody (Sigma) to a FLAG epitope present in the C-terminal part of I
B
-DN. For detection of endogenous I
B
or total (endogenous and I
B
-DN) I
B
s, an anti-I
B
antibody (sc-847) from Santa Cruz was used. To check for equal loading and transfer, the filters were probed with an anti-ß-tubulin antibody (N357, Amersham Life Sciences, Arlington Heights, IL). For detection, enhanced chemiluminescence (Amersham) was used. The intensities of the bands were quantified by a luminescent image analyzer (LAS-1000, Fuji Photo Film Co., Ltd, Stamford, CT).
Northern Blotting
Cells were treated with or without 100 nM Dex in the presence or absence of 5 µg/ml CHX for 6 h. The CHX was added 1 h before hormone treatment. Total RNA was prepared by the RNeasy kit from QIAGEN (Chatsworth, CA). Northern blotting using 10 µg of total RNA was performed as previously described (24). [32P]UTP-labeled cRNA probes were used using CMV-IB
-DN (see above) and ß-actin cloned into pGEM3 (24) as templates. Hybridization was quantified by phosphor imager analysis using a Bas111 phosphor imager (Fuji Photo Film Co.)
Cell Proliferation
Cells (n = 5000) were seeded in 24-well plates and treated with or without 100 nM Dex or 2 µg/ml Dox for the indicated time points. Cells were trypsinized and counted in a Bürker chamber. Five wells were counted per time point, and experiments were repeated two to three times.
Flow Cytometric Analysis of Cell Cycle
Fixation, PI staining of cells, and flow cytometric analysis of cell cycle were performed as described previously (24). For analyzing the cell cycle after GFP/IB
-DN transfection, cells were first fixed with 1% formaldehyde at 4 C for 1 h, washed in PBS, and kept in 70% ethanol at 4 C overnight before staining with PI. Gated GFP-positive cells (n = 2000) were analyzed by flow cytometry.
Statistical Analysis
Unless otherwise stated, results are expressed as mean ± SD; n is given for each experiment. For analysis of significance, two-tailed homoscedastic Students t test was used.
Gene Expression Profiling and Identification of Regulated Genes
GtttG- and GttgG-expressing cells were treated with or without 100 nM Dex for 6 h. For nontreated cells, a similar volume of solvent was added. Three separate experiments were performed on different days. Total RNA was isolated using the RNeasy kit from QIAGEN, and its quality was determined using the Nano 6000 Chip in the Bioanalyzer from Agilent Technologies, Inc. (Palo Alto, CA). Total RNA (8 µg) was used for target cDNA synthesis according to the Affymetrix manual (www.affymetrix.com). Hybridization of final targets to Human Gene Focus arrays representing 8500 transcripts followed by probing and scanning was performed according to the Affymetrix manual. The scanned CEL-files from Affymetrix Microarray Suite 5.0 were used for quantile normalization and quantification of expression using the robust multichip analysis (44). For statistical analysis of significantly changed genes, the R software from BioConductor (www.bioconductor.org) was employed. In Quantile t plots, genes with higher experimental t scores than 5 and lower experimental t scores than 5 were scored as having significantly altered expression in Dex-treated vs. untreated cells. A minimal mean ratio of 1.7-fold was used as threshold for Dex-dependent induction of t score-selected genes in the two cell lines, and 0.7 was used as threshold for repressed genes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 4, 2004
Abbreviations: ALP, Alkaline phosphatase; AP-1, activator protein 1; CHX, cycloheximide; CMV, cytomegalovirus; DBD, DNA-binding domain; Dex, dexamethasone; Dox, doxycycline; GC, glucocorticoid hormone; GFP, green fluorescence protein; GR, GC receptor; GRE, glucocorticoid response element; HEK, human embryonic kidney; MMP1-luc, matrix metalloproteinase-1 luciferase; MMP-1, matrix metalloproteinase-1 collagenase-1; NF-B, nuclear factor-
B; PI, propidium iodide; rtTA, tetracycline-regulated reverse tTA transactivator; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; TRß, thyroid hormone receptor-ß; wtGR, wild-type GR.
Received for publication July 22, 2004. Accepted for publication October 26, 2004.
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REFERENCES |
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