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Original Studies |
Department of Medicine (C.M.B., F.U.B.) and Institute for Pathology (A.-M.B.), University Hospital Eppendorf, 20246 Hamburg; and the IHF Institute for Hormone and Fertility Research, University of Hamburg (C.M.B., T.E., H.M.S.), 22529 Hamburg, Germany
Address all correspondence and requests for reprints to: Christoph M. Bamberger, M.D., Department of Medicine, University Hospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: bamberger{at}uke.uni-hamburg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the molecular level, the effects of GC are mediated by the
intracellular glucocorticoid receptor (GR). GR is a ligand-dependent
transcription factor, which, upon hormone binding, translocates to the
cell nucleus, where it binds to glucocorticoid response elements (GREs)
in the promoter regions of target genes, resulting in
trans-activation of these genes (4, 5, 6, 7, 8, 9).
Trans-activation is probably the predominant mechanism by
which GC exert many of their metabolic and cardiovascular side-effects
(10, 11, 12). In contrast, the antiinflammatory/immunosuppressive effects
of GC involve trans-repression of target genes not
containing any GR-binding sites (2, 3, 9, 13). The human interleukin-2
(IL-2) gene is the prototype of a key immune gene that is repressed by
GC (14, 15, 16). GC-mediated repression of IL-2 gene expression is thought
to be due to direct interaction of GR with other transcriptional
enhancers, such as activating protein-1 (AP-1) and nuclear factor-
B
(NF-B) (14, 15, 16, 17, 18, 19, 20).
Conventional GC do not dissociate trans-activation and trans-repression. Strategies to develop improved GC aim to maintain trans-repression of immune genes in the absence of significant trans-activation of GRE-dependent promoters. In the current study, we tested whether the progesterone receptor (PR) agonist medroxyprogesterone acetate (MPA), which also binds to GR (21, 22), would fulfill these criteria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral venous blood was drawn from 10 healthy volunteers, in 3 cases repeatedly for different experiments. Peripheral blood leukocytes were isolated using Ficoll-Isopaque density centrifugation (13). Cells were cultured in RPMI 1640 containing 10% FCS, antibiotics and 1 µg/mL phytohemaglutinin to improve transfection efficiency, as recently described by Hughes and Pober (23). After 20 h in culture, the lymphocyte-enriched population of nonadherent cells was collected and used for transfection.
Jurkat human lymphoma T cells and HeLa cells were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 or DMEM (Life Technologies, Inc.-BRL, Gaithersburg, MD) with 10% FCS and antibiotics, and passaged twice weekly.
Plasmids
The pGL3-IL-2-luciferase construct (described in Ref. 24)
contains a 595-bp fragment of the human IL-2 gene (-548/+47) that has
been shown to mediate full induction and repression of IL-2 gene
transcription (14, 15, 16). The IL-2 promoter contains binding sites for
AP-1, NF-B, and NF-AT (nuclear factor of activated T cells), but no
GREs (14, 15, 16). pGL3-GRE-tk81-luciferase contains the GRE sequence of
the human tyrosine aminotransferase promoter linked to a minimal herpes
simplex virus type 1 thymidine kinase (tk) promotor (24).
The hPR-B expression vector (25) was a gift from Dr. P. Chambon.
pRShGR
contains the full-length coding region of the human GR
under the control of the constitutively active Rous sarcoma virus
promoter. The plasmid pRSv-erbA-1 that contains
a thyroid receptor complementary DNA in inverse orientation but is
otherwise similar to pRShGR was used as carrier DNA to yield a
constant amount of transfected DNA. Both plasmids were donated by Dr.
R. Evans (The Salk Institute, La Jolla, CA).
Transfection, luciferase assays, and enzyme-linked immunosorbent assays (ELISAs)
Normal human lymphocytes and Jurkat cells were transfected by electroporation (250 V; capacitance, 960 µF). Jurkat cells were electroporated with 10 µg reporter vector and 5 µg total expression vector/8 x 106 cells in a volume of 400 µL. The highest transfection efficiency for normal lymphocytes was achieved by electroporating 50 µg reporter vector/5 x 106 cells in a volume of 250 µL. After transfection, cells were plated in 12-well plates at a density of 500,000 cells/well. After 12 h, cells were stimulated with phorbol ester [tetradecanoyl phorbol acetate (TPA); 0.5 x 10-7 mol/L; Sigma Chemical Co., Deisenhofen, Germany] and ionomycin (1 µg/mL; Sigma Chemical Co.) in the absence or presence of dexamethasone (2.5 x 10-7 mol/L), hydrocortisone (2.5 x 10-7 mol/L), MPA (2.5 x 10-7 mol/L), or progesterone (2.5 x 10-7 mol/L). After 8 h, cells were lysed with a reporter lysis buffer (Promega Corp., Madison, WI), and luciferase activity in the lysate was determined in a luminometer (Lumat LB 9501, Berthold, Wildbad, Germany). HeLa cells were transfected by lipofection as previously described (26).
IL-1, IL-2, and IL-6 concentrations in cell culture supernatants were determined using specific ELISAs (Genzyme, Cambridge, MA) according to the manufacturer’s instructions.
All experiments were performed at least twice, in triplicate each time. The transfection experiments in normal lymphocytes were performed at least 10 times. Statistical analysis (unpaired t test) was performed using Macintosh StatView software (SAS Institute, Cary, NC).
RT-PCR and Southern hybridization
Total ribonucleic acid (RNA) from normal human lymphocytes, Jurkat cells, HeLa cells (positive control for GR), T47D cells (positive control for PR), and normal human testis [positive control for androgen receptor (AR)] was extracted using standard methods and quantified by UV absorption. Complementary DNA was synthesized from 5 µg total RNA with 100 U SuperScript polymerase (Life Technologies, Inc.), using oligo(deoxythymidine) primers (Life Technologies, Inc.). PCR was carried out with 7 pmol 5'- and 3'-primers, 0.2 mmol/L deoxy-NTPs, and 0.5 U Taq polymerase (Genecraft, Germany) in a reaction volume of 50 µL. Initial denaturation was performed for 2 min at 95 C, followed by two cycles each at 68, 66, 64, and 62 C and 35 cycles at 60 C (annealing temperature). Primer sequences were as follows: human GR sense primer, 5'-TGG GGT AAT TAA GCA AGA GA-3'; human GR antisense primer, 5'-AGA TCA GGA GCA AAA CAC AG-3'; human PR sense primer, 5'-AGC CAG AGA TTC ACT TTT TCA C-3'; human PR antisense primer, 5'-TCA TCC GCT GTT CAT TTA GTA TTA-3'; human AR sense primer, 5'-CCC CGA GAG AGG TTG CGT CCC-3'; and human AR antisense primer, 5'-CTC CAA CGC CTC CAC ACC CAG-3'. Glyceraldehyde-3-phosphate dehydrogenase-specific primers were used as a positive control and to exclude contamination with genomic DNA. PCR products were electrophoresed in a 2.0% agarose gel and visualized by UV light.
Southern blotting of the RT-PCR products was performed by standard methods, using a Hybond N+ membrane (Amersham Pharmacia Biotech, Braunschweig, Germany). Blots were hybridized with a digoxigenin-labeled GR- or PR-specific complementary DNA probe, which was generated by PCR, using the Boehringer DIG-PCR Labeling Kit (Roche Molecular Biochemicals, Mannheim, Germany). Signals were visualized by incubation with antidigoxigenin alkaline phosphatase-conjugated antibodies (Boehringer Mannheim, Mannheim, Germany), subsequent application of CDP-Star luminescence substrate (Boehringer Mannheim), and exposure to Hyperfilm ECL films (Amersham Pharmacia Biotech).
PAGE and Western blotting
Cells were harvested in ice-cold sample buffer b1 [50 mmol/L
Tris (pH 6.8), 1% SDS, and 10% sucrose]. Protein concentrations were
determined following standard protocols and using BSA protein standards
diluted with sample buffer b1. Samples were diluted in a 1:1 mixture of
sample buffer b1 and b2 [containing 50 mmol/L Tris (pH 6.80, 3% SDS,
10% sucrose, 10% ß-mercaptoethanol, and 0.01% bromophenol blue]
in a final volume of 100 µL and a final protein concentration of 300
µg/mL. Electrophoresis was performed in a 10% polyacrylamide
separating gel and a 3% stacking gel. Proteins were transferred to a
polyvinylidene difluoride membrane (Immobilon P, Millipore Corp., Eschborn, Germany). Membranes were stained with Ponceau S
to determine transfer efficiency and homogeneity of protein lanes.
After destaining with TBS [Tris-buffered saline: 20 mmol/L Tris-HCl,
(pH 7.6), and 137 mmol/L NaCl] for 5 min, membranes were incubated
overnight at 4 C in blocking solution [0.1 mol/L maleic acid (pH 7.5),
0.15 mol/L NaCl, 0.005% thimerosal, and 1% blocking reagent;
Roche Molecular Biochemicals]. Membranes were washed for
10 min in TBST (TBS plus 0.05% Tween-20) and incubated with the
primary antibody. The polyclonal rabbit antihuman GR antibody was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA), and used at a dilution of 1:500 in 9:1 TBST-blocking solution. The
monoclonal mouse antihuman PR antibody was obtained from Santa Cruz Biotechnology, Inc., and used at a dilution of 1:500. Blots
were incubated for 1 h at room temperature, washed three times for
10 min each time in TBST, and incubated with the IgG-peroxidase-labeled
second antibody (1:1000; Sigma Chemical Co.) for 1 h
at room temperature. The second antibody was visualized by enhanced
chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech) and Hyperfilm ECL films (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal human peripheral blood lymphocytes were transfected with a
human IL-2 luciferase construct, using a recently developed
electroporation protocol. Stimulation of transfected cells with the
phorbol ester TPA and the calcium ionophore ionomycin caused a
267.2 ± 27.5-fold increase in luciferase activity (mean ±
SD; Fig. 1a
). Both 2.5
x 10-7 mol/L hydrocortisone and 2.5 x
10-7 mol/L dexamethasone caused a significant
reduction of TPA/ionomycin-induced luciferase activity in most
experiments [22.9 ± 3.6% inhibition (P = 0.024)
and 38.4 ± 10% inhibition (P = 0.006),
respectively]. Trans-repression of IL-2 promoter activity
in response to 2.5 x 10-7 mol/L MPA was at
least equal to that produced by dexamethasone (up to 73.3 ± 7.2%
repression; P = 0.0005), whereas the natural progestin
progesterone had no significant effect (P = 0.47). The
androgen dihydrotestosterone did not cause any significant effect
(data not shown). The trans-repressive effect of MPA was not
influenced by the presence of estradiol (not shown). The
trans-repressive effects of dexamethasone or MPA could be
partially reversed (30–50% reduction of trans-repressive
activity) by the addition of RU 486, which by itself did not
significantly suppress IL-2 promoter activity (data not shown).
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Transfection of normal lymphocytes with a GRE-driven luciferase
construct in normal human lymphocytes resulted in 6.4 ± 0.5-fold
(P = 0.001) and 8.2 ± 0.4-fold (P
= 0.0002) induction of promoter activity in response to 2.5 x
10-7 mol/L hydrocortisone or 2.5 x
10-7 mol/L dexamethasone, respectively (Fig. 1b
). In contrast, 2.5 x 10-7 mol/L MPA had
only a marginal effect on GRE-dependent promoter activity (2.4 ±
0.4-fold induction; P = 0.008). Progesterone did not
influence luciferase activity to a significant extent
(P = 0.28). Therefore, a
trans-repression/trans-activation ratio can be
calculated for MPA. This ratio is 1.0 for the standard glucocorticoid
dexamethasone (trans-repression =
1.0/transactivation = 1.0). As trans-repression induced
by MPA is 1.91 times stronger than that induced by dexamethasone in a
typical experiment, and as MPA-mediated trans-activation is
only 29% (0.29) of that caused by dexamethasone, the
trans-repression/trans-activation ratio for
MPA is 6.6 (trans-repression =
1.91/transactivation = 0.29).
MPA suppresses IL-2 and IL-6 production by normal human lymphocytes in a dose-dependent manner
TPA/ionomycin-induced IL-2 release in normal human lymphocytes, as
determined by an IL-2-specific ELISA, was strongly reduced by 2.5
x 10-7 mol/L hydrocortisone or 2.5 x
10-7 mol/L dexamethasone (69.4 ± 4.4% and
78.8 ± 2.3% inhibition, respectively) and somewhat less by
2.5 x 10-7 mol/L MPA (62.4 ± 6.9%
inhibition), indicating more pronounced posttranscriptional effects of
the classic GC (not shown). Progesterone inhibited IL-2 release by
9 ± 11.7% (not shown). In dose-response experiments, both
dexamethasone and MPA significantly suppressed IL-2 release at
concentrations of 10-7 mol/L or more (Fig. 2a). Similar results were obtained for
IL-6 (Fig. 2b) and IL-1 (not shown).
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To determine whether the dissociative effect of MPA depends on the
GR expression level, normal lymphocytes were cotransfected with the GRE
reporter construct and the GR expression vector. The results of
these experiments are shown in Fig. 3a.
Trans-activation in response to MPA was significantly
stronger in GR-transfected compared to mock-transfected lymphocytes
[hydrocortisone, 17.2 ± 2.1-fold vs. 6.4 ±
0.5-fold stimulation (P = 0.005); dexamethasone,
18.7 ± 2.3-fold vs. 8.2 ± 0.4-fold
(P = 0.007); MPA, 12.5 ± 2.1-fold vs.
2.4 ± 0.4-fold (P = 0.006); progesterone,
2.8 ± 0.7-fold vs. no effect (P =
0.02)]. This effect was observed at all hormone concentrations tested
(10-9–10-5 mol/L; not
shown).
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). In HeLa cells
transfected with the GRE reporter plasmid only, MPA-induced
trans-activation was weak. Again, the
trans-activating effect of MPA (and the other steroids) was
dramatically enhanced in cells cotransfected with GR
[hydrocortisone, 35.8 ± 6.2-fold vs. 7.8 ±
1.4-fold stimulation (P = 0.0099); dexamethasone,
39.9 ± 3.1-fold vs. 11.1 ± 1.4-fold
(P = 0.00045); MPA, 33.4 ± 1.4-fold
vs. 2.1 ± 0.1-fold (P = 0.00006);
progesterone, 1.7 ± 0.2-fold vs. no stimulation
(P = 0.0016)].
To determine whether the effects of receptor cotransfection are related
to the level of GR expression, Jurkat T lymphoma cells, which do not
express functional GR endogenously, were transfected with the above
reporter plasmids and with different amounts of GR expression
vector. In these experiments, the maximum trans-activating
effect of MPA reached that of dexamethasone only at maximum GR
concentrations (10 µg expression vector/8 x
106 cells), but not at 2 and 5 µg transfected
GR expression vector, where it was below 50% of maximum
dexamethasone-induced trans-activation (not shown).
These results confirm the importance of using cells that express physiological levels of GR when analyzing potentially dissociative GC (see also Discussion).
GR, PR, and AR mediate the effects of MPA in Jurkat T lymphoma cells
To test whether the dissociative GC-like effects of MPA
(i.e. strong trans-repression, weak
trans-activation) observed in normal human lymphocytes could
also be mediated by other steroid receptors, such as PR and AR, Jurkat
T lymphoma cells were transfected with the respective expression
vectors and the luciferase constructs indicated above (Fig. 4).
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In Jurkat cells transfected with the GR plasmid, IL-2 promoter
repression rates were as follows (hormone concentration = 2.5
x 10-7 mol/L in all experiments):
hydrocortisone, 69 ± 6.9% (P = 0.007);
dexamethasone, 69.4 ± 6.2% (P = 0.007); MPA,
67.6 ± 5.2% (P = 0.008); progesterone, 30.6
± 11.4% (P = 0.08; Fig. 4a). In some experiments, the
repressive effect of progesterone on IL-2 promoter activity reached
significance. The GRE construct was induced 12.6 ± 0.8-fold by
hydrocortisone (P = 0.002), 10.9 ± 1.7-fold by
dexamethasone (P = 0.004), 5.8 ± 0.7-fold by MPA
(P = 0.004; P(dexamethasone vs.MPA) = 0.0095), and 1.8 ± 0.1-fold by
progesterone (P = 0.0008) in GR-transfected Jurkat
cells (Fig. 4b).
In PR-transfected Jurkat cells, hydrocortisone repressed the IL-2
promoter by 38.6 ± 7.3% (P = 0.0086),
dexamethasone by 8 ± 18% (P = 0.19), MPA by
73.1 ± 0.6% (P = 0.0007), and progesterone by
59 ± 8.5% (P = 0.001; Fig. 4a). The GRE reporter
plasmid was induced 2.1 ± 0.1-fold by hydrocortisone
(P = 0.007), 1.1 ± 0.1-fold by dexamethasone
(P = 0.25), 4.4 ± 0.6-fold by MPA
(P = 0.006), and 6.5 ± 1.7-fold by progesterone
(P = 0.007; Fig. 4b).
In summary, trans-repression of the IL-2 construct by
MPA was similar in normal human lymphocytes and in GR-expressing Jurkat
cells. MPA also suppressed the IL-2 construct in PR-transfected Jurkat
cells, indicating that both GR and PR are able to mediate this effect.
Compared to normal lymphocytes, the dissociative effect of MPA was less
pronounced, yet still highly significant in GR-expressing Jurkat
cells (trans-repression/trans-activation ratio,
1.83). Finally, the AR also mediated the effects of MPA in Jurkat cells
(not shown).
GR, but not PR, messenger RNA (mRNA) and protein are expressed in normal human lymphocytes
To confirm that the dissociative effects of MPA on
trans-repression and trans-activation in normal
human lymphocytes are mediated by GR, we studied the expression of GR,
PR, and AR in these cells at both the mRNA (RT-PCR) and protein
(Western blot) levels. As shown in Fig. 5a, GR mRNA
was expressed in normal lymphocytes of male and female donors and in
Jurkat cells. The correct sequence of the amplification products was
confirmed by Southern hybridization. Using a highly sensitive touchdown
protocol, no band was obtained with the PR-specific primers. When the
number of cycles was increased to 40 or more, a faint and inconsistent
band was obtained in human lymphocytes and Jurkat cells, indicating
extremely low, if any, expression of PR mRNA in these cells. AR mRNA
was weakly expressed in all samples tested.
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. GR
protein was detectable in all protein extracts from normal human
lymphocytes. Surprisingly, untransfected Jurkat cells expressed
more immunoreactive protein than normal lymphocytes. As untransfected
Jurkat cells do not respond to GC, and transfection with a GR
expression vector can reestablish GC responsiveness (see above), the GR
protein present in these cells must be defective. Consistent with the
PCR experiments, no PR protein was detectable in any of the lymphocyte
preparations. As opposed to AR mRNA, no AR protein was detectable in
human lymphocytes. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As early as 1965, Hulka et al. reported that progestins can exert immunosuppressive effects (27). Later, it became clear that progesterone can inhibit T cell responsiveness, and this effect was thought to be important in tolerance of the fetal allograft (28). However, the immunosuppressive action of progesterone itself is rather weak, as indicated by the massive doses that are needed to exert this effect (29). There are few reports analyzing the effects of the synthetic progestin MPA on the immune system. Mallmann et al. observed decreased T cell numbers in breast cancer patients who received high doses of MPA (30). Another group showed that MPA could inhibit lymphocyte proliferation in vitro (21). Our study is the first to analyze the effects of MPA on IL-2 gene expression and thus represents the first quantitative approach to the immunosuppressive action of MPA at the molecular level. Surprisingly, we found that the suppressive effect of MPA on IL-2 gene expression is as strong as that of the classic glucocorticoids, dexamethasone and hydrocortisone. IL-1 and IL-6 were also strongly suppressed by MPA. In contrast, progesterone did not exert any immunosuppressive effect at physiological concentrations in our study.
Whether the immunosuppressive effects of progestins are mediated by PR or other steroid receptors has been a matter of dispute. Indeed, PR expression in human lymphocytes, which would be a prerequisite for any PR-mediated effect, is controversial (29, 31). Using a highly sensitive touchdown PCR protocol, we did not detect any PR mRNA in unstimulated or stimulated human lymphocytes under the conditions described. Furthermore, we did not detect any PR protein in these cells by Western blotting. We conclude that if PR is expressed in human lymphocytes, the levels are far too low to exert any significant effect. In contrast, we and others have shown that GR mRNA and protein are clearly expressed in human lymphocytes. It has also been shown that MPA can bind to GR (21, 22). Finally, Jurkat cells, which are devoid of functional GR, are rendered MPA sensitive by transfection of a GR expression vector. AR does not play a role in mediating the effects of MPA in human lymphocytes, as its expression is below the detection limit of our Western blot, and dihydrotestosterone, which is a much stronger AR agonist than MPA, has no effect on IL-2 promoter activity in these cells.
The most important finding in our study is that MPA can dissociate
between trans-repression and trans-activation in
normal human lymphocytes. It is noteworthy that this effect is most
pronounced in lymphocytes expressing normal levels of GR endogenously.
MPA-induced trans-activation was much stronger in both
lymphocytes and HeLa cells transfected with a GR expression vector.
These data are consistent with findings reported by Szapary et
al., who showed that the percentage of maximal GC-induced
trans-activation is dramatically increased in transiently
GR-transfected cells (32). Furthermore, antiglucocorticoids can be
converted to GC under these conditions. Both our data and the study by
Szapary et al. underline the importance of using cells that
express normal levels of GR when analyzing potentially dissociative GC
and/or antiglucocorticoids.
It has been the long-standing goal of pharmacological research to develop GC that dissociate trans-repression and trans-activation, because many of the side-effects of conventional GC can be attributed to trans-activation (10, 11, 12). In 1997, Vaysierre et al. reported that a novel class of synthetic GC could differentiate between transactivation and AP-1 trans-repression (33). We now demonstrate similar effects of MPA. As opposed to the novel agents described by Vaysierre et al., the clinical experience with MPA is extensive; it has been used for the treatment of metastatic endometrial, breast, and renal cancer. Consistent with our in vitro findings, the development of Cushingoid features is less pronounced than that in patients treated with conventional GC and only occurs at excessive doses (34, 35). It has even been shown that MPA can reverse one of the most devastating side-effects of conventional GC, the development of osteoporosis (36). We conclude that MPA is a highly promising substance for the treatment of autoimmune/inflammatory diseases, and that our data justify further studies in animals and humans affected with these diseases.
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Acknowledgments |
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Footnotes |
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Received January 28, 1999.
Revised May 20, 1999.
Revised July 1, 1999.
Accepted July 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by activated glucocorticoid receptors. Mol
Cell Biol. 15:943–953.[Abstract]
and ß isoforms of the glucocorticoid receptor. Mol Cell
Endocrinol. 131:233–240.[CrossRef][Medline]
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