©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Negative Interaction between the RelA(p65) Subunit of NF-B and the Progesterone Receptor (*)

(Received for publication, September 12, 1995; and in revised form, December 26, 1995)

Eric Kalkhoven(§)(¶) Sacha Wissink (§) Paul T. van der Saag Bart van der Burg (**)

From the Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interactions between transcription factors are an important means of regulating gene transcription. The present study describes the mutual repression of two transcription factors, the RelA(p65) subunit of NF-kappaB and the progesterone receptor (PR). This trans-repression is shown to occur independent of PR isoform, reporter construct, or cell type used. Together with the demonstration of an interaction between PR and RelA in vitro, these findings suggest that the mutual repression is due to a direct interaction between these proteins. Furthermore, activation of NF-kappaB by tumor necrosis factor-alpha also results in repression of PR, while PR is able to repress tumor necrosis factor-alpha-induced NF-kappaB activity. Since NF-kappaB-regulating cytokine receptors are expressed in progesterone target tissues, like breast and endometrium, the mutual repression of PR and RelA could play an important role in a wide variety of physiological processes in these tissues, including maintenance of pregnancy, immunosuppression, and tumorigenesis.


INTRODUCTION

The human progesterone receptor (PR) (^1)belongs to the superfamily of steroid/thyroid hormone receptors(1, 2, 3) . The transcription factors of this family share (at least) two structurally related functional domains, the DBD, which contains the so-called zinc finger motif, and the more C-terminally located HBD. Two transactivation domains have been mapped in the PR, of which one is located N-terminal to the DBD. This transactivation domain, named AF-1, functions autonomously, but the level of activity depends on the cell type and reporter construct used(4, 5) . The second transactivation domain AF-2 lies within the HBD, and its activity is strictly dependent on the presence of ligand(4) . Detailed analysis of the function of these transactivation domains of the PR has also led to more insight in anti-hormone action. Anti-progestins bind to the receptor without activating AF-2, and the partial agonistic activity evoked by the anti-progestin RU486 is therefore the result of its stimulation of AF-1 activity(4, 5) .

Within the superfamily of steroid receptors, PR is unique in that it exists in two isoforms, named A and B(6) , which differ in their N terminus. Differences in transcriptional activity between the PR(B) (amino acids 1-933) and PR(A) (amino acids 164-933) isoforms have been observed, depending on the cell type and the reporter construct used(4, 5) .

Functioning of transcription factors can be modified by interplay with transcription factors of a different type, resulting in either an inhibitory or stimulatory effect. In particular, interactions between steroid receptors and members of the AP1 family of transcription factors have been studied extensively. AP1 family members, like c-Jun and c-Fos, and GR have been shown to repress each others functioning (7, 8, 9, 10, 11) , but the actual mechanism of repression is currently controversial. In vitro, a direct interaction between GR and AP1 was shown, resulting in impaired DNA binding(7, 9) , while others failed to detect a direct interaction (10, 11) or found the two proteins to interact without DNA binding being affected(8, 12) . It has clearly been demonstrated that the magnitude of the repression of AP1 by GR and PR is cell type- and promoter-specific(11, 13, 14) , suggesting that intermediary proteins are likely to be involved, the expression of which can vary between different cell types. The transcriptional activity of PR was shown to be affected by c-Jun in a stimulatory or an inhibitory fashion, depending on the cell type(11) , while c-Fos was shown to inhibit PR in all cases(11) . Recently, GR (15, 16, 17, 18) , and also estrogen receptor(19, 20) , have been shown to be inhibited by members of a second family of transcription factors, the NF-kappaB family. These repressions were found to be mutual, and GR (15, 17, 18) and estrogen receptor (20) were shown to interact physically with NF-kappaB proteins in vitro. Several studies have indicated that, in the case of GR, this interaction was shown to result in impaired DNA binding(15, 16, 17) , but others failed to find this (21) .

The NF-kappaB family consists of DNA-binding proteins that share homology in an N-terminal region of 300 amino acids, termed the Rel homology region. Amongst others, this family includes RelA(p65), NFKB1(p50), and c-Rel(22, 23) . NF-kappaB exists as a dimer, typically a heterodimer of RelA and NFKB1(24) , but homodimers as well as heterodimers of different composition are also possible. NF-kappaB is present in an inactive form in the cytoplasm, where it is associated to an inhibitory protein, IkappaB. Exposure of cells to a plethora of stimuli, including cytokines (TNF-alpha and IL-1), lipopolysaccharide, UV radiation, and oxidative stress, results in the dissociation of IkappaB from the NF-kappaB complex, probably through a phosphorylation event(25) , upon which NF-kappaB translocates to the nucleus. Subsequently, binding to specific DNA sequences and activation of transcription can occur. For some members of the NF-kappaB family, like RelA (26) and c-Rel(27, 28) , C-terminally located transactivation functions have been found.

Since NF-kappaB sites have been identified in the promoters of numerous genes that play a role in cell proliferation and immune response(22, 23) , and since progestins also play a role in these processes(29, 30) we have investigated the effects of NF-kappaB family members and PR on each others transcriptional activity. Here we show the RelA subunit to specifically inhibit the hormone-induced transactivation of PR, independent of receptor isoform, reporter construct, or cell type used. Furthermore, the repression is mutual, since PR is shown to repress the transcriptional activity of RelA. Both the DBD and the HBD of PR are shown to contribute to the repression. This trans-repression between RelA and PR could play an important role in a large variety of processes like maintenance of pregnancy, immunosuppression, and tumorigenesis.


EXPERIMENTAL PROCEDURES

Materials

A phenol red-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DF) was obtained from Life Technologies, Inc. FCS was purchased from Integro (Linz, Austria), and bovine serum albumin was from Sigma, recombinant human TNF-alpha was from Boehringer Mannheim. Trypsin and EDTA used for cell culture were obtained from Flow Laboratories (Irvine, UK). The progestin Org2058 was provided by Organon International (Oss, The Netherlands), the anti-progestins RU486 and ZK98299 were obtained from Roussel-Uclaf (Romainville, France) and Schering AG (Berlin, Germany), respectively. Monoclonal antibody against the AB region of PR (AB-52; 32) was a kind gift of Dr. K. B. Horwitz (Denver, CO), while monoclonal antibody against the E region (C262; 33) of PR was purchased from StressGen (Victoria, Canada). The polyclonal antibody against the N terminus of RelA (SC-109) was purchased from SantaCruz (Santa Cruz, CA). Dextran-coated charcoal-FCS was prepared by treatment of FCS with dextran-coated charcoal to remove steroids, as described previously(31) . HeLa 229 and COS-1 cells were obtained from American Type Culture Collection (Rockville, MD); T47D cells were originally provided by Dr. R. L. Sutherland (Sydney, Australia). Cells were cultured in bicarbonate-buffered DF medium containing phenol red, supplemented with 7.5% FCS in a 7.5% CO(2) humidified atmosphere.

Plasmid Constructs

The pSG5 expression vector containing human PR(B) has been described previously(34) . To create an expression vector containing the human PR(A) isoform, PCR was performed with a primer containing the second ATG in the coding region of the human sequence and a Kozak sequence (5`-ggaattcgatatccacCATGAGCCGGTCCGGGTGCAA-3`) and 5`-ggaattcGAGGCAGGATAGGCACGTGG-3` as reverse primer. This fragment was exchanged for the corresponding region in the pSG5-PR(B) construct using unique EcoRI and BalI sites. The DeltaE construct of PR(B) lacks amino acids 674-933 (numbering refers to original cDNA(34, 35) , originally designated hPR5(5) , was a kind gift of Dr. Gronemeyer (Strasbourg, France). The DeltaAB1 construct was generated by cutting the PR(B) cDNA with BamHI and SauI and religating it in the presence of the oligonucleotide 5`-GGATCCTATCTCAACTACCTGAGG-3`, and it lacks amino acids 26-537. The DeltaAB2 construct, lacking amino acids 26-453, was generated by cutting with BamHI and HincII, filling-in, and religation. The DeltaAB3 construct, lacking amino acids 252-489, was generated by digestion with SacII and religation. The DeltaC construct of PR(B) was made by introducing two EcoRI sites into the original cDNA by site-directed mutagenesis (5`-CCTGAGGCCGAATTCAGAAGC-3` and 5`-AGTCAGAGTTGTGAATTCACTGGATGCTGTTG-3`). As a consequence of the mutagenesis, amino acid 548 is changed from Asp to Asn and amino acid 650 from Ala to Ser. By cutting out the 300-base pair EcoRI fragment, amino acids 549-649 are lacking.

The CMV-4 expression vectors containing the cDNAs encoding human RelA, NFKB1, and c-Rel have been described before(18) . The DeltaTA1 construct of RelA, lacking amino acids 515-550 (numbering refers to original cDNA(37) ), was made by cutting the cDNA at a unique SmaI site and ligating it into CMV-4.

The reporter plasmids PRE(2)tkCAT(38) , MMTVCAT(39) , and PRE(2)TATACAT (40) were kind gifts of Drs. Muller (Martinsried, Germany), Evans (San Diego, CA), and Gronemeyer (Strasbourg, France), respectively. The luciferase reporter containing three NF-kappaB sites from the ICAM-1 promoter(21) , and PDMLacZ (41) have been described before.

GST-PR (amino acids 457-933) was made by cutting the PR(B) cDNA at an internal HincII site and a BamHI site in the pSG5 vector and, after filling-in, ligated into SmaI-cut PGEX-2T vector (Pharmacia, Uppsala, Sweden). GST-PRDeltaC was cloned similarly, using pSG5-PR(B)DeltaC to isolate the HincII-BamHI fragment. To create GST-NFKB1, PCR was performed with 5`-tcccccgggcaccATGGCAGAAGATGATCC-3` as forward primer and T3 as reverse primer on the SK-NFKB1 vector. This fragment was cut with SmaI and ligated into SmaI-cut PGEX-2T. For in vitro translation, the coding region of RelA was cut from the CMV4-RelA vector, using XbaI and HindIII sites outside the coding region, and cloned into XbaI-HindIII-cut pBluescript SK.

Transient Transfections

Cells were cultured in six-well tissue culture plates in DF+, supplemented with 5% dextran-coated charcoal-FCS at a density of 2 times 10^4/cm^2 or 3 times 10^4/cm^2 for T47D cells. Cells were transfected by calcium phosphate co-precipitation using 5 µg of CAT reporter or 2 µg of luciferase reporter, 3 µg of PDMLacZ plasmid, and 100 ng of eukaryotic expression plasmid pSG5 containing the PR cDNAs or CMV-4 plasmids containing the NF-kappaB cDNAs. pBluescript SK plasmid was added to obtain a total amount of 10 µg of DNA/well. After 16 h, or 6 h in the case of T47D cells, the medium was refreshed and (anti-) hormones were added. Cells were harvested 24 h later and assayed for CAT activity (34, 42) or luciferase activity (18) as described. Values were corrected for transfection efficiency by measuring beta-galactosidase activity (43) .

In Vitro Binding Assays

Recombinant RelA in pBluescript SK was transcribed and translated in vitro in reticulocyte lysate (Promega, Madison, WI) in the presence of [S]methionine according to the manufacturer's description. GST-fusion vectors were transformed into Escherichia coli BL21. Expression and purification with glutathione-coated Sepharose beads (Pharmacia) was performed as described previously(44) . The fusion proteins loaded on Sepharose beads were subsequently incubated with in vitro translated protein in NETN (20 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol) containing protease inhibitors (0.5 mg/ml bactracin, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 2 mM phenylmethylsulfonyl fluoride) for 2 h in the absence or presence of 1 µM Org2058. Beads were washed 4 times with NETN, dried under vacuum, resuspended in sample buffer, and analyzed by SDS-polyacrylamide gel electrophoresis. Finally, gels were dried and, after amplification by fluorography (Amplify, Amersham Corp.), exposed at -70 °C.

Western Blotting

COS-1 cells were transfected as described above. For hormone-dependent phosphorylation of PR, cells were treated with Org2058 (10 nM) for 4 h. Subsequently, cells were harvested immediately in SDS sample buffer. Samples were separated on 8% SDS-polyacrylamide gels and transferred onto Immobilon (Milipore, Bedford, MA). Blots were blocked with Blotto (phosphate-buffered saline containing 4% nonfat died milk powder and 0.05% Tween 20) for 1 h. All subsequent steps were carried out in Blotto/phosphate-buffered saline (1:1). Blots were probed with monoclonal antibodies AB-52 or C262 against PR or the SC-109 antibody against RelA. After vigorous washing, blots were incubated with peroxidase-conjugated antibodies (1:10,000; Amersham Corp.). Subsequently, blots were washed again and immunoreactive bands were visualized with ECL (Amersham Corp.).

RNA Isolation and Northern Blotting

Total RNA was isolated by the acid-phenol method(45) . Northern blotting and (pre-) hybridization with the fatty acid synthase cDNA (46) was carried out as described previously(47) . For control hybridizations, a rat glyceraldehyde 3-phosphate dehydrogenase cDNA was used(48) . Probes were labeled with [alpha-P]dCTP using the Multiprime DNA labeling system (Amersham Corp.). Final (most stringent) washing was done in 0.1 times SSC, 0.1% SDS at 68 °C. Subsequently filters were exposed for autoradiography and quantified using a Molecular Dymanics PhosphorImager with Image Quant software.


RESULTS

Mutual Repression of PR and RelA

To study the effects of NF-kappaB on progesterone-mediated gene transcription, we transfected HeLa cells with a reporter construct containing two progesterone responsive elements (PREs) in front of the thymidine kinase promoter coupled to CAT, in combination with expression constructs expressing the A and B isoform of the human PR and expression constructs containing the RelA(p65) subunit of NF-kappaB. As shown in Fig. 1A, RelA is clearly capable of repressing the transcriptional activity of PR(A) and PR(B) induced by the synthetic progestin Org2058 (10 nM). Only marginal repressive effects of RelA were observed in the absence of hormone (data not shown). The repression of activated PR is specific for RelA, since co-transfection with other NF-kappaB family members, like NFKB1(p50) or c-Rel, did not affect the activity of PR(A) or PR(B) (Fig. 1B).


Figure 1: Repression of the hormone-induced activity of the human PR by RelA. A, HeLa cells were transiently transfected with 100 ng of PR(A) (white bars) or PR(B) (black bars) and 1.0 µg of empty expression vector (CMV-4; -), 0.5 µg of empty vector and 0.5 µg of RelA or 1.0 µg of RelA. PRE(2)tkCAT (5 µg) was used as reporter construct. Fold induction indicates reporter activity in cells treated with 10 nM Org2058 over untreated cells. Bars represent the mean of four to five independent experiments ±S.E. B, HeLa cells were transfected with PR(A) (white bars) or PR(B) (black bars), as under A, but now with 1.0 µg of empty expression vector (CMV-4; -) or the same vector containing the RelA, NFKB1, or c-Rel cDNAs, as indicated. C, cells were transiently transfected as under A, using MMTV-CAT (5 µg) as reporter construct.



Next, transient transfections were performed with a reporter construct containing the MMTV promoter, which contains numerous PREs(38) . As shown before(5) , a marked difference between the activities of PR(A) and PR(B) is observed, with PR(A) being a weak activator of this reporter construct (Fig. 1C). Again, hormone-induced transactivation by both PR(B) and PR(A) is repressed by RelA (Fig. 1C). Similar results were obtained when a reporter construct was used in which two PREs are located directly upstream of a TATA box (data not shown). We conclude therefore that the RelA subunit of NF-kappaB is capable of repressing both PR(A) and PR(B), independent of the context of the PRE.

To examine whether PR is also able to repress RelA, HeLa cells were transfected with a reporter construct containing three NF-kappaB sites from the human ICAM promoter(40) , coupled to the thymidine kinase promoter in front of luciferase(21) . Co-transfection with RelA expression vector (100 ng) resulted in a modest but significant activation of this construct (Fig. 2). When expression vectors containing PR(A) or PR(B) (1 µg) were added, the RelA-mediated activity was already reduced in the absence of hormone, while hormone addition resulted in a further repression (Fig. 2). Similar results were obtained when 10-fold lower amounts of RelA and PR expression vectors were used (data not shown). Taken together, these results show that the repression between PR and RelA is mutual and occurs on a variety of promoters.


Figure 2: PR represses RelA-mediated transactivation. HeLa cells were transiently transfected with 100 ng of RelA, 1.0 µg of empty expression vector (pSG5; -), 0.5 µg of empty vector, and 0.5 µg of PR(A) or PR(B), or 1 µg of the PR constructs. 3xNF-kappaBtkLuc (2 µg) was used as reporter construct. Cells were treated with vehicle (white bars) or with 10 nM Org2058 (black bars), harvested and luciferase activity was measured. Depicted is the induction of luciferase activity evoked by RelA over cells transfected with empty expression vector.



Cell Type Specificity of the Mutual Repression of PR and RelA

Since the mutual repression between PR and RelA could depend on cell type-specific factors, transient transfection assays were performed not only in HeLa cells but also in COS-1 cells and in the human breast tumor cell line T47D, which expresses high endogenous levels of both PR(A) and PR(B) in approximately equal amounts (data not shown). As was also shown in HeLa cells (Fig. 1), the transactivation potential of PR(A) and PR(B) is different in COS-1 cells (Table 1). In both cell lines, however, RelA clearly inhibited the hormone-induced transcriptional activity of both PR(A) and PR(B). RelA is also capable of repressing endogenous PR, as shown in T47D cells.



In addition, the effects of PR(A) and PR(B) on RelA-induced reporter activity were studied in the same cell lines. The activation by RelA was much greater in COS-1 cells than the modest activation in HeLa cells (Fig. 2), but also in these cells both PR isoforms inhibited RelA transactivation and progestin treatment resulted in a further repression of RelA-induced reporter activity (Table 1). Furthermore, endogenous PR also represses RelA, as shown in T47D cells. Although the transactivation and repression potential of RelA and the PR isoforms differ between cell lines, the mutual repression between these proteins is observed clearly in all cell lines tested.

Domains of PR and RelA Involved in the Mutual Repression

To identify the regions of PR involved in the negative cross-talk with RelA, deletion constructs of PR were used which lack (part of) the AB region, the C region, or the E region (Fig. 3A). Expression of these PR deletion constructs was found to be approximately equal, as detected on Western blots, using monoclonal antibodies directed against the AB region (AB-52; 31) or the E region (C262; 32) (data not shown). First, the effect of RelA on transactivation mediated by either PRDeltaAB1 or PRDeltaE was examined. Deletion of the E region results in higher basal activity of AF-1, when compared with unliganded intact receptor (data not shown, (5) ). The activity of PRDeltaE, which functions independent of hormone since it lacks AF-2, was still repressed by RelA (Fig. 3B). RelA also repressed the activity of PRDeltaAB1, which is strictly hormone-dependent since it only contains AF-2 (Fig. 3B). These findings suggest that not the two AFs of PR, but different domains are essential for the repression by RelA. To examine whether the same holds true for the repressor function of PR, the same PR constructs were used to examine their effect on RelA-induced activity of the 3xNF-kappaB reporter. For this, COS-1 cells were used, in which co-transfection of RelA results in a stronger activation of this reporter (Table 1) than in HeLa cells (Fig. 2). Unliganded PR and PR already repress RelA to some extent (Fig. 3C). This effect is lost when amino acids 26-537 of the AB region of PR are deleted (PRDeltaAB1; Fig. 3), but not when amino acids 26-453 (PRDeltaAB2) or 252-489 (PRDeltaAB3) are deleted. Therefore, hormone-independent repression by PR requires a region between amino acids 489 and 537, but not AF-1, since this encompasses amino acids 455-546(40) . All PR constructs mentioned above lower the transcriptional activity of RelA even more upon hormone treatment (Fig. 3C). When the HBD was deleted (PRDeltaE), the resultant receptor repressed RelA-induced transcription independent of hormone, to a level comparable with the hormone-independent repression by PR or PR (Fig. 3C), showing that the HBD is essential for hormone-dependent repression. Both the hormone-dependent and -independent repressive activity were lost when the DBD of PR was deleted (DeltaC). These findings show that while (part of) the AB domain and the E domain are required for hormone-independent and -dependent repression, respectively, both types of repression require the C domain.


Figure 3: Domains of PR and RelA involved in repression. A, schematic representation of PR deletion constructs. Numbers refer to original amino acid sequence(35, 36) . B, HeLa cells were transfected with 100 ng of expression vector containing PR(B)DeltaE or PRDeltaAB1 as indicated in the absence or presence of 1.0 µg of RelA expression vector. Assays were performed as described in Fig. 1A. Fold induction indicates reporter activity in cells transfected with expression vector containing PR(B)DeltaE over empty expression vector or reporter activity in cells treated with 10 nM Org2058 over untreated cells, in the case of PRDeltaAB1. C, COS-1 cells were transfected with 100 ng of RelA expression vector and 2 µg of 3xNF-kappaBtkLuc reporter, in the presence of various PR deletion constructs (1 µg). Cells were treated with vehicle (white bars) or with Org2058 (10 nM) and assayed for luciferase activity. D, HeLa cells were transiently transfected with 100 ng of PR(A) (white bars) or PR(B) (black bars) in the presence of 1 µg of empty expression vector (CMV-4; -), RelA, or RelADeltaTA1, using PRE(2)tkCAT as a reporter (5 µg). Depicted is the transactivation evoked by 10 nM Org2058.



To examine whether the transactivation function of RelA is essential for the repression of PR, a deletion construct of RelA was used that lacks the 30 most C-terminal amino acids, which encode transactivation domain TA1(26) . Only a marginal difference between the repressive activity on PR(A) and PR(B) of this construct compared with the full-length RelA protein was observed (Fig. 3D). Similar results were obtained in COS-1 and T47D cells (data not shown). Therefore, TA1 is not essential for the repressive activity of RelA.

Effects of Anti-progestins on the Mutual Repression of PR and RelA

Like progestins(11) , the anti-progestin RU486 has been shown to be capable of inducing PR-mediated repression of AP1(49) , while it has antagonistic and partial agonistic activity with respect to PR-mediated transcription, depending on the cell type and reporter construct used(4, 5) . Therefore, we used this anti-progestin to examine the mutual repression between PR and RelA in more detail. As described before(5) , RU486 behaved as a weak agonist on PR(B) when PRE(2)tkCAT is used as a reporter (Fig. 4A), while PR was unaffected ((5) ; data not shown). Co-transfection of RelA resulted in repression of the transcriptional activity of the RU486-occupied PR (Fig. 4A).


Figure 4: Effects of anti-progestins on PR-RelA interaction. A, HeLa cells were transfected with full-length PR(B) in the presence of CMV-4 or CMV-4/RelA as in Fig. 1A and treated with 1 µM RU486. PRE(2)tkCAT (5 µg) was used as reporter construct. B, COS-1 cells were transfected with RelA vector in the presence of empty expression vector (pSG5; -), PR(A), PR(B), or PRDeltaAB1, as in Fig. 3C, and treated with 1 µM RU486 (black bars) or 1 µM ZK98299 (striped bars). 3xNF-kappaBtkLuc was used as reporter construct.



To investigate whether antagonist-occupied PR could also inhibit RelA-mediated transcription, transfections in COS-1 cells were performed with the 3xNF-kappaB reporter described above. RU486 was able to induce PR(A)- or PR(B)-mediated repression of RelA (Fig. 4B), albeit to a lesser extent than the agonist Org2058 (Table 1). The RU486-evoked repression was more easily observed when the PRDeltaAB construct of PR is used (Fig. 4B), which lacks the hormone-independent repression observed with PR(A) and PR(B) (Fig. 3C). While RU486 only exhibits weak agonistic effects under certain specific conditions (Fig. 4A), the ``pure'' antagonist ZK98299 is unable to evoke PR-mediated transactivation (data not shown). Like RU486, this anti-progestin was able to repress RelA (Fig. 4B). Together with RelA-repression by RU486-occupied PRDeltaAB, which lacks AF-1 through which RU486 is thought to exert its agonistic action(5) , these results indicate that the enhancer and repressor functions of the two PR isoforms are separate functions. This hypothesis is confirmed by our findings that the transcriptional activity of the two PR isoforms differs in HeLa and COS-1 cells, while the repressive effect on RelA is independent of receptor-isoform and cell type (Table 1).

Direct Physical Interaction between PR and RelA in Vitro

To investigate the possibility that RelA and PR repress each others transcriptional activity via a direct physical interaction, the PR cDNA (amino acids 457-933) and the PR cDNA lacking the C region (PRDeltaC) were fused to GST. The complete coding region of NFKB1, a protein that can associate directly with RelA(24) , was also fused to GST. Upon overexpression in E. coli, GST-fusions were purified with glutathione-coated agarose beads(44) , and tested for their ability to bind in vitro translated, [S]methionine-labeled RelA. As shown in Fig. 5, the RelA protein could not be precipitated by GST alone (lane 2), while the GST-PR fusion protein clearly precipitated RelA (lane 3), indicating that the two proteins can bind directly to each other, while GST-PRDeltaC hardly bound RelA (lane 5). In all cases, the additional presence of hormone had no effect (lanes 4 and 6). Bacterially expressed NFKB1 was clearly capable of interacting with in vitro translated RelA (lane 7). Therefore, we conclude that at least in vitro PR and RelA are capable of physically interacting with each other. Furthermore, the C region of PR, which was shown to be essential for the repression of RelA (Fig. 3C), is also essential for the in vitro interaction with PR.


Figure 5: RelA and PR are capable of direct interaction. GST, GST-PR, GST-PRDeltaC, or GST-NFKB1, bound to glutathione-agarose beads, were incubated with radiolabeled in vitro translated RelA in the absence or presence of Org2058 (1 µM), as indicated. After extensive washing, proteins were loaded on a 10% SDS-PAA gel. In lane 1, one-tenth of the total input of in vitro translated RelA was loaded. Numbers on the right indicate molecular weight markers; arrowhead on the left indicates the position of radiolabeled RelA.



Effects of RelA on PR Phosphorylation

Since PR becomes sequentially hyperphosphorylated upon hormone and DNA binding, reflected by reduced mobility in SDS-polyacrylamide gels(50, 51) , the effect of RelA on this process was examined. COS-1 cells were transiently transfected with PR(A) or PR(B) in the absence or presence of RelA. Subsequently, cells were treated with Org2058 for 4 h. Treatment with Org2058 results in reduced mobility of PR(A) and PR(B), but co-transfection of RelA has no effect (Fig. 6), as was also true for RU486-treated cells (data not shown), indicating that hyperphosphorylation of PR, and therefore probably DNA binding, are not prevented by RelA.


Figure 6: Effects of RelA on PR phosphorylation. COS-1 cells were transfected with PR(A) or PR(B) expression vectors (1 µg) in combination with CMV-4 or CMV-4/RelA (9 µg), as indicated. Subsequently, cells were treated with vehicle(-) or 10 nM Org2058 (P), harvested, and loaded on a 8% SDS-polyacrylamide gel. PR and RelA were visualized on Western blot as described under ``Experimental Procedures.''



Negative Interaction between Cytokine and Progesterone Signaling

To determine whether the mutual repression between RelA and PR can also be observed when NF-kappaB is activated by cytokines, several experiments were performed. First, the effects of TNF-alpha on PR activity were studied in HeLa and T47D cells. Gel retardation assays showed that TNF-alpha treatment of these cells results in binding of proteins to the NF-kappaB site located in the ICAM-1 promoter (data not shown). When transient transfections were performed in HeLa cells, a clear inhibition of the hormone-induced transcriptional activity of PR(A) and PR(B) was observed when TNF-alpha was added (Table 2). Transcriptional activity of PR was also inhibited by TNF-alpha in T47D (Table 2), although less pronounced than in HeLa cells. This may be due to the relatively weak induction of NF-kappaB, as assessed by measuring transactivation of a 3xNF-kappaBtkLuc reporter (Table 2). Alternatively, other TNF-alpha induced factors, like AP1 family members, could prevent effective repression of PR through their ability to bind to NF-kappaB(17, 52) . The effect of PR(A) and PR(B) on TNF-alpha-induced NF-kappaB activity was also examined. Treatment with progestins resulted in a repression of the NF-kappaB activity evoked by TNF-alpha, both in HeLa and T47D cells (Table 2).



To study if the PR/NF-kappaB interaction could have relevance for the in vivo situation, the influence of NF-kappaB inducing reagents on the expression of a PR target gene was examined. To this end, T47D cells were treated with Org2058 (10 nM) for 16 h in the absence or presence of TNF-alpha (250 units/ml) or H(2)O(2) (150 µM), and total RNA was isolated. Northern blot analysis showed that the induction of the progestin-induced fatty acid synthetase mRNA was decreased both by TNF-alpha and H(2)O(2) (Fig. 7A), with TNF-alpha treatment resulting in a 30% decrease and HO in a 60% decrease of induction (Fig. 7B). Although the reduction by TNF-alpha was relatively small, it is in line with the repression of PR mediated transactivation by TNF-alpha in these cells (Table 2), which is probably caused by their inherent low sensitivity to TNF-alpha. From these results, we conclude that TNF-alpha-induced NF-kappaB activity can repress PR functioning and vice versa and that this trans-repression is likely to be operational in vivo.


Figure 7: Repression of progestin-induced fatty acid synthetase mRNA expression by RelA inducing agents in T47D cells. A, cells were treated with the progestin Org2058 (P; 10 nM) in the absence or presence of TNF-alpha (250 units/ml) or H(2)O(2) (150 µM) for 16 h. Total RNA was isolated, and Northern blotting was performed. Blots were probed with a cDNA encoding the fatty acid synthetase gene and subsequently with a glyceraldehyde 3-phosphate dehydrogenase probe as a control for equal loading (10 µg/lane). B, graphic representation of the repression of progestin-induced (P) fatty acid synthetase mRNA expression by TNF-alpha (T) and H(2)O(2) (H), as determined by PhosphorImager quantification after correction for the internal glyceraldehyde 3-phosphate dehydrogenase control. The results are presented as the level of induction with reference to the induction by progestin (100%). The bar diagram represents the average of two independent experiments.




DISCUSSION

In the present study, we show mutual repression of the hormone-activated PR and the RelA(p65) subunit of NF-kappaB. This mutual repression could be caused by several mechanisms. The first possibility is that RelA and PR are able to bind to their respective cognate DNA elements. However, treatment of T47D cells with progestins did not result in specific complex formation on the NF-kappaB site from the ICAM-1 promoter. (^2)Also in the case of the mutual inhibition of GR and AP1, no evidence was found for this mechanism(7, 9, 10, 11) . Second, PR and RelA could compete for common co-activators or transcription intermediary factors, a process referred to as transcriptional interference or squelching(53, 54) . This is unlikely, since we found the repression of PR by RelA to occur independent of receptor isoforms, transactivation functions of PR, reporter construct, and cell type and since repression of PR by RelA was independent of the main transactivation domain TA1 of RelA. A third possibility is that a direct interaction between the two proteins, resulting in a heterodimer, could account for the repression. In vitro association assays showed that the two proteins are indeed capable of a direct physical interaction. Such a complex could either (i) be unable to bind DNA or (ii) result in the formation of inactive complexes on the DNA by preventing the interaction with essential co-factors or the basal transcription machinery. Our data are in line with the second mechanism, since TNF-alpha-induced DNA binding of NF-kappaB was found to be unaffected by the presence of PR in gel retardation assays.^2 Furthermore, we also found the hormone-induced change in mobility of PR, which is caused by DNA-dependent phosphorylation events(51, 52) , to occur irrespective of the presence of RelA, indicating that binding of PR to DNA is not prevented by RelA. Similarly, GR was shown to interfere with AP1 activity without altering its DNA binding, as shown by in vivo footprinting (12) .

As was also shown for the repression of AP1 by GR(7, 8, 9) , we found the C and E domains of PR to be essential for repression of RelA. With respect to the HBD, this domain is likely to function differently with respect to the enhancer and repressor functions of PR. The ability of this domain to enhance transcription is probably not essential for the repression of PR, since anti-progestin-occupied receptors, which are unable to evoke transactivation, still induced repression of RelA. Together with the cell type-specific differences between the two PR isoforms in transcriptional activity, but not in repressive function, these findings show that the ability to enhance or repress transcription are separate functions within the steroid receptor proteins.

Although the interactions of steroid receptors with transcription factors of the AP1 and NF-kappaB families share several features, notable differences are also evident. The repressive action of c-Fos was mainly directed against AF-2 of PR(11) , while we show RelA to inhibit the transactivation of a PR construct lacking this domain (PR(B)DeltaE). Second, the repression of AP1 proteins by PR was shown to be cell type- and promoter-specific(11) , indicating that additional, promoter-specific proteins are involved also, the expression of which could be different in various cell types. While the transactivation potential of RelA and the PR isoforms was shown to differ, the repression of RelA by PR appeared to be independent of cell type and promoter context. Together with the association between PR and RelA in vitro, these data indicate that the mutual repression between PR and RelA could be due to a direct interaction between the proteins, without the additional involvement of other proteins.

Deletion analysis of RelA showed that the region corresponding to TA1 (amino acids 515-550; (26) ) is not required for the repression of PR, indicating that other regions of the protein are involved in the interaction with PR. Preliminary results indeed indicate that regions outside the TA1 domain of RelA are essential for repression of PR and GR. (^3)This could explain why the NFKB1(p50) and c-Rel proteins, which are substantially different from RelA outside the Rel homology region, are unable to repress PR (this study) and GR (18) .

The interaction between PR and RelA is potentially important in organs in which the PR together with NF-kappaB-regulating cytokine receptors is expressed, like mammary gland, ovarium, and endometrium. During pregnancy, progesterone levels are high, and the presence of progesterone is essential for the maintenance of pregnancy(30) . In endometrial cells, cytokines, which induce NF-kappaB, like TNF-alpha and IL-1, and their receptors are expressed, as well as PR(55) . Progesterone-induced decidualization of endometrial cells, which is thought to be important in maintaining pregnancy, is inhibited in vitro by TNF-alpha and IL-1 in endometrial cells(56, 57) . Second, the expression of IL-8, which is both a chemotactic factor for neutrophils and causes them to secrete lysosomal enzymes, is repressed by progesterone(58) . Since these effects of IL-8 may, besides playing a role in inflammation, also be an early step in the initiation of labor(59) , suppression of its secretion prevents premature birth. The recent finding that in mice that carry a null mutation of the PR, uterine inflammations occur frequently (60) is in line with this hypothesis. Recently, NF-kappaB-induced expression of IL-8 was shown to be repressed by GR (16) in a mechanism similar to what we propose for PR. Another important function of progesterone during pregnancy is its immunosuppressive effect, to prevent activation of an immune response directed against the embryo(30) . A number of genes that are important in the immune system have been shown to be regulated by NF-kappaB(22, 23) . It is therefore possible that the immunosuppressive action of progesterone during pregnancy is partly due to the inhibition of the transcriptional activity of NF-kappaB.

The negative cross-talk between RelA and PR could also play a role in cell proliferation, since both NF-kappaB (23) and progestins (29) have been implicated in this process. Several lines of evidence suggest that constitutive activation of NF-kappaB contributes to the malignant phenotype of tumor cells. First, a naturally occurring splice variant of RelA, named p65Delta, was shown to transform Rat-1 cells(61) . Higgins et al.(62) have shown proliferation and tumorigenicity of several tumor cell lines, including the human breast tumor cell lines MCF7 and T47D, to be inhibited by antisense oligonucleotides to RelA. In addition, activation of NF-kappaB through the disruption of IkappaBalpha regulation, was shown to result in malignant transformation(63) . In contrast to their different ability to transactivate, both progestins and anti-progestins can be used to treat breast tumors(64) . Therefore, it seems reasonable to propose that trans-repression of NF-kappaB (this study) and AP1(11) , which is induced by both types of ligand, could be relevant for tumor inhibition.


FOOTNOTES

*
This work was supported by Grant 92.96 from the Netherlands Asthma Foundation (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work.

Present address: Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

**
To whom correspondence should be addressed. Tel.: 30-2510211; Fax: 30-2516464.

(^1)
The abbreviations used are: PR, progesterone receptor; GR, glucocorticoid receptor; TNF-alpha, tumor necrosis factor-alpha; IL, interleukin; FCS, fetal calf serum; PCR, polymerase chain reaction; PRE, progesterone response element; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; CMV, cytomegalovirus; DBD, DNA binding domain; HBD, hormone binding domain; MMTV, mouse mammary tumor virus; ICAM-1, intercellular adhesion molecule-1.

(^2)
E. Kalkhoven, S. Wissink, P. T. van der Saag, and B. van der Burg, unpublished results.

(^3)
S. Wissink, E. Kalkhoven, B. van der Burg, and P. T. van der Saag, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. R. M. Evans and M. Muller for reporter constructs, Dr. P. Baeuerle for the RelA(p65) and NFKB1(p50) cDNAs, Dr. M. Misrahi for the human PR cDNA, Dr. H. Gronemeyer for reporter and expression constructs, Dr. D. Chalbos for the fatty acid synthetase cDNA, and Dr. K. B. Horwitz for the AB-52 antibody. We also thank E. C. van Heerde and G. Folkers for technical assistance and J. Heinen and F. Vervoordeldonk for photographic reproductions.


REFERENCES

  1. Evans, R. M. (1988) Science 240, 889-895 [Medline] [Order article via Infotrieve]
  2. Green, S., and Chambon, P. (1988) Trends Genet. 4, 309-314 [CrossRef][Medline] [Order article via Infotrieve]
  3. Beato, M. (1989) Cell 56, 335-344 [Medline] [Order article via Infotrieve]
  4. Gronemeyer, H. (1991) Annu. Rev. Genet. 25, 89-123 [CrossRef][Medline] [Order article via Infotrieve]
  5. Meyer, M.-E., Pornon, A., Ji, J., Bocquel, M.-T., Chambon, P., and Gronemeyer, H. (1990) EMBO J. 9, 3923-3932 [Abstract]
  6. Schrader, W. T., and O'Malley, B. W. (1972) J. Biol. Chem. 247, 51-59 [Abstract/Free Full Text]
  7. Schüle, R., Rangarajan, P., Kliewer, S., Ransone, L. J., Bolado, J., Yang, N., Verma, I. M., and Evans, R. M. (1990) Cell 62, 1217-1226 [Medline] [Order article via Infotrieve]
  8. Jonat, C., Rahmsdorf, H. J., Park, K. K., Cato, A. C., Gebel, S., Ponta, H., and Herrlich, P. (1990) Cell 62, 1189-1204 [Medline] [Order article via Infotrieve]
  9. Yang-Yen, H.-F., Chambard, J.-C., Sun, Y.-L., Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990) Cell 62, 1205-1215 [Medline] [Order article via Infotrieve]
  10. Lucibello, F. C., Slater, E. P., Jooss, K. U., Beato, M., and Müller, R. (1990) EMBO J. 9, 2827-2834 [Abstract]
  11. Shemshedini, L., Knauthe, R., Sassone-Corsi, P., Pornon, A., and Gronemeyer, H. (1991) EMBO J. 10, 3839-3849 [Abstract]
  12. König, H., Ponta, H., Rahmsdorf, H. J., and Herrlich, P. (1992) EMBO J. 11, 2241-2246 [Abstract]
  13. Zhang, X. K., Wills, K. N., Husman, M., Herman, T., and Pfahl, M. (1991) Mol. Cell. Biol. 11, 6016-6025 [Medline] [Order article via Infotrieve]
  14. Tzukerman, M., Zhang, X. K., and Pfahl, M. (1991) Mol. Endocrinol. 5, 1983-1992 [Abstract]
  15. Ray, A., and Prefontaine, K. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 752-756 [Abstract]
  16. Mukaida, N., Morita, M., Ishikawa, Y., Rice, N., Okamoto, S., Kasahara, T., and Matsushima, K. (1994) J. Biol. Chem. 269, 13289-13295 [Abstract/Free Full Text]
  17. Scheinman, R. I., Gualberto, A., Jewell, C. M., Cidlowski, J. A., and Baldwin, A. S. (1995) Mol. Cell. Biol. 15, 943-953 [Abstract]
  18. Caldenhoven, E., Liden, J., Wissink, S., van der Stople, A., Raaijmakers, J., Koenderman, L., Okret, S., Gustafson, J.-Å., and van der Saag, P. T. (1995) Mol. Endocrinol. 9, 401-412 [Abstract]
  19. Ray, A., Prefontaine, K. E., and Ray, P. (1994) J. Biol. Chem. 269, 12940-12946 [Abstract/Free Full Text]
  20. Stein, B., and Yang, M. X. (1995) Mol. Cell. Biol. 15, 4971-4979 [Abstract]
  21. Van de Stolpe, Caldenhoven, E., Stade, B., Koenderman, L., Raaijmakers, J. A. M., Johnson, J. P., and Van der Saag, P. T. (1994) J. Biol. Chem. 269, 6185-6192 [Abstract/Free Full Text]
  22. Siebenlist, U., Fransozo, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455 [CrossRef]
  23. Baeuerle, P., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179 [CrossRef][Medline] [Order article via Infotrieve]
  24. Urban, M. B., Schreck, R., and Baeuerle, P. A. (1991) EMBO J. 10, 1817-1825 [Abstract]
  25. Beg, A. A., Ruben, R. I., Scheinman, S., Haskill, Rosen, C. A., and Baldwin, A. S., Jr. (1993) Genes & Dev. 6, 1899-1913
  26. Schmitz, M. L., and Baeuerle, P. A. (1991) EMBO J. 10, 3805-3817 [Abstract]
  27. Ishikawa, H., Asano, M., Kanda, T., Kumar, S., Gelinas, C., and Ito, Y. (1993) Oncogene 8, 2889-2896 [Medline] [Order article via Infotrieve]
  28. Sarkar, S., and Gilmore, T. D. (1993) Oncogene 8, 2245-2252 [Medline] [Order article via Infotrieve]
  29. Clarke, C. L., and Sutherland, R. L. (1990) Endocr. Rev. 11, 266-301 [Medline] [Order article via Infotrieve]
  30. Siiteri, P. K., and Stites, D. P. (1982) Biol. Reprod. 26, 1-14 [Abstract]
  31. Van der Burg, B., Rutteman, G. R., Blankenstein, M. A. de Laat, S. W., and van Zoelen, E. J. J. (1988) J. Cell. Physiol. 123, 101-108
  32. Estes, P. A., Suba, E. J., Lawler-Heavner, J., Elashry-Stowers, D., Wei, L. L., Toft, D. O., Sullivan, W. P., Horwitz, K. B., and Edwards, D. P. (1987) Biochemistry 26, 6250-6252 [Medline] [Order article via Infotrieve]
  33. Weigel, N. L., Beck, C. A., Estes, P. A., Prendergast, P., Altmann, M., Christensen, K., and Edwards, D. P. (1992) Mol. Endocrinol. 6, 1585-1597 [Abstract]
  34. Kalkhoven, E., Kwakkenbos-Isbrücker, L., de Laat, S. W., van der Saag, P. T., and van der Burg, B. (1994) Mol. Cell. Endocrinol. 102, 45-52 [CrossRef][Medline] [Order article via Infotrieve]
  35. Misrahi, M., Atger, M., d'Auriol, L., Loosfelt, H., Mériel, C., Fridlansky, F., Guiochon-Mantel, A., Galibert, F., and Milgrom, E. (1987) Biochem. Biophys. Res. Commun. 143, 740-748 [Medline] [Order article via Infotrieve]
  36. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H., and Chambon, P. (1990) EMBO J. 9, 1603-1614 [Abstract]
  37. Ruben, S. M., Dillon, P. J., Schreck, R., Henkel, T., Chen, C.-H., Maher, M., Baeuerle, P., and Rosen, C. A. (1991) Science 251, 1490-1493 [Medline] [Order article via Infotrieve]
  38. Schüle, R., Muller, M., Kaltschmidt, C., and Renkawitz, R. (1988) Science 242, 1418-1420 [Medline] [Order article via Infotrieve]
  39. Hollenberg, S. M., and Evans, R. (1988) Cell 55, 899-906 [Medline] [Order article via Infotrieve]
  40. Meyer, M.-E., Quirin-Stricker, C., Lerouge, T., Bocquel, M.-T., and Gronemeyer, H. (1992) J. Biol. Chem. 267, 10882-10887 [Abstract/Free Full Text]
  41. Boer, P. H., Potten, H., Adra, C. N., Jardine, K., Mulhofer, G., and McBurney, M. W. (1990) Biochem. Genet. 28, 299-308 [Medline] [Order article via Infotrieve]
  42. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051 [Medline] [Order article via Infotrieve]
  43. Pfahl, M., Tzukerman, M., Zhang, X.-K., Lehmann, J. M., Hernann, T., Wills, K. N., and Graupner, G. (1990) Methods Enzymol. 189, 256-270 [Medline] [Order article via Infotrieve]
  44. Cavaillès, V., Dauvois, S., Danielian, P. S., and Parker, M. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10009-10013 [Abstract/Free Full Text]
  45. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  46. Chalbos, D., Westley, B., May, F., Alibert, C., and Rochefort, H. (1986) Nucleic Acids Res. 14, 965-982 [Abstract]
  47. Van der Burg, B., van Selm-Miltenburg, A. J. P., de Laat, S. W., and van Zoelen, E. J. J. (1989) Mol. Cell. Endocrinol. 64, 223-228 [CrossRef][Medline] [Order article via Infotrieve]
  48. Fort, P., Marty, L., Piechaczyk, M., Elsabrouty, S., Dnai, C., Jeanteur, P., and Blanchard, J. M. (1985) Nucleic Acids Res. 13, 1431-1442 [Abstract]
  49. Chen, J.-Y., Penco, S., Ostrowski, J., Balaguer, P., Pons, M., Starrett, J. E., Reczek, P., Chambon, P., and Gronemeyer, H. (1995) EMBO J. 14, 1187-1197 [Abstract]
  50. Bagchi, M. K., Tsai, S. Y., Tsai, J.-M., and O'Malley, B. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2664-2668 [Abstract]
  51. Takimoto, G. S., Tasset, D. M., Eppert, A. C., and Horwitz, K. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3050-3054 [Abstract]
  52. Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993) EMBO J. 12, 3879-3891 [Abstract]
  53. Gill, G., and Ptashne, M. (1988) Nature 334, 721-724 [CrossRef][Medline] [Order article via Infotrieve]
  54. Meyer, M.-E., Gronemeyer, H., Turcotte, B., Bocquel, M.-T., Tassett, D., and Chambon, P. (1989) Cell 57, 433-442 [Medline] [Order article via Infotrieve]
  55. Tabibzadeh, S. (1991) Endocr. Rev. 12, 272-290 [Medline] [Order article via Infotrieve]
  56. Inoue, T., Kanzaki, H., Iwai, M., Narukawa, S., Higuchi, T., Katsuragawa, H., and Mori, T. (1994) Hum. Reprod. 9, 2411-2417 [Abstract]
  57. Jikihara, H., and Handwerger, S. (1994) Endocrinology 134, 353-357 [Abstract]
  58. Ito, A., Imada, K., Sato, T., Kubo, T., Matsushima, K., and Mori, Y. (1994) Biochem. J. 301, 183-186 [Medline] [Order article via Infotrieve]
  59. Kelly, R. W. (1994) Endocr. Rev. 15, 684-706 [Medline] [Order article via Infotrieve]
  60. Lydon, J. P., DeMayo, F. J., Funk, C. R., Mani, S. K., Hughes, A. R., Montgomery, C. A., Jr., Shyamala, G., Conneely, O. M., and O'Malley, B. W. (1995) Genes & Dev. 9, 2266-2278
  61. Narayanan, R., Klement, J. F., Ruben, S. M., Higgins, K. A., and Rosen, C. A. (1992) Science 256, 367-370 [Medline] [Order article via Infotrieve]
  62. Higgins, K. A., Perez, J. R., Coleman, T. A., Dorshkind, K., McComas, W. A., Sarmiento, U. M., Rosen, C. A., and Narayanan, R. (1993) Proc. Natl. Acad, Sci. U. S. A. 90, 9901-9905 [Abstract]
  63. Beauparlant, P., Kwan, I., Bitar, R., Chou, P., Koromilas, A. E., Sonenberg, N., and Hiscott, J. (1994) Oncogene 9, 3189-3197 [Medline] [Order article via Infotrieve]
  64. Henderson, B. E., Ross, R. K., and Pike, M. C. (1993) Science 259, 633-638 [Medline] [Order article via Infotrieve]

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