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J Biol Chem, Vol. 273, Issue 36, 23567-23574, September 4, 1998


Glucocorticoids Repress Transcription from a Negative Glucocorticoid Response Element Recognized by Two Homeodomain-containing Proteins, Pbx and Oct-1*

Nanthakumar Subramaniam, William CairnsDagger , and Sam Okret§

From the Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, F60 Novum, S-141 86 Huddinge, Sweden

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Several studies have established that the prolactin (PRL) gene is expressed not only in lactotrophs and somatotrophs of the anterior pituitary but, albeit to a lesser extent, in non-pituitary cells like human thymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in the pituitary for the pituitary-specific transcription factor Pit-1/GHF-1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. This prompted us to investigate the transcription factors in non-pituitary cells which are involved in controlling expression and glucocorticoid repression of a previously characterized negative glucocorticoid response element from the bovine prolactin gene (PRL3 nGRE). Here we have demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly because of the binding of the ubiquitously expressed POU-homeodomain-containing octamer transcription factor-1 (Oct-1) to an AT-rich sequence present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-containing protein, Pbx, previously shown to bind cooperatively with several homeotic selector proteins. The Pbx binding site in the PRL3 nGRE, located just upstream of the Oct-1 binding site, showed a strong sequence similarity with known Pbx binding sites and bound Pbx with an affinity similar to that of other established Pbx target sequences. Interestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro translated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvement of the homeobox protein Pbx in glucocorticoid repression via an nGRE identifies a new role for this protein.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The peptide hormone prolactin (PRL)1 has been shown to regulate milk production, cause cell proliferation, and to play an important role in maintaining normal immune responses together with various cytokines (1-3). It is synthesized mainly in lactotrophs of the anterior pituitary (4). It has been demonstrated that the presence of the pituitary-specific transcription factor Pit-1/GHF-1 in lactotrophs is indispensable but not sufficient to activate the PRL gene in those cells (5, 6). A number of studies have demonstrated that the PRL gene is also expressed, albeit to a lesser extent, in non-pituitary cells that lack Pit-1/GHF-1 (for review, see Ref. 3). RNA blot analysis by O'Neal et al. (7) detected PRL message in human thymocytes and in several non-pituitary human cell lines including Jurkat, HeLa, and JEG cells. In addition, the uterine sarcoma cell line SKUT-IB-20 and B-lymphoblastoid cell line IM-9-P3 express the human PRL gene. In all of these cases expression was initiated from the pituitary-specific transcriptional start site, even though these cells are devoid of Pit-1/GHF-1 (8, 9). Also in the primate decidua and human lymphoid cells the PRL gene is transcribed. However, in these cases an alternative upstream promoter is utilized (9 and refs. therein). In cells devoid of Pit-1/GHF-1 expression, it has been suggested that the transcription factor Oct-1 or CREB contributes to the PRL gene expression (10-12).

Rat PRL release and synthesis are inhibited by glucocorticoids, thyroid hormone, and dopamine, whereas estradiol, thyrotropin-releasing hormone, epidermal growth factor, vasoactive intestinal polypeptide, cAMP, and insulin have been reported to have a stimulatory effect on rat PRL expression (8, 13, 14). Similar regulation, including repression by glucocorticoids, has been described for the human PRL gene (15). However, for this gene thyroid hormone resulted in a 3-fold induction. In addition, the bovine PRL promoter has been shown to be a target for negative regulation by glucocorticoids, whereas epidermal growth factor and thyrotropin-releasing hormone stimulated expression of the downstream gene controlled by this promoter (16, 17). A negative glucocorticoid response element (PRL3 nGRE) in the bovine PRL promoter important for glucocorticoid repression has been identified (17). This nGRE by itself conferred both increased expression and glucocorticoid-mediated repression in pituitary GH3 cells when fused to a heterologous promoter (17, 18). Interestingly, this recombinant gene, when introduced into non-pituitary cells lacking Pit-1/GHF-1 expression, also showed an increased expression and repression by glucocorticoids (17, 19).

Several mechanisms by which the glucocorticoid receptor (GR) is able to repress transcription have been documented (20-22). The mechanism of GR-mediated transcriptional repression involves either a direct interaction between the GR and nGRE, leading to displacement of transcription factors or interference with their transcriptional activation (18, 23-26), or alternatively a protein-protein interaction between the GR and a second transcription factor independent of GR binding to the DNA (27-29). In the case of the PRL3 nGRE, we have shown previously that GR binding to this element was required for glucocorticoid repression to occur (18, 19). Because the PRL3 nGRE increased the expression in the absence of hormone in both pituitary and non-pituitary cells, it was speculated that the PRL3 element bound one or more positively acting transcription factors that were repressed by the GR (17). Previously we have shown that the PRL3 nGRE is composite in nature and that the enhanced expression in pituitary GH3 cells was mainly the result of the binding of Pit-1/GHF-1. Furthermore, glucocorticoid-mediated repression of transcription occurred when the GR displaced this factor with the help of a second unknown DNA-binding factor present in a complex B, which we called XTF (18). In this study, we have investigated which factor(s) in cells devoid of Pit-1/GHF-1 bind to the PRL3 nGRE, mediate the increase in expression, and are involved in the repression by glucocorticoids. We report that the ubiquitously expressed POU-homeodomain protein Oct-1 together with a second ubiquitously expressed Pbx protein (30, 31) are responsible for maximal expression. Binding of both proteins to the nGRE is required for glucocorticoids to repress the enhanced expression.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Constructions and Oligonucleotides-- The sequences of the PRL3 nGRE and its mutants are shown in Fig. 2B. The oligonucleotide OCT spans the octamer element from an immunoglobulin heavy chain promoter (32). RRE is a repressor element from the mouse mammary tumor virus promoter (33). The Pbx-binding CRS1 oligonucleotide is from the 17alpha -hydroxylase cytochrome P450 promoter (34). PRS is a Pbx1 binding site oligonucleotide selected by random polymerase chain reaction-mediated binding site selection ((35) and see Fig. 2B). All oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer and purified by high performance liquid chromatography. The GR expression vector SVGR1 has been described previously (36). PRL3CAT and the other plasmid constructs containing the mutant PRL3 elements PX, PX1, and PX5 (see Fig. 2B) have also been described previously (18, 19). The in vitro transcription translation vector for GR DNA binding domain (DBD) was from Lindebro et al. (37).

Cell Culture and Transfections-- Monkey kidney COS-7, rat pituitary GH3, and embryonic kidney 293 cells were grown as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Inc.) at 37 °C in 10% CO2. Mouse hepatoma Hepa1c1c7 cells were grown as monolayers at 37 °C in 5% CO2 in minimum essential medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µl/ml streptomycin. COS-7 cells were plated at 50% confluence on 60-mm plates overnight, and the next day cells were transfected with the indicated amount of receptor and reporter plasmids by Lipofectin, essentially following the protocol of the manufacturer (Life Technologies, Inc.). After a 6-h incubation at 37 °C in 10% CO2, the medium containing the DNA-Lipofectin mix was removed, and fresh medium was added. Cells were allowed to recover overnight, and fresh medium, supplemented with 10% fetal calf serum containing either ethanol (final concentration, 0.01%) or 0.1 µM dexamethasone (Sigma) in an equal volume of ethanol, was added. Hepa1c1c7 cells were transfected with the indicated amount of reporter plasmids by the calcium phosphate-DNA method (38) at 60% confluence on 60-mm tissue culture plates. Transfection of GH3 cells with reporter and expression vector constructs was performed as described previously (18). Cells were harvested 36 h after the start of the hormone treatment, extracts prepared, and chloramphenicol acetyltransferase (CAT) activity assayed as described by Gorman et al. (39). All CAT assays were performed such that the rate of acetylation of chloramphenicol was in the linear range (less than 30% conversion). Acetylated chloramphenicol was quantified by PhosphorImaging analysis (FUJIX BAS 2000). The values given represent the mean ± S.D. from at least three experiments. A minimum of two plasmid preparations was used for each plasmid construction, and the same results were obtained.

Nuclear Extract Preparation, in Vitro Translation, and Gel Retardation Analysis-- COS-7, Hepa1c1c7, GH3, and 293 cells were washed once with phosphate-buffered saline and harvested by scraping in 1 ml of phosphate-buffered saline by a rubber policeman. Nuclear extracts from all of these cells were prepared as described by Gough (40). The oligonucleotides were end labeled by T4 polynucleotide kinase using [gamma -32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech). DNA binding reactions of 20 µl were carried out in buffer containing 20 mM Tris-Cl, pH 8.0, 10% (w/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 25 mM KCl, 3% bovine serum albumin, 0.1-0.3 ng of radiolabeled oligonucleotide, and 3-5 µg of nuclear extract. Binding reactions were performed at room temperature for 20 min. Free and bound DNA were separated on 4% polyacrylamide (acrylamide:bisacrylamide, 29:0.5) gels, which were run at a constant voltage of 200 V in 22 mM Tris borate, 0.5 mM EDTA. Where indicated, unlabeled competitor oligonucleotides (50-fold excess), rabbit polyclonal antiserum against Oct-1 (41), Pbx1 (42), or nonimmune rabbit serum was included in the binding reactions. GR DBD was translated in vitro using the rabbit reticulocyte lysate system from Promega.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The PRL3 Element Confers Increased Expression and Glucocorticoid-mediated Repression in Both Pituitary and Non-pituitary Cells-- The PRL3 nGRE, when cloned in front of the heterologous thymidine kinase promoter (-105 to +52) driving the expression of the CAT reporter gene (PRL3CAT), was compared with regard to its ability to mediate enhanced expression and to confer repression by glucocorticoids in a cell line containing the pituitary-specific transcription factor Pit-1/GHF-1 (pituitary GH3 cells) and in cells devoid of Pit-1/GHF-1 expression (hepatoma Hepa1c1c7 and COS-7 cells). As can be seen in Fig. 1, the level of expression in the absence of hormone of PRL3CAT transfected into the GH3 cells was approximately 7-fold higher compared with the parent pBLCAT2 vector lacking the PRL3 element. This enhanced expression was slightly higher compared with the 4-5-fold increase observed in the Hepa1c1c7 and COS-7 cells. The addition of dexamethasone resulted in a 50-60% repression of CAT expression in all three cell types. Dexamethasone did not repress the expression of the pBLCAT2 itself, demonstrating that the negative regulation of PRL3CAT was mediated through the PRL3 nGRE sequence. This experiment demonstrated that Hepa1c1c7 and COS-7 cells, which lack the pituitary-specific factor Pit-1/GHF-1, are regulated in the same manner as Pit-1/GHF-1-containing GH3 cells. This suggests that the non-pituitary cells Hepa1c1c7 and COS-7 contain factor(s) other than Pit-1/GHF-1 which act via the PRL3 element to enhance CAT expression and can be repressed by glucocorticoids.


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  		Fig. 1.   PRL3 nGRE functions in both pituitary and non-pituitary cell lines. The Hepa1c1c7, COS-7, and GH3 cells were transfected with 4 µg of either PRL3CAT or pBLCAT2. In the case of COS-7 or GH3 cells, cotransfection with 2 µg of pSVGR1 was performed. After transfection, cells were incubated with (dark bars) or without (light bars) 0.1 µM dexamethasone for 36 h. Cellular extracts were prepared and CAT activity measured. CAT activity was calculated as the percentage of chloramphenicol converted to acetylated forms, and the data are presented taking the expression of pBLCAT2 in each cell line in the absence of hormone as 1. Each bar gives the average and standard deviation for four to six experiments.

Identification of Protein-DNA Complexes Formed between the PRL3 Element and Nuclear Factors from the Hepatoma Hepa1c1c7 and COS-7 Cells-- Most likely one or more nuclear factor(s) from the non-pituitary cells interacting with the PRL3 element are responsible for the enhanced expression that PRL3 conferred upon the thymidine kinase promoter. To identify specific protein-DNA complexes formed on the PRL3 nGRE, electrophoretic mobility shift assays (EMSA) with nuclear extracts from Hepa1c1c7 cells were performed. Incubation of PRL3 with Hepa1c1c7 nuclear extracts resulted in the formation of three major (A1, A2, and B) protein-DNA complexes (Fig. 2A). The A1, A2, and B complexes were all specific because their formation was abolished by the addition of an excess of unlabeled PRL3 (lane 3) but not by an excess of an unrelated sequence (lane 5). Similar results were obtained with COS-7 cells, although complex A1 was less pronounced and seen only when higher amounts of nuclear extracts were used in the binding reaction (see Fig. 2C).


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  		Fig. 2.   Analysis of nuclear factors binding to PRL3 and PX. Panel A, radiolabeled PRL3 was incubated with Hepa1c1c7 nuclear extracts, and the DNA binding activity was monitored by EMSA. Lanes 2-5 contained 5 µg of nuclear extract. Competition experiments were performed with a 50-fold excess of unlabeled PRL3 (lane 3), OCT (lane 4), or a nonrelated oligonucleotide RRE (lane 5). The three specific complexes formed on PRL3 were designated A1, A2, and B. Panel B, the mutated PRL3 oligonucleotides used in binding or competition experiments and two Pbx binding sites, CRS1 and PRS. Panel C, the PRL3 nGRE deleted in the 3'-end (PX, panel B) was incubated with 5 µg of COS-7 nuclear extracts and analyzed by EMSA. Unlabeled competitors (50-fold excess) were included in the figure as indicated.

To identify sequences in the PRL3 element responsible for formation of the observed protein-DNA complexes, mutations were introduced into the PRL3 element (see Fig. 2B). The PX element, which is identical to the PRL3 element except that it lacks 14 base pairs in the 3'-end of PRL3, gave rise to the same three specific complexes (A1, A2, and B, Fig. 2C) as the PRL3 element when using COS-7 nuclear extract, indicating that the 3'-sequence deleted was not responsible for the formation of the protein-DNA complexes. All three complexes formed on the PX element were specific because competition with an excess of unlabeled PX was able to abolish the complexes (Fig. 2C, lane 3). When an A/T-rich site found in the PX element was mutated (PX5, see Fig. 2B), it was no longer able to compete efficiently for the formation of complex A2 but reduced both complex B and A1 formation (Fig. 2C, lane 5). A sequence 5' to the AT-rich region was shown to be important for complex B formation because addition of an excess of an oligonucleotide mutated in this region (PX1, see Fig. 2B) led to a loss of its ability to compete for formation of the B complex (lane 4). Because both PX1 and PX5 were able to compete for the formation of complex A1, this complex possibly consists of the proteins responsible for forming the B and A2 complexes, respectively.

Complex A Contains Oct-1-- In pituitary GH3 cells, two major proteins bind to the PRL3 nGRE, both of which contribute to the enhanced expression and the repression by glucocorticoids (18). One of the proteins was identified as Pit-1/GHF-1. Binding of Pit-1/GHF-1 occurred to the same AT-rich sequence present in the PRL3 or PX which is responsible for the formation of complex A1 and A2 (see above). Because the ubiquitously expressed Oct-1 belongs to the same family of POU-homeodomain-containing proteins and binds to similar AT-rich sequences as does Pit-1/GHF-1 (41, 43), we suspected that it may be present in complexes A1 and A2 as observed in the EMSA using Hepa1c1c7 or COS cell nuclear extracts. To substantiate this possibility, we included an oligonucleotide (OCT) containing a known binding site for Oct-1 in competition experiments. This OCT element completely blocked the formation of complexes A1 and A2 while the B complex remained intact (Fig. 2A, lane 4). Hence it was possible that the formation of complex A1 and A2 involved the binding of Oct-1 or an Oct-1-related protein to the PRL3 element. To support this possibility further an EMSA was performed where radiolabeled PRL3 and OCT elements were incubated with COS-7 cell nuclear extract. As can be seen from Fig. 3, lanes 2 and 6, the mobility of the A2 complex formed on PRL3 was identical to the mobility of the complex formed on the Oct-1-binding OCT oligonucleotide. Because the oligonucleotides were of equal length, this suggested that the two elements bound the same protein, possibly Oct-1. The demonstration that the A2 complex did indeed contain Oct-1 was performed by an antibody supershift experiment. Addition of an Oct-1 antiserum to the reaction mixture before running the EMSA supershifted or removed the A2 complex (Fig. 3, lane 3), as was the case for the complex formed on the OCT element (lane 7), whereas a nonimmune serum had no effect (lanes 4 and 8). Identical experiments with Hepa1c1c7 nuclear extracts produced similar results (data not shown).


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  		Fig. 3.   Oct-1 binds to the PRL3 element. Radiolabeled PRL3 (lanes 1-4) and OCT (lanes 5-8) oligonucleotides were used as probes in the EMSA. All lanes except lanes 1 and 4 contained 5 µg of COS-7 nuclear extracts. 1 µl of Oct-1 antiserum (Oct-1 A.S., lanes 3 and 7) or nonimmune serum (N.I.S., lanes 4 and 8) was included during the incubation period between probe and nuclear extracts. Supershifted complexes are marked with S.S.

The Sequence Responsible for Complex B Formation Resembles a Pbx Binding Site-- The mutational studies of the PRL3 element identified the sequence 5'-TCACCA-3' (5'-TGGTGA-3', lower strand) to be important for complex B formation (see above). Closer examination of this and surrounding sequences in the PRL3 element identified a sequence present in PRL3 (lower strand, 5'-TGATGGTG-3'), which shows a high conservation with the sequence TTGATGGAC (homology underlined) in an element (CRS1) of the 17alpha -hydroxylase cytochrome P450 gene and in the Hox B1 promoter repeat 3, known to bind the homeodomain-containing DNA-binding factor Pbx1 (34, 44). The PRL3 sequence that shows homology to the CRS1 or the Hox B1 elements includes part of the sequence involved in the formation of complex B (see above). The PRL3 sequence also shows a strong homology to the sequence 5'-TTGATTGAT-3' (lower strand, homology underlined), identified by random polymerase chain reaction-mediated binding site selection to interact with Pbx1 (35). In addition, the 5'-GA nucleotides in this latter sequence have been demonstrated by mutational studies to be important for Pbx1 binding, and these two nucleotides are conserved in the PRL3 element (35). Thus, we speculated that Pbx1 or another member of this family of homeodomain-containing proteins (for review, see Ref. 45) binds to the PRL3 nGRE and is responsible for the formation of complex B. As can be seen from Fig. 4A, incubation of a labeled CRS1 element with nuclear extract from embryonic kidney 293 cells known to contain the Pbx proteins formed a specific Pbx-CRS1 complex as described previously (46). A similar complex was formed when COS-7 nuclear extract was incubated with CRS1 (not shown). Interestingly, the Pbx-CRS1 complex was abolished when an excess of unlabeled PX was added to the reaction before the EMSA (Fig. 4A, lane 3). However, when the oligonucleotide with a mutation in the sequence responsible for formation of complex B was used as a competitor (PX1, see above), competition was no longer observed (lane 4). As expected, the Oct-1 binding site mutant oligonucleotide (PX5, see above) did compete (lane 5).


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  		Fig. 4.   Panel A, the PRL3 element competes for Pbx binding to the CRS1 element. 3 µg of nuclear extract from 293 cells was incubated with the labeled Pbx-binding CRS1 element from the P45017alpha promoter. A 50-fold excess of unlabeled PX, PX1, or PX5 oligonucleotide was included in the incubation before the EMSA, as indicated in the figure. Panel B, known Pbx binding sites compete for complex B formation. Radiolabeled PX oligonucleotide was incubated with 5 µg of COS-7 nuclear extracts and increasing concentrations of unlabeled PX (), CRS1 (diamond ), and PRS (open circle ) oligonucleotides followed by EMSA analysis. Complex B abundance was measured using a PhosphorImager and expressed relative to the complex B abundance in the absence of competitor.

To test whether known binding sites for Pbx can compete for the formation of complex B formation on the PRL3 nGRE, the reverse experiment was performed. COS-7 nuclear extract was incubated with labeled PX in the absence or presence of increasing concentrations of two unlabeled oligonucleotides known to bind Pbx1, namely CRS1 and a Pbx1 binding site selected by random polymerase chain reaction-mediated binding site selection (PRS (35), see Fig. 2B). Both CRS1 and PRS competed with affinity similar to that of unlabeled PX for the formation of complex B (Fig. 4B). This suggests that PRL3 indeed binds Pbx1 or a related family member through the site responsible for formation of complex B. In addition, complex B formed on PX and the Pbx-CRS1 complex showed the same mobility (data not shown). Because PX and CRS1 oligonucleotides are of similar length, this further supports the idea that the protein responsible for the formation of complex B is identical to Pbx1 or a related family member (30), all of which recognize the same sequence (47).

Complex B Contains Pbx-- To investigate whether complex B formed by COS-7 nuclear extract and the PX element contained a Pbx protein, nuclear extract and labeled PX oligonucleotide were incubated in the presence of an anti-Pbx antiserum or a nonimmune serum before analysis by EMSA. As can be seen in Fig. 5, the anti-Pbx antiserum caused a supershift of complex B (lane 2), whereas the nonimmune serum did not (lane 3). A similar pattern of supershifted bands was observed when the anti-Pbx antiserum was incubated with COS-7 nuclear extract and the established Pbx binding site oligonucleotide PRS (Fig. 5, lane 5). This demonstrated that complex B indeed contains Pbx. The anti-Pbx antiserum also supershifted complex B when using nuclear extracts from 293 or GH3 cells (data not shown). In addition, South-Western blotting showed that the molecular mass of the protein involved in complex B formation was approximately 45 kDa (data not shown). This is very close to the described molecular mass of 46.5 kDa for Pbx1a (30), further indicating that Pbx1 is responsible for the formation of complex B on PRL3.


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  		Fig. 5.   A Pbx1 antiserum supershifts complex B and Pbx-PRS complexes. 5 µg of COS-7 nuclear extract was incubated with radiolabeled PX or PRS (except in lanes 1 and 4) and analyzed by EMSA. 1 µl of Pbx1 antiserum (Pbx1 A.S.) was included in lanes 2 and 5, whereas lanes 3 and 6 contained 1 µl of nonimmune serum (N.I.S). Supershifted complexes are marked with S.S.

Role of Oct-1 and Pbx for Enhanced Expression and Glucocorticoid Repression via PRL3-- To determine the in vivo role of Oct-1 and Pbx for the increased expression and glucocorticoid repression from the PRL3 nGRE, PRL3, PX (lacking the 3'-end of the PRL3 element), and the described mutants PX1 (Pbx binding-deficient) and PX5 (Oct-1 binding-deficient) (see above) were cloned into the polylinker of pBLCAT2 and transfected into COS-7 cells. As can be seen in Fig. 6, the effect of PX on the enhanced activity of the thymidine kinase promoter in the absence of hormone was almost in the same range as PRL3, demonstrating that the 3'-end of the PRL3 nGRE was not significantly involved in producing the enhanced expression. PX could also confer negative regulation by dexamethasone, and this effect was as efficient as seen for PRL3. When the Oct-1 binding site in PX was mutated (PX5), approximately 80% of the enhanced expression was lost (Fig. 6), indicating that Pbx alone conferred only 20% of the transcriptional activity (p < 0.05). Mutation of the Pbx binding site (PX1) showed that Oct-1 contributes about half of the transcriptional activity (p < 0.005). Interestingly, negative regulation by dexamethasone was lost not only when the Oct-1 site was mutated but also after mutation of the Pbx binding site, suggesting an important role for both Oct-1 and Pbx in the repressive mechanism.


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  		Fig. 6.   Effects on expression and glucocorticoid repression after specific mutations of the Pbx and Oct-1 binding sites in the PRL3 nGRE. The mutated PX oligonucleotides were introduced into the pBLCAT2 vector and transfected into COS-7 cells as described under "Experimental Procedures." After transfection, the cells were incubated with (dark bars) or without (light bars) 0.1 µM dexamethasone for 36 h before harvesting. All CAT activities were related to the activity determined for pBLCAT2 in the absence of dexamethasone, which was given the arbitrary value of 1. Each bar gives the average and standard deviation for four to six experiments.

GR DBD Prevents Oct-1 and Pbx Binding to the PRL3 nGRE-- For a better understanding of the mechanism by which the GR represses the enhanced expression conferred by Oct-1 and Pbx binding to the PRL3 nGRE, we performed an EMSA in which in vitro translated GR DBD was added to the COS-7 nuclear extract before the EMSA. As can be seen in Fig. 7 (lanes 3-5), addition of an increasing amount of reticulocyte lysate containing in vitro translated GR DBD gradually prevented binding of both Oct-1 and Pbx to the DNA. In contrast, reticulocyte lysate alone, when added to the binding reaction (lanes 6-8), did not affect complex formation. This experiment suggests that the glucocorticoid-mediated repression of the enhanced PRL3 nGRE expression involves prevention by the GR of Pbx and Oct-1 binding to the element.


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  		Fig. 7.   Effects of GR DBD on the binding of Oct-1 and Pbx to PRL3 nGRE. 5 µg of COS-7 nuclear extract was incubated with radiolabeled PXL (lanes 2-8) and increasing amounts of reticulocyte lysate containing in vitro translated GR DBD (lanes 3-5) or reticulocyte lysate alone (lanes 6-8).). The PXL element is identical to the PX element except that it contains a 6-base pair extension in the 5'end (18). Complex formation was analyzed by EMSA.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The repression of PRL gene expression by glucocorticoids in both pituitary and non-pituitary cells is well documented (8, 15-17, 48). In the bovine PRL gene a sequence between -247 and -214 of the promoter, termed PRL3, was found to be important for glucocorticoid repression (17). This nGRE alone, when cloned in front of a heterologous promoter, enhances expression and is repressed by glucocorticoids in both pituitary and non-pituitary cells (Fig. 1). The intrinsic enhancing activity of the PRL3 nGRE in the absence of glucocorticoids suggests that transactivating factor(s) bind to the element and that these factors are targets for the repressive activity exerted by glucocorticoids. We have shown previously that in pituitary cells, the main factor interacting with the PRL3 nGRE and responsible for the enhanced expression is the pituitary-specific transcription factor Pit-1/GHF-1 (18). In this report we identify the ubiquitously expressed Oct-1 as one of the factors responsible for the enhanced expression in cells devoid of Pit-1/GHF-1. The Oct-1 protein was found to bind to an AT-rich sequence in the PRL3 element. This was the same binding site to which Pit-1/GHF-1 from pituitary cells binds (18). The ability of Oct-1 to bind to PRL3 is anticipated because the Oct-1 and Pit-1/GHF-1 factors both belong to the same family of POU-homeodomain-containing transcription factors with similar binding specificity to AT-rich DNA elements (49). In non-pituitary cells, prevention of Oct-1 binding by mutation of the Oct-1 binding site in PRL3 abolished most of the enhanced expression as well as the repression by glucocorticoids. In line with our finding of a role for Oct-1 in bovine PRL gene regulation, Rosenfeld and collaborators (10) have shown that the Oct-1 is capable of transactivating the expression of the rat PRL gene. Furthermore, based on polymerase chain reaction cloning, DiMattia et al. (11) suggested that Oct-1 controls the decidual PRL gene expression. However, the PRL3 nGRE being a target for Oct-1 binding has not been demonstrated previously.

The relatively weak 3-fold increase in expression conferred by Oct-1 on PRL3 is in agreement with Oct-1 being a weak transcriptional activator by itself (50). In many cases, it has been demonstrated that the weak transcriptional activity of Oct-1 is potentiated by the interaction of Oct-1 with other proteins. Examples of cellular gene products that associate with Oct-1 and potentiate its transcriptional activity include Pit-1/GHF-1 (10) and Jun family proteins (51), which contribute to the induction of the rat PRL and the interleukin-2 gene, respectively. Furthermore, transcriptional enhancement of interleukin-3 and granulocyte-macrophage colony-stimulating factor genes by Oct-1 are potentiated by its association with 43- and 45-kDa proteins (52). As in the case for the above genes, we have identified a second protein, Pbx, which binds to the PRL3 nGRE and contributed together with Oct-1 to the regulation of the expression (Fig. 6). The Pbx proteins belong to an important family of vertebrate homeobox genes. Genetic and biochemical studies have suggested a role for Pbx proteins and their Drosophila homolog extradenticle (exd) as a cofactor for the Hox homeotic selector genes (53-55). The Pbx/exd factors have been proposed to stimulate the sequence-specific DNA binding of Hox proteins cooperatively and to select different consensus binding sites in association with different Hox proteins (35 and refs. therein, 56-59). Unlike the Hox homeotic selector genes, which are for the most part expressed temporally during development and differentiation, the Pbx proteins are expressed ubiquitously (30). This ubiquitous expression of Pbx proteins may indicate a more generalized role for the Pbx factors. Recently, Pbx proteins were shown to interact with and modulate the function of other homeodomain proteins, as for example in the activation of the somatostatin gene by acting as a cofactor for STF-1 (60). Pbx also interacts with Meis proteins present in a number of cell types and binds to a subset of Pbx-Hox sites (61). Furthermore, Kagawa et al. (34) demonstrated that Pbx1 (one of the Pbx family members) binds to the cAMP-regulatory sequence CRS1 of the P45017alpha promoter. In our study, we demonstrate that the bovine PRL gene through the PRL3 nGRE is a target for Pbx. Several experiments established that Pbx binds to the PRL3 nGRE. First, the CRS1-Pbx1 complex could be abolished by competition with the PX oligonucleotide (Fig. 4A). Second, the nucleotides in PRL3 involved in Pbx binding were part of the PRL3 sequence which showed homology to other Pbx binding sequences (Fig. 4A). Furthermore, Pbx binding affinity to the PX element was similar to Pbx binding affinity to CRS1 or PRS because all three elements competed for complex B with the same efficiency (Fig. 4B). Finally, the antibody supershift experiment demonstrated that endogenous Pbx protein(s) bind to the PRL3 nGRE as demonstrated by the ability of a Pbx1 antibody to supershift complex B formed by nuclear extracts and the PX element in a fashion similar to Pbx bound to the prototypic Pbx-binding element PRS (Fig. 5). Because the anti-Pbx1 antibody shows some cross-reactivity with Pbx2 and Pbx3,2 the exact nature of the Pbx protein(s) present in complex B cannot be resolved. Furthermore, complex B on PX was formed with nuclear extracts from most cell types, in line with Pbx being expressed ubiquitously. Interestingly, the Pbx binding sequence is conserved in the rat and human PRL genes, suggesting that Pbx may play a role in the expression of these genes as well.

Mutation of the Pbx site in PX reduced expression by approximately 50%, although Pbx itself in the absence of Oct-1 binding is only responsible for about 20% of the transcriptional activity (Fig. 6). This is indicative of Pbx cooperation with Oct-1 in regulating transcriptional activity from the PRL3 nGRE. Pbx also seems to cooperate with other POU-homeodomain-containing proteins. This conclusion is based on the fact that the Pbx-containing complex B, also formed when nuclear extract from pituitary GH3 cells is incubated with the PRL3 nGRE (data not shown) concomitant with binding of Pit-1/GHF-1, contributes to Pit-1/GHF-1-dependent transactivation of PRL3 nGRE (18). In pituitary cells, Pbx binding alone to the PRL3 nGRE shows no transcriptional activity but enhances Pit-1/GHF-1-dependent transactivation, suggesting a true cooperativity (18). Whether Pbx and the POU-homeodomain-containing proteins interact directly is not known but presently under investigation.

Interestingly, both Pbx and Oct-1 binding to PRL3 were found to be required for glucocorticoids to be able to repress the expression in non-pituitary cells (Fig. 6). As a comparison, a sole GR-Pit-1-GHF-1 or GR-Oct-1 interaction has been suggested to be the mechanism responsible for glucocorticoid repression of the rat PRL gene or the Oct-1-dependent expression from the H2B promoter, respectively (48, 62). This suggests that the mechanism for GR-mediated repression of different genes, including Oct-1 or Pit-1/GHF-1-dependent genes, may vary. This variation in repressive mechanisms is exemplified further by experiments by Wieland et al. (63), who demonstrated an ability of GR to repress Oct-2A- but not Oct-1-dependent expression and that this repressive activity was dependent on cell type. In analogy to our previous observation that the GR prevented both complex B (Pbx) and Pit-1/GHF-1 from pituitary GH3 cells from binding to the PRL3 nGRE (18), the GR DBD was able to prevent Pbx and Oct-1 from COS-7 cells from binding to the PRL3 nGRE (Fig. 7). To note is that the GR DBD was sufficient in preventing Pbx and Oct-1 binding. This is in line with transfection results demonstrating the ability of the GR DBD to repress PRL3-dependent transcriptional activity in vivo (64). The exact mechanism by which the GR prevents Pbx and Oct-1 from binding to the PRL3 element is not clear, but previous results have suggested that GR binding to the PRL3 nGRE is required (18, 19). Furthermore, GR-mediated displacement of Pit-1/GHF-1 required complex B (Pbx) (18). One possibility is that the GR interferes with Pbx binding or activity because the activity of the wild type PRL3 nGRE in the presence of glucocorticoids is similar to the activity of PRL3 nGRE in the absence of glucocorticoids when the Pbx binding site is mutated (Fig. 6). This may include a direct GR-Pbx interaction. In fact, preliminary results have indicated that GR and Pbx1 can physically interact in vitro in pull down experiments.3

The results presented in this paper show that the ubiquitously expressed Oct-1 in non-pituitary cells seems to replace the role of Pit-1/GHF-1 in pituitary cells in enhancing the expression conferred by the PRL3 nGRE. In addition, a second homeodomain protein, Pbx, expressed in both pituitary and non-pituitary cells, binds to an adjacent site and enhances transcriptional activation further. Both Oct-1 and Pbx binding are required for glucocorticoid repression. The requirement for more than one protein to bind to the PRL3 nGRE to obtain negative glucocorticoid regulation resembles the situation for glucocorticoid activation from so-called glucocorticoid-responsive units, where binding of one or more factors in addition to the GR is necessary for an effect (65). Thus, in parallel to this situation, the PRL3 nGRE can be designated as a negative glucocorticoid responsive unit, where a complex interaction among several proteins is required for an effect to be obtained. Future studies will address a possible direct physical interaction among the involved proteins. Furthermore, it would be interesting to know if Pbx, possibly through an interaction with the POU homeodomain proteins, may not only contribute to the terminally differentiated tissue-specific expression and glucocorticoid control of the prolactin gene, but also to the development of the anterior pituitary gland cell phenotypes.

    ACKNOWLEDGEMENT

We thank Dr. Winship Herr for the gift of the Oct-1 antiserum. We also thank Drs. Mark P. Kamps, Norio Kagawa, Larry Bishop, and Anthony Wright for providing anti-Pbx1 antiserum and for invaluable discussions. We thank Dr. L. Poellinger for providing the GR DBD in vitro translation vector and Dr. Joseph Rafter for valuable comments on the manuscript.

    FOOTNOTES

* This work was supported by grants from the Swedish Cancer Society and the Magnus Bergvall Foundation (to S. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: SmithKline Beecham, New Frontiers Science Park, Harlow, Essex CM19 5AD, United Kingdom.

§ To whom correspondence should be addressed. Tel.: 46-8-5858-3728; Fax: 46-8-711-6659; E-mail: Sam.Okret{at}mednut.ki.se.

The abbreviations used are: PRL, prolactin; Oct-1, octamer transcription factor-1; nGRE, negative glucocorticoid response element; GR, glucocorticoid receptor; DBD, DNA binding domain; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay.

2 M. Kamps, personal communication.

3 N. Subramaniam, unpublished data.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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