Transcription Factor AP1 Is Involved in Basal and Okadaic Acid-Stimulated Activity of the Human PRL Promoter

Laure Caccavelli1, Isabelle Manfroid1, Joseph A. Martial and Marc Muller

Laboratoire de Biologie Moléculaire et de Génie Génétique (I.M., J.A.M., M.M.) Université de Liège Institut de Chimie B6 B-4000 Sart-Tilman, Belgium
Laboratoire d’immunopathologie (L.C.) INSERM U430 Hopital Broussais 75014 Paris, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The tumor promoter, okadaic acid (OA), an inhibitor of protein phosphatases, stimulates the activity of the human PRL (hPRL) proximal promoter. We analyzed in detail the effects of OA on transcription factor binding to elements P1 and P2 of this promoter, sequences known to contain at least one Pit-1 binding site each. OA treatment induces binding of an AP1-related transcription factor to the P1 site. This effect is specific, as protein binding to the P2 site is not altered by the treatment. Specific antibodies were used to confirm that the OA-induced complex is related to AP1 and to show that it contains JunD and c-fos, but not Pit-1. The increase in AP1 binding to P1 and to a canonical AP1 site correlates to an increase in cellular JunD and c-fos content. Transient transfection experiments showed that both AP1 and Pit-1 are involved in the regulation of basal and OA-stimulated promoter activity. Our results demonstrate that a member of the AP1 family, containing JunD and c-fos, can bind to the proximal element P1 within the hPRL promoter. In addition, they show that AP1 is involved in both basal and OA-stimulated expression of the hPRL gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human PRL (hPRL), a polypeptide hormone synthesized by pituitary lactotroph cells, regulates many physiological functions, such as lactation and growth. In the anterior pituitary, hPRL secretion and gene expression are regulated by various hormones and growth factors, such as dopamine, TRH, and epidermal growth factor (1, 2). These factors bind to transmembrane receptors and modulate different signaling pathways (cAMP, Ca2+, protein kinase C, mitogen-activated protein kinases), which have been shown to regulate hPRL gene transcription. Ultimately, these various second messengers regulate hPRL gene activity via the proximal promoter region (3). In lactotroph cells, the activity of the hPRL proximal promoter depends mainly on two types of elements: binding sites for the tissue-specific factor Pit-1 and a binding site (sequence A) for ubiquitous proteins (4, 5). These elements are required for the basal activity of the promoter and for its regulation by different signal transduction pathways. Since second messengers were shown to activate protein kinases, it is likely that the activities of transacting factors binding to the proximal hPRL promoter are regulated by their phosphorylation status. It has been shown that Pit-1 is phosphorylated on Ser115 and Thr220 by protein kinase A and protein kinase C both in vitro and in vivo (6), although studies concerning the influence of the Pit-1 phosphorylation status on its activities have provided conflicting results. Phosphorylation of Pit-1 can modulate its DNA-binding activity (6, 7), but this effect appears to be promoter specific (8, 9) and not to affect the Pit-1 transcriptional activity (10, 11).

In addition, although less exhaustively studied, PRL gene expression was shown to be regulated by protein phosphatases (12, 13). Protein phosphatases are involved in the interruption of signal transduction by reverting phosphorylation events on defined target proteins. Integration of external signals in a cell is partially based on the equilibrium between the activities of protein kinases and phosphatases (14, 15, 16). Thus, understanding regulation of gene transcription in a cell would not be complete without studying the effects of protein phosphatases. The growing numbers of protein phosphatases are classified according to their specificity as serine/threonine phosphatases, tyrosine phosphatases, or dual specificity phosphatases. About half of them are serine/threonine phosphatases, four major types (PP1, PP2A, PP2B, and PP2C) of which have been genetically defined (17). The Ca2+-dependent protein phosphatase, PP2B, and okadaic acid (OA)-sensitive phosphatases, such as PP1 or PP2A, regulate hPRL gene transcription via the proximal region of the promoter (12, 13).

The tumor promoter OA inhibits the two major intracellular protein phosphatases, PP1 and PP2A; the in vitro inhibition of PP2A occurs at nanomolar concentrations and that of PP1 at micromolar doses (18). Recently, it was shown that low concentrations of OA also inhibit other serine/threonine protein phosphatases including PP3, PP4, and PP5 (19, 20, 21). Despite this limitation, the use of OA has provided useful information on the implication of protein phosphatases in many cellular regulations. In rat pituitary GC cells, low OA concentrations (30 nM) stimulate the basal activity and potentialize the cAMP stimulation of the proximal PRL promoter region, linked to the thymidine kinase (TK) promoter. Both effects were shown to involve sequence A (12).

The aim of our work was to further analyze the effect of protein phosphatases on the hPRL proximal promoter. We investigated the OA regulation of the natural hPRL promoter activity in GH3B6 cells that express the endogenous PRL gene. OA stimulation was studied at the level of transcription factor binding, using nuclear extracts from OA-treated and untreated cells, and at the functional level, using transient transfection experiments. Our results show that OA induces binding of JunD and c-fos to a newly identified AP1 motif within sequence P1 of the hPRL proximal promoter and in parallel increases JunD and c-fos cellular content. Moreover, we demonstrate that basal activity and OA stimulation of the hPRL proximal promoter require this AP1 site. Although both AP1 and Pit-1 are needed to obtain OA stimulation of the hPRL proximal promoter activity, the binding of each to sequence P1 is independent.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OA Stimulates Transcription of the hPRL Proximal Promoter
To examine the OA effect on both binding and activity of the transcription factors involved in the regulation of the hPRL promoter activity, we used the rat pituitary cell line GH3B6, since it expresses the endogenous rat PRL gene. As a significant OA induction of the TK promoter activity is observed in these cells, we chose to study the natural hPRL promoter and to use a minimal hPRL promoter containing only the TATA box, as a control.

Transient transfection experiments were performed using a reporter plasmid containing the luciferase gene under the control of the proximal 164 bp of the pituitary hPRL promoter (164P-Luc). Within this part of the promoter (Fig. 1AGo), footprint experiments have defined two protected regions (P1 and P2) containing Pit-1 binding sites (4). In addition, gel retardation assays have revealed binding of a ubiquitous protein to sequence A (5). Moreover, the 164- bp region is sufficient to mediate the response to almost all second messengers and is the target for phosphatase inhibitor effects (12). OA stimulation of this construct was analyzed in GH3B6 cells and compared with a control construct containing only 40 bp upstream of the hPRL start site (40P-Luc), which comprise only the TATA box. In this system, OA stimulates transcription of the 164P-Luc construct dose-dependently with the maximal stimulation (45-fold) when cells are treated with 80 nM of OA (Fig. 1BGo). At concentrations lower than 20 nM, we observed a similar OA stimulation of both the 164P-Luc and the 40P-Luc. These effects were considered as nonspecific and may result from OA action on the basal transcription machinery, as previously discussed by Wera et al. (12). At higher concentrations, stimulation of the 164P-Luc was much stronger than stimulation of the control, which reflects the OA action on the hPRL promoter.



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Figure 1. OA Stimulation of the Proximal hPRL Promoter Activity

A, Sequence of the region [-164; +1] of the hPRL promoter. Sequences P1 and P2, containing two high-affinity Pit-1 binding sites, and sequence A are underlined. Consensus motifs for AP1 and Pit-1 within the P1 sequence are framed. B, GH3B6 cells were electroporated with 6 pmol of plasmid 164P-Luc ({blacktriangleup}) or 40P-Luc ({triangleup}). Three hours after transfection, OA was added at different concentrations as indicated. Twenty hours after transfection, the cells were harvested, and the luciferase activity was measured as described. Fold induction ± SD relative to untreated cells is shown. Triplicate experiments were performed at least twice.

 
OA Induces AP1 Binding on Sequence P1 of the hPRL Promoter
To examine whether OA treatment affects binding of transcription factors to the hPRL promoter, gel retardation assays were performed using nuclear extracts from OA treated or untreated GH3B6 cells. As Pit-1 represents a potential target for protein phosphatases, we focused our study on sequences P1 and P2, which both contain Pit-1 binding sites. Figure 2AGo shows DNA-protein complexes formed on oligonucleotides P1 and P2. When untreated nuclear extracts are used (lanes 2 and 5), two major complexes (C1 and C2) appear on P1 and P2. As demonstrated by competition experiments (see below) and as already shown by others (22), the C1 complex corresponds to the monomer of Pit-1 (actually a double band is observed due to the presence of a shorter Pit-1 variant, initiated at an alternative ATG, in pituitary cells). The C2 complex corresponds to the dimer of Pit-1. Treatment of the cells with OA does not change the binding of Pit-1 on sequence P2 (lane 6). In contrast, an additional complex (C3), with a slower migration, appears on P1 when nuclear extracts from OA-treated cells are used (Fig. 2AGo, lane 3). This complex is not observed on P2 (lane 6), indicating that its formation is specific for P1 and suggesting that its appearance does not depend on Pit-1 alone.



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Figure 2. OA Treatment Induces Binding of an Additional, AP1- or CREBP-Like Factor to the P1 Sequence, but Not to the P2 Site

A, Nuclear extracts from OA-treated (lanes 3 and 6) or untreated (lanes 2 and 5) GH3B6 cells were incubated with 32P-labeled DNA fragments corresponding to the P1 or P2 sequence. The positions of Pit-1 monomer (C1), Pit-1 dimer (C2), and of the OA-inducible complex (C3) are indicated. B, complex C3 corresponds to AP1 or CREB transcription factor. Nuclear extracts of OA-treated GH3B6 cells were incubated with 32P-labeled DNA fragments corresponding to P1. Competition experiments were performed using a 100-fold excess of unlabeled oligonucleotides corresponding to the P1 or P2 sequences, or containing consensus sequences for AP1, CREB, Oct-1, or the unrelated factor NFY (nonspecific). The complex C4 observed in this experiment corresponds to a Pit-1 related factor, probably Oct-1, which was not induced upon OA treatment and was not further analyzed.

 
Competition experiments were performed using extracts from OA-treated cells to determine the nature of the C3 complex (Fig. 2BGo). Disappearance of all complexes is observed upon addition of a 50-fold molar excess of unlabeled P1 oligonucleotide (lane 2), whereas only C1, C2, and C4 complexes are displaced by an excess of unlabeled fragment P2 (lane 3).

Appearance of a C4 complex on the P1 sequence is observed in some experiments. This complex is displaced by oligonucleotides P1, P2, and by an oligonucleotide containing the consensus binding site for Oct-1. Oct-1 is a transcription factor related to Pit-1 and able to bind similar sequences (Fig. 2BGo, lane 6). Thus, this C4 complex corresponds to a protein related to these transcription factors. The C4 complex is not induced by OA treatment, as it is observed in treated and untreated extracts (data not shown). Furthermore, the C3 complex is not affected by oligonucleotides that inhibit C4 binding, clearly indicating that the two complexes are distinct. Thus, the C4 complex was not investigated further.

Binding of the C3 complex is not affected by an excess of oligonucleotide P2 (Fig. 2BGo lane 3), again indicating that it does not correspond to the binding of Pit-1. Similarly, complex C3 is not displaced by an unlabeled oligonucleotide containing a binding site for Oct-1, whereas this oligonucleotide displaces both C1 and C2 complexes (lane 6). An oligonucleotide containing a consensus site for the transcription factor NFY, used as a nonspecific competitor, does not displace any of the complexes (lane 7). These observations show that complexes C1, C2, and C4 contain Pit-1 or a related protein, while C3 clearly involves a different transcription factor.

A careful analysis of the P1 sequence revealed a degenerated putative binding site for cAMP response element-binding protein (CREB) or AP1 transcription factors (Fig. 1AGo). Therefore, competition experiments were performed using DNA fragments containing a consensus site for these two factors. Both unlabeled oligonucleotides displace the C3 complex from P1 specifically (Fig. 2BGo, lanes 4 and 5), without affecting Pit-1 binding (monomeric or dimeric form). Moreover, a complex migrating as the C3 complex is observed when gel retardation assays are performed using labeled oligonucleotides containing CREB or AP1 consensus sequences (data not shown). These data strongly suggest that this additional C3 complex corresponds to CREB or AP1.

Specific antibodies were then used to further investigate the nature of the factors involved. Antibodies against Pit-1 strongly affected complexes C1 and C2, both in untreated (Fig. 3AGo, compare lanes 2 and 3) or OA-treated cells (compare lanes 5 and 6), confirming that they consist of Pit-1. The supershift in this case migrates at a position similar to complex C3 (lane 3). A sharp band with a very low mobility was also present when Pit-1 antibody was used alone (lane 11). Anti-CREB antibodies directed against the entire protein (anti-CREB 253) or against the kinase-inducible domain (anti-CREB 244) did not alter formation of the C3 complex (lanes 8 and 9). However, they were able to inhibit binding of CREB to a consensus cAMP response element (CRE)-binding site (data not shown). Thus, complex C3 does not contain CREB. On the contrary, antibodies directed against the DNA-binding domain of c-jun inhibit formation of the C3 complex, indicating that it corresponds to AP1 (Fig. 3AGo, lane 7). As these antibodies recognize c-jun, JunB, and JunD, more specific antibodies directed against the more variable C-terminal region of these proteins were used to determine the precise nature of the C3 complex. Figure 3BGo shows that only anti-JunD antibody is able to form a supershifted complex (lane 3). Specific antibodies directed against members of the fos family were used to demonstrate that a supershift was obtained using anti-c-fos antibody, but not using anti-fosB antibody (Fig. 3CGo, lanes 2 and 3). Appearance of both the junD and c-fos supershift correlates with a slight decrease of complex C3 formation. Complete disappearance of complex C3 was not obtained, due to the low concentration of the antibodies used in these experiments. The supershifts were not obtained using the antibodies alone (Fig. 3BGo and C, lane 4) or using untreated extracts, further showing that they are specific and that they arise from complex C3. Thus, OA induces binding of an AP1-related complex, containing JunD and c-fos, on the P1 site of the hPRL promoter.



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Figure 3. The OA-Induced Complex Is Formed by AP1 and Contains JunD and c-fos

A, Untreated nuclear extracts were preincubated with anti-Pit-1 antibodies or preimmune serum (pre. serum) before addition of the P1 probe and analysis of the complexes by gel retardation (lanes 3 and 4). OA-treated GH3B6 cell nuclear extracts (lane 5) were preincubated with antibodies directed against Pit-1, c-jun, CREB, or with preimmune serum (pre. serum), as indicated. Incubation of the probe with anti-Pit1 alone is shown (lane 11). B, Nuclear extracts of OA-treated GH3B6 cells were incubated with specific antibodies directed against JunB and JunD. The position of the JunD supershift is indicated (JunD ss). Incubation of the probe with anti-JunD alone is shown (lane 4). C, Nuclear extracts of OA-treated GH3B6 cells were incubated with specific antibodies directed against c-fos and fosB. C-fos ss indicates the supershifted complex. Lane 4 shows preincubation with anti-c-fos alone.

 
Appearance of the AP1 Complex Correlates with the OA Induction of the hPRL Promoter
The appearance of the C3 complex as a function of OA concentration was assessed using gel retardation experiments. Probes corresponding to both P1 and a consensus AP1-binding site were used. The amount of AP1 bound to its consensus sequence increases with the OA concentration used to treat the GH3B6 cells (Fig. 4AGo).



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Figure 4. OA Induction of the hPRL Promoter Correlates with an Increase in AP1 Binding, both to P1 and to Its Consensus Sequence, and in junD and c-fos Cellular Amount

Nuclear extracts from GH3B6 cells treated with increasing OA concentrations were incubated with 32P-labeled DNA fragment corresponding to A] an AP1 consensus sequence (A) or to the hPRL promoter P1 element (B). C, Western blot analysis was performed using Pit-1, JunD, or c-fos-specific antibodies on nuclear extracts from GH3B6 cells treated with OA at the indicated concentrations.

 
Similarly, formation of the C3 complex on P1 increases with OA concentration (Fig. 4BGo). In both cases, maximal complex formation is observed at 80–100 nM concentrations. Moreover, the amount of AP1 or C3 complex formation correlates with the fold induction achieved by OA in the transfection experiments (Fig. 1B). In parallel, formation of the Pit-1 complexes C1 and C2 appears to decrease upon OA treatment.

Western blot experiments were performed to determine whether modification of the binding on the P1 site reflects OA regulation of the cellular concentration of Pit-1, JunD, or c-fos proteins. Figure 4CGo shows that the cellular amounts of JunD and c-fos proteins increase upon OA treatment of the cells. These increases are dose-dependent and could account for the OA induction of AP1 binding to P1 sequence and formation of the C3 complex. Western blot experiments reveal a decrease of the Pit-1 binding after OA treatment (Fig. 4CGo), correlating with the decrease of the dimer complex in gel retardation experiments (Fig. 4BGo). This decrease could be due to a long-term decrease of Pit-1 content, which has been previously described (22, 23). Nevertheless it should be noted that the C2 complex is much more affected by OA treatment than C1, which could also reflect the lower binding affinity of C2 on site P1 (24).

OA Stimulation of the 164P-Luc Construct Is Mediated by Pit-1 and AP1 Binding Sequences
To study the functional implication of AP1-like factors in the OA induction of the hPRL gene, different reporter constructs, containing the first 164 bp of the hPRL promoter, were transiently transfected in GH3B6 cells. Wild-type 164P-Luc and constructions mutated within the P1 site, either in the AP1 or in the Pit-1 consensus sequence (Fig. 5AGo), were transfected in parallel to determine the respective role of each transcription factor. The abolition of the respective Pit-1 or AP-1 binding was verified using gel retardation assays. A P1 oligonucleotide mutated in the Pit-1 motif showed that this mutation inhibits binding of Pit-1 (Fig. 5BGo, lanes 5 and 6) without affecting AP1 binding. Conversely, a P1 oligonucleotide mutated in the AP1 motif does not bind AP1 and is still able to bind Pit-1 (Fig. 5BGo, lanes 8 and 9).



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Figure 5. OA Stimulation of the Proximal hPRL Promoter Is Abolished when Point Mutations Are Generated in the Consensus Sites for AP1 or for Pit-1

A, Sequence of the P1 element. Consensus sequences are boxed, and the mutations generated in each binding site are indicated in the top line. B, Nuclear extracts of OA-treated (+) or untreated (-) GH3B6 cells were incubated with 32P-labeled DNA fragments corresponding to wild-type P1 sequence (wild type), P1 mutated in the Pit-1 consensus (Pit-1 m), or P1 mutated in the AP1 consensus (AP1 m). C, GH3B6 cells were electroporated with 6 pmol of the indicated plasmid. Three hours after transfection, OA was added at the indicated concentration. Twenty hours after transfection, cells were harvested and the luciferase activity was measured. Fold induction ± SD relative to untreated cells is shown. Triplicate experiments were performed at least twice.

 
For the transfection experiments, the 40P-Luc construct, containing the first 40 bp of the hPRL promoter, was used as a control. Its stimulation by OA represents effects on the basal transcription machinery and was considered as nonspecific. Both basal expression and stimulation by OA will be considered.

Basal activity of the 164P-Luc construct (3750 ± 847) is reduced 3-fold when either the AP1 (164AP1 m-Luc) or the Pit-1 (164Pit-1 m-Luc) site is mutated (1141 ± 335 and 1212 ± 122, respectively), but remains higher than the activity of 40P-Luc (449 ± 22). This indicates that both transcription factors are required for the basal transcription of the hPRL promoter. Moreover, both mutations lead to a similar reduction of basal activity, suggesting that AP1 is as important as Pit-1 in basal hPRL gene expression.

To analyze OA stimulation, the fold induction at each OA concentration was represented in Fig. 5CGo and basal values at 0 nM OA were set to 1 for each curve. OA stimulation of the 164P-Luc construction is abolished when either the Pit-1 or the AP1 consensus sites are mutated. Both mutated constructs respond to OA stimulation to the same level as the 40P-Luc (Fig. 5CGo) at each OA concentration tested.

In conclusion, both Pit-1 and AP1 sites are required for an efficient stimulation of the hPRL proximal promoter by OA.

AP1 and Pit-1 Bind to Sequence P1 Independently
The functional importance of both Pit-1 and AP1 in OA stimulation prompted us to investigate more precisely their interactions with sequence P1. First, we used specific antibodies to analyze the complexes formed and to determine whether the two transcription factors associate to form a heterodimer. The anti-Pit-1 antibody is directed against the N-terminal part of Pit-1 and displaces C1 and C2 complexes, which both correspond to Pit-1 binding (Fig. 3AGo). Unfortunately, the supershift obtained comigrates with the C3 complex, thus preventing a conclusion about the presence of Pit-1 in the C3 complex. To circumvent this problem, gel retardation assays were performed using unlabeled P1 oligonucleotide and blotted on a nylon membrane. The complexes obtained were revealed using specific anti-Pit-1 or anti-c-jun antibodies (Fig. 6Go). Anti-Pit-1 antibody is able to detect complexes C1 and C2, but most importantly does not recognize the C3 complex (lanes 3 and 4). On the contrary, anti-c-jun antibody specifically detects complex C3, but not C1 or C2 (lanes 5 and 6). Most of the anti-c-jun immunoreactive compounds remained at the top of the gel, probably reflecting the lower affinity of sequence P1 for AP-1. Note also that the c-jun antibody binds weakly to the jun-DNA complexes, since it is directed against the DNA-binding domain. We conclude that the C3 complex contains jun-related factors, but no significant amounts of Pit-1, suggesting that C3 does not consist of an AP-1/Pit-1 heterocomplex.



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Figure 6. The OA-Induced AP-1 Complex Does Not Contain Pit-1

Nuclear extracts of OA-treated GH3B6 cells were incubated with 32P-labeled DNA fragment corresponding to the P1 sequence (lanes 1 and 2) as a control. In parallel, nuclear extracts of OA-treated GH3B6 cells were incubated with unlabeled P1 fragment. The unlabeled complexes were blotted onto nitrocellulose membrane and revealed with specific antibodies directed against Pit-1 (lanes 3 and 4) or c-jun (lanes 5 and 6).

 
To further analyze the binding characteristics of these transcription factors to sequence P1, oligonucleotides corresponding to the P1 sequence mutated in the consensus AP1 or Pit-1 motifs (Fig. 5AGo) were used in gel retardation assays. Complex C3 does not form when the AP1 site is mutated, whereas Pit-1 is still able to bind as monomer and dimer (Fig. 5BGo, lane 9). On the other hand, mutation of the Pit-1 consensus site inhibits binding of Pit-1, but does not affect formation of complex C3 in OA-induced extracts (Fig. 5BGo, lane 6). These results were confirmed by competition experiments. An oligonucleotide corresponding to P1 mutated in the AP1 consensus sequence (P1,AP1 m) competes Pit-1 binding to P1, but does not affect formation of the C3 complex to the P1 site (Fig. 7Go, lane 6). Conversely, an oligonucleotide mutated in the Pit-1 consensus sequence within the P1 site (P1,Pit-1 m) completely and specifically inhibits formation of the C3 complex (lane 4), very similar to a consensus AP1 sequence (lane 5). Gel retardation experiments using the AP1 consensus sequence as a probe (Fig. 7Go, lanes 8–14) were performed in parallel (lane 9). Wild-type P1 oligonucleotide is able to inhibit AP1 complex formation nearly as well as the AP1 oligonucleotide (lanes 10 and 12). The P1 oligonucleotide mutated in the Pit-1 consensus site competes AP1 binding (lane 11), whereas the P1 oligonucleotide mutated in the AP1 consensus sequence does not (lane 13).



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Figure 7. AP1 and Pit-1 Binding to Sequence P1 Is Independent

Nuclear extracts of OA-treated GH3B6 cells were incubated with 32P-labeled DNA fragments corresponding to the P1 sequence (lanes 1–7) or to the AP1 consensus sequence (lanes 8–14). Competition experiments were performed with unlabeled oligonucleotides corresponding to wild-type P1 sequence, P1 mutated in the Pit-1 motif (P1,Pit-1 m), a consensus AP1 site, P1 mutated in the AP1 motif (P1,AP1 m), or a consensus site for NFY (nonspecific).

 
Taken together, these results confirm that both transcription factors bind independently to the P1 sequence in the hPRL promoter, as blocking of the binding of one factor by site mutation or specific competition does not change the binding of the other.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we have demonstrated that: 1) OA stimulation of the hPRL gene transcription results in the formation of an additional, AP1-related complex on site P1; 2) this new complex contains JunD and c-fos; 3) OA increases the AP1 binding activity and the cellular amount of JunD and c-fos proteins; 4) basal level expression and OA stimulation require binding of both AP1 and Pit-1; 5) binding of AP1 and Pit-1 occur independently on the P1 sequence.

We analyzed OA regulation of transcription factor binding to the hPRL proximal promoter [-164/+1], shown to be sufficient for OA stimulation (Ref. 12 and Fig. 1BGo). Gel retardation experiments using sequences P1 and P2, previously known to bind Pit-1, show that OA treatment induces the appearance of an additional complex (C3) specifically on sequence P1, but not on P2. Supershift experiments show that only anti-junD and anti-c-fos antibodies bind to complex C3 (Fig. 3Go); competition experiments show that Pit-1-specific oligonucleotides (P2 or P1,AP1 m) (Figs. 2Go and 7Go) or mutation of the Pit-1 site (Fig. 5BGo) do not affect formation of the complex C3, strongly suggesting that the OA-induced P1 complex is composed of the AP1-related transcription factors JunD and c-fos.

The functional implication of AP1 in OA stimulation is supported by several observations. First, we have observed an increase of AP1 binding to a consensus AP1 binding site and to P1 (Fig. 4Go, A and B), which perfectly correlates with the stimulation of the hPRL promoter (Fig. 1Go). Second, a parallel, clear and specific increase in JunD and c-fos protein is observed in Western blot analysis. Finally, mutation of the AP1 binding site in sequence P1 reduces OA stimulation to the level of the minimal promoter 40P-Luc (Fig. 5CGo).

The c-jun and c-fos protooncogenes were originally identified as components of the AP1 transcription complex. Through protein-protein interactions within the leucine zipper domain, c-jun can form either homodimers or heterodimers with other leucine zipper-containing proteins, such as other Jun family proteins (JunB, JunD), members of the Fos (c-fos, fosB, fra1, fra2), or members of the CREB/activation transcription factor (ATF) families. Phosphorylation has been implicated in the regulation of AP1 function. Phosphorylation of residues located in the C-terminal domain of c-jun, by the CKII and the GSK-3, inhibits its DNA-binding activity (25, 26). Conversely, phosphorylation of serine residues located in the N-terminal part of c-jun by Ha-ras (27, 28) or JNK (29) leads to the increase of c-jun transactivating properties. Although these residues are well conserved in JunD, it has been recently shown that both hypophosphorylated or hyperphosphorylated forms of JunD are able to bind DNA (30). Thus, phosphorylation at the C-terminal sites of junD may not account for the increase in binding to the P1 site observed in OA-treated cells. Although we cannot completely rule out an increase of the AP1 transactivation function by phosphorylation of the N-terminal region, OA stimulation of the hPRL promoter activity appears to result from up-regulation of AP1 expression.

In other systems, two complementary mechanisms have been shown to lead to accumulation of c-jun and could also account for JunD accumulation. First, a positive feedback by activated c-jun leading to enhancement of c-jun gene expression, through an AP1 site located in the c-jun promoter, has been reported (31, 32). Moreover, in human T-cell derived Jurkat cells, OA treatment increases AP1 binding to its response element and is associated with an increase in mRNA levels of c-jun, JunB, and JunD (33). Thus, increasing jun phosphorylation after OA treatment could result in positive autoregulation of JunD expression, as has been described in mouse keratinocytes (34). Second, an increase in protein stability by phosphorylation could also lead to junD accumulation, such as has been postulated in the OA induction of the urokinase-type plasminogen activator gene by c-jun (35). However, involvement of such mechanisms remains to be established, but they may lead to the clear increase of jun-like factors observed in Western blot experiments (Fig. 4CGo) and could account for OA stimulation of the hPRL promoter activity.

The c-fos accumulation (Fig. 4CGo) may be due to activation of the independent ERK1/2 pathway (36) by inhibition of protein phosphatases. In addition, carboxy-terminal phosphorylation of ser362 and 374 also increases c-fos transactivating properties (37) and may contribute to AP1 stimulation of the hPRL promoter activity in OA-treated cells. However, c-fos is a labile protein, and the hyperphosphorylated form appears to be unstable (38). Thus, accumulation of c-fos after OA treatment rather reflects an increase in c-fos expression.

OA stimulation of the hPRL proximal promoter not only involves AP1, but also requires binding of Pit-1 to sequence P1. Our results (Fig. 5Go) clearly show that mutation of the Pit-1 site in sequence P1 reduces OA stimulation to the same extent as mutation of the AP1 motif. In contrast to the increase of AP1 binding to sequence P1, a decrease of Pit-1 complex formation is observed upon OA treatment, especially considering the dimeric form. As competition experiments show that both transcription factors bind independently to the P1 site, this decrease could be due to the decrease in Pit-1 protein amounts observed in Western blot experiments, which would mainly affect the homodimeric complex due to its lower affinity (Fig. 4CGo).

Recently, a decrease in protein stability was shown after hyperphosphorylation of Pit-1 after activin treatment of somatotropic cells (22). Furthermore, a 2-fold repression of the human Pit-1 promoter by AP1 was shown in GH3 cells (23). Thus, the increase of Pit-1 phosphorylation and the increase in AP1 activity upon OA treatment might result in a long-term decrease in Pit-1 protein amounts, leading to the observed decrease in dimer formation. Such a secondary decrease in Pit-1 amounts would most likely result in a decrease in hPRL transcription and cannot account for the observed involvement of Pit-1 in OA induction. Therefore, we can conclude that the mechanism for Pit-1 stimulation of the hPRL gene by OA does not occur via regulation of Pit-1 binding activity but rather relies on the modulation of its transcriptional activity.

The fact that mutation of the AP1 or the Pit-1 motif both resulted in the abolition of the OA induction suggests a cooperative action of these two factors. Pit-1 and AP1 do not associate to form a heterodimer, as complex C3 (containing AP1) is not recognized by Pit-1-specific antibody (Fig. 6Go), and the same complex is obtained in gel retardation experiments using nuclear extracts from heterologous cells that do not express Pit-1 (data not shown). Moreover, gel retardation experiments using mutated oligonucleotides either as a DNA probe or competitor demonstrate that AP1 and Pit-1 binding to sequence P1 are independent. Thus, the observed synergistic induction requires functional interaction between Pit-1 and AP1. Such an interaction between Pit-1 and an AP1-like transcription factor has been shown to be involved in the hormone-regulation of the human TSHß promoter in HeLa cells (8). Both transcription factors are required for stimulation of the human TSHß promoter activity, but they do not cooperate to bind to their target sequences.

Interestingly, our results show that AP1 is also involved in basal activity of the proximal hPRL promoter. Mutation of the AP1 motif, which does not alter Pit-1 binding, reduces the activity of 164P-Luc to the same extent as mutation of the Pit-1 motif, suggesting that the AP1 synergism with Pit-1 operates at the basal level as well. Moreover, cotransfection of c-jun and c-fos expression vectors in HeLa cells was shown to increase the proximal hPRL promoter activity (39). It is unclear at present whether such a synergism would require the phosphorylation of one or both of the factors involved.

Our results clearly show that mutation of the AP1 or of the Pit-1 motif in element P1 abolishes OA induction of the hPRL promoter in GH3B6 cells. A previous report suggested that OA stimulation of hPRL was mediated by sequence A without involvement of the P1 site in GC cells (12). This discrepancy could be due to the different experimental procedures used. The previous study was performed using constructs containing part of the hPRL upstream region cloned in front of the TK promoter, which by itself responds to OA. Moreover, the GC pituitary cells secrete endogenous GH, but no more endogenous PRL. In contrast, our experiments were performed using hPRL promoter constructs in PRL-producing GH3B6 cells. Thus, it is possible that regulation of PRL synthesis is different in these two cell lines, due to their different intracellular protein content (i.e. transcription factors, protein phosphatases... ).

Interestingly, AP1 regulation of the rat PRL (rPRL) proximal promoter appears to differ from AP1 regulation of the human promoter. It was shown that c-jun inhibits the rat PRL promoter activity in GH4 pituitary cells (40). No AP1 binding site is found in the rat PRL proximal promoter, and it has been shown recently that this repression depends on the presence of the inhibitory sequence FPII, the c-jun {delta}-domain, and the cellular context (41). In the hPRL promoter, sequence FPII is missing, and a AP1 binding site is present in sequence P1, resulting, in this case, in an activation by AP1. Our results represent the first description of a differential regulation between rat and human PRL gene transcription.

In conclusion, we propose a mechanism by which AP1 binding to sequence P1 results in synergism with Pit-1, located at the most proximal binding site, leading to an increase in hPRL basal level expression. Activation by the protein phosphatase inhibitor OA increases the cellular amount of AP1-related factors (JunD/c-fos) resulting in an increase of the synergism with Pit-1 and, ultimately, induction of the hPRL transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
OA and D-luciferin were purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal rabbit antibodies, directed against c-jun, JunB, JunD, c-fos, or fosB were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Plasmid Constructs
The first 164 bp of the hPRL promoter were obtained after digestion with BglII and HindIII of the p164PRL-CAT (24). Fragments were purified on polyacrylamide gel and then inserted in the pXP2 vector (42) in front of the luciferase reporter gene. Mutants in Pit-1 or in AP1 consensus sites of the sequence P1 were built using the 164P-Luc plasmid and adequate mutated oligonucleotides (Pit-1 m and AP1 m, see below), with the Chameleon double-stranded, site-directed mutagenesis kit from Stratagene (La Jolla, CA). The correct sequence of all these mutant plasmids was confirmed by dideoxy chain termination sequencing. All plasmids were prepared by alkaline lysis and purified by centrifugation in CsCl-ethidium bromide gradients.

Oligonucleotides
Single- and double-stranded oligonucleotides used for plasmid mutations and gel shift assays were obtained from Eurogentec (Seraing, Belgium). The AP1 oligonucleotide contains the AP1 site of human collagenase promoter (43). The CRE oligonucleotide contains the CREB binding site of the human CG gonadotropin {alpha}-subunit promoter (44). The NFY oligonucleotide contains the NFY consensus site of the rat albumin promoter (45). For each oligonucleotide, the sequence of one strand is presented below:

[Pit-1 m: 5'-CCTTTGATATCTTCACAGATATAATGAATCAGGC-3'

AP1 m: 5'-CTTCATGAATATAATGCAGCAGGCATTCGTTTCCC-3'

P1: 5'-CTAGAATGCCTGAATCATTATATTCATGAAGATATC-3'

P1, Pit-1 m: 5'-CTAGAATGCCTGAATCATTATATCTGTGAAGATATC-3'

P1, AP1 m: 5'-CTAGAATGCCTGCTGCATTATATTCATGAAGATATC-3'

P2: 5'-CTTCCTGAATATGAATAAGAAATAAAATACC-3'

AP1: 5'-AGCTTAAAGCATGAGTCAGACACCT-3'

CRE: 5'-CTAGAAATTGACGTCATGGTAA-3'

Oct-1: 5'-TGTCGAATGCAAATCACTAGAA-3'

NFY: 5'-GGGGTAGGAACCAATGAAATGAAAGGTTA-3'

Cell Cultures and Electroporation
GH3B6 cells were grown in monolayer, in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 10% FCS (GIBCO-BRL), 1% penicillin/streptomycin solution (GIBCO-BRL), and 5 x 10-10 M estradiol (E2, Sigma). For transient transfection experiments, cells were harvested using trypsin-EDTA and resuspended in DMEM supplemented with FCS and E2, at a concentration of 15 x 106 cells/ml. Then, 800 µl of cell suspension were mixed with 6 pmol of plasmid and submitted to a single pulse of 250 V/4 mm and 1500 µFarads capacitance, using a "cellject" apparatus (Equibio, Seraing, Belgium). Electroporated cells were transferred in six-well tissue culture dishes and treated 3 h later with the appropriate drugs for about 18 h. Cells were then lysed in 25 mM Tris/phosphate, pH 7.8; 8 mM MgCl2; 1 mM EDTA; 1% Triton X-100; 15% glycerol; 1 mM dithiothreitol (DTT). Protein concentrations were determined using the Bradford assay (46). Before lysis of the cells, toxicity of treatments was tested using the trypan blue exclusion assay.

Cell Extracts and Western Blot Assay
Nuclear cell extracts were prepared from OA-treated or control cells. The collected cells were incubated for 15 min in hypotonic buffer ( 10 mM HEPES; 10 mM KCl; 0.1 mM EDTA; 0.1 mMEGTA; 1 mM DTT; 0.5 mM phenylmethylsulfonylfluoride) before addition of 25 µl of NP40 10%. After centrifugation for 30 s at 14,000 rpm, the pellet was resuspended in binding buffer (20 mM HEPES; 400 mM KCl; 20% glycerol 20%; 2 mM DTT) containing an antiprotease mixture (Boehringer Mannheim, Indianapolis, IN). Then it was frozen in liquid nitrogen, thawed, and submitted to a second centrifugation for 30 min at 14,000 rpm. The supernatant was stored at -70 C, and the protein concentration was determined using a Bradford assay (46) with BSA as a standard.

Proteins were separated by electrophoresis on a 8% SDS-polyacrylamide gel. After migration, the gel was electroblotted on to a nylon membrane and probed with the appropriate antibody, and the bands were revealed by chemiluminescence (ECL, RPN2106 kit, Amersham, Arlington Heights, IL).

Luciferase Assay
For each sample, protein concentration was normalized in 350 µl of lysis buffer. Luciferase activity was measured in a Lumat LB951 apparatus (EG&G Berthold, Wildbad, Germany) after addition of 100 µl of a 0.3 mM luciferin/0.8 mM ATP solution.

Results are expressed as the ratio of luciferase activity measured in treated vs. untreated cells. As both treated and untreated cells originate from the same pool of electroporated cells, then variations in luciferase activity are only due to OA treatment. Results are expressed as the mean ± SD of three independent pools of electroporated cells.

Gel Retardation Assays
Gel retardation analysis was performed using 5 µg of nuclear protein extract, 2.5 µg of poly(deoxyinosinic-deoxycytidylic)acid and 10,000–15,000 cpm of 32P-labeled DNA fragments, in the following buffer: 5 mM HEPES; 100 mM KCl; 5% glycerol. The resulting DNA-protein complexes were resolved by electrophoresis on a prerun 5% native-polyacrylamide gel, with 0.5xTris-borate-EDTA as running buffer. The gel was dried and then exposed overnight to x-ray sensitive films (Fuji, Stamford, CT).

Nuclear cell extracts were prepared in the same way as the extracts used in Western blot assays, and double-stranded oligonucleotides were labeled by the T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) with [{gamma}-32P]ATP.

When antibodies were used to reveal the DNA-protein complexes, gel retardation assays were performed as described, except that nuclear extracts were incubated with unlabeled DNA fragments. After migration, gel was electroblotted on to a nylon membrane, probed with anti-Pit-1, anti-junD, or anti-c-fos antibody and revealed by chemiluminescence (ECL, RPN2106 kit, Amersham).


    ACKNOWLEDGMENTS
 
We are grateful to Professor M. Montminy for providing anti-CREB antibodies. We would like to thank Dr. B. Viollet for the gift of NFY oligonucleotide and for helpful discussions and advice.


    FOOTNOTES
 
Address requests for reprints to: M. Muller, Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Institut de Chimie B6, B-4000 Sart-Tilman, Belgium. E-mail: m.muller{at}ulg.ac.be

This work was supported by Grant 93811–28/A000/DRET/DS/SR from the Direction des Recherches et Etudes Techniques (DRET); Grant PAI P4–30 from the Services Federaux des Affaires Scientifiques, Techniques et Culturelles; Grants V6/5-ILF.379, -3.4537.93, and -9.4569.95 from the Fonds National de la Recherche Scientifique (FNRS); Grant 95/00–193 from Actions de Recherche Concertees; and Grant ASTF 8413 from the European Molecular Biology Organization (EMBO).

1 Equal contributors to this manuscript. Back

Received for publication February 4, 1998. Revision received April 27, 1998. Accepted for publication April 28, 1998.


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