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Endocrinology, doi:10.1210/en.2002-0183
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Endocrinology Vol. 144, No. 9 3995-4007
Copyright © 2003 by The Endocrine Society

The Rat Pituitary Promoter of the Neuronal Nitric Oxide Synthase Gene Contains an Sp1-, LIM Homeodomain-Dependent Enhancer and a Distinct Bipartite Gonadotropin-Releasing Hormone-Responsive Region

Lydia K. Bachir, Ghislaine Garrel, Anne Lozach, Jean-Noël Laverrière and Raymond Counis

Signalisation Cellulaire, Régulation de Gènes et Physiologie de l’Axe Gonadotrope, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 7079, Physiologie et Physiopathologie, Université Pierre et Marie Curie, 75252 Paris, France

Address all correspondence and requests for reprints to: Dr. Raymond Counis, Unité Mixte de Recherche 7079 Centre National de la Recherche Scientifique, Physiologie et Physiopathologie, Université Pierre et Marie Curie, 4 Place Jussieu, Case 256, 75252 Paris cedex 05, France. E-mail: raymond.counis{at}snv.jussieu.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neuronal nitric oxide synthase (NOS I) is expressed and hormonally regulated in rat anterior pituitary gonadotropes. In the present study, we investigated the mechanisms that underlie the constitutive and GnRH up-regulated activity of the pituitary exon 1p promoter of the NOS I gene in these cells. Through the use of 5'-deletions and transient transfections in LßT2, a gonadotrope-derived cell line, we delineated a NOS I cell-specific (NCS) enhancer region (-73/-59) that is required for constitutive activity. Independently of the NCS enhancer, GnRH responsiveness is supported by a bipartite regulatory domain referred to as the GnRH response element I and II located between -33/-10 and -4/+4, the latter consisting of a cAMP-like response element. By combining transient transfections, gel shift, and supershift assays, we demonstrate that Sp1 and LIM-homeodomain-related protein bind the NCS enhancer, whereas cAMP response element binding protein and cAMP regulatory element modulator-like factors bind the GnRH response element II motif. We further show that factors involved in GnRH regulation are also implicated in constitutive activity, suggesting intimate links between constitutive and regulated promoter activity. We speculate that specific expression of the NOS I gene in gonadotropes together with its regulation by GnRH is suggestive of a critical participation of NOS I in gonadotrope function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NITRIC OXIDE (NO) synthase (NOS) enzyme family is composed of three major isoforms, the neuronal (NOS I), inducible (NOS II), and endothelial NOS (NOS III). These enzymes convert L-arginine to L-citrulline, producing NO, an important signaling molecule involved in a variety of physiological and pathological processes (1). Most of the effects of NO are mediated through cGMP formation (2). The NOS I isoform is widely distributed in the central and peripheral nervous system and induces the highest levels of NO in the body (3). NOS I is also expressed in a number of nonneuronal tissues including the kidney, skeletal muscle, small intestine, and testis (4, 5, 6, 7, 8, 9, 10), as well as in the anterior, intermediate, and posterior lobes of the pituitary (11, 12, 13, 14).

The NOS I gene is the most complex and the largest of the three NOS genes and, despite its wide tissue expression, paradoxically the less studied. The sequence of the human gene, the only one that has been fully characterized, spans a region greater than 240 kb on chromosome 12 and the transcription unit contains 29 exons with initiation and termination sites located in exon 2 and 29, respectively (15, 16, 17). Numerous transcripts have been isolated from various tissues, differing in exon splicing, cassette insertion/deletion, alternate promoter usage and 3'-untranslated region cleavage and polyadenylation. The most complex region is obviously the 5'-untranslated region that displays great diversity in humans, rats, and mice due to several alternate exon 1 (17, 18, 19, 20, 21). Indeed, nine distinct exon 1 have been identified in humans, directly spliced to exon 2 or 3 (22). Various promoter usage and tissue-specific exons are thought to generate a higher degree of diversity (23). To date, three different human promoters have been characterized (24, 25), whereas in rats, five exon 1 have been described and shown to be expressed in a tissue- and/or a developmental stage-specific manner (20, 21). We have ourselves recently described a novel exon 1 in the rat pituitary gland referred to as exon 1p that corresponds to a truncated version of the previously described ubiquitous exon 1a (26). The novel initiation sites of transcription in the pituitary have been localized at position +370 and +385 in reference to exon 1a.

In the anterior pituitary gland, NOS I has been specifically detected in gonadotrope and folliculo-stellate cells by nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry, immunohistochemistry, and/or in situ hybridization (12, 27). In gonadotropes, NOS I is regulated by GnRH, the primary regulator of gonadotrope function. GnRH stimulates not only the catalytic activity of NOS I, resulting in the NO-mediated enhanced production of cGMP, but also the steady-state levels of NOS I protein and mRNA (27, 28). Accordingly, the level and activity of NOS I, and consequently cGMP, is up-regulated in gonadotropes during proestrus (29), a critical GnRH-dependent phase in reproduction. A recent study from our laboratory has provided evidence that NOS I protein levels are also enhanced by cAMP and the hypothalamic neuropeptide pituitary adenylate cyclase-activating polypeptide, exclusively in gonadotropes (28). Likewise, we showed that both GnRH and cAMP were able to stimulate NOS I promoter activity in the gonadotrope-derived cell line LßT2, whereas direct activation of the protein kinase C pathway or induction of calcium entry was inoperative (26). Response elements were found to reside within the -73/+60 proximal promoter region, which was also involved in cell-specific activity.

In the present study, we delineate a novel enhancer element that confers gonadotrope cell-specific activity through the binding of Sp1 (and possibly, also Sp3) and a LIM homeodomain-related protein. We also provide further evidence for protein kinase A (PKA)-mediated stimulation of NOS I gene transcription by GnRH and demonstrate that a bipartite regulatory domain that includes a cAMP response element binding protein (CREB)-related motif, is required for GnRH responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructions
The series of 5' deletions in exon 1p promoter, termed -1523, -246, -73, and +60, were inserted upstream to the firefly luciferase (Luc) reporter gene into the pGL3-basic vector (Promega Corp., Lyon, France) as previously described (26). As in previous study, numbering is mentioned relative to the exon 1a transcriptional start site described by Lee et al. (20). The plasmids termed -59, -33, -10, +19, and +37 were obtained by amplifying the entire exon 1p promoter using antisense primer A coupled to sense primer B, C, D, E, or F, respectively (Table 1Go). PCR was carried out in a 50 µl volume using 1.5 U Expend High Fidelity Taq DNA polymerase (Roche Molecular Biochemicals, Meylan, France) and 100 pmol primers. The PCR program included an initial denaturation of 2 min at 94 C, followed by 30 cycles consisting of successive incubations at 94 C for 15 sec, 55 C for 30 sec, and 72 C for 1 min, and a final extension at 72 C for 5 min. The PCR products were then digested with restriction enzymes HindIII and KpnI and ligated into the pGL3-basic vector (Promega Corp.).


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  		TABLE 1. Oligonucleotides used in plasmid construction and EMSA

 
Block-replacement mutations (mutA, mutB, mutC, and mutD) within the -246 construct, were generated by PCR amplification using a series of sense/antisense primers (overlapping over 14 bp) designed to place an EcoRI restriction site with an additional cytosine at the 3' end (GAATTCC) instead of the original sequence. Two separate PCR reactions were performed using the mutated sense primers G, H, I, and J coupled to the antisense primer A, and the mutated antisense primers K, L, M, and N coupled to sense primer O for mutA, mutB, mutC, and mutD, respectively. The amplified products obtained from the two PCRs were combined and submitted to a second round of PCR using primers A and O. The resulting amplified products were then digested with restriction enzymes HindIII and KpnI and cloned into the pGL3-basic vector (Promega Corp.). Constructs mutCRE and consCRE were generated according to the same protocol, using as a template the -1523 construct and primers P and Q coupled to primer A and primers R and S coupled to sense primer T for consCRE and mutCRE, respectively. The -80/+73 promoter region of the NOS I gene was generated by PCR using the sense primer U and the antisense primer V, subjected to digestion by HindIII and KpnI restriction enzyme and then inserted into the pLucPRL vector upstream of the minimal PRL promoter. The pcDNA3PKI and the control pcDNA3ßgal vectors as well as the minimal PRL promoter containing vector (pLucPRL) were previously prepared in our laboratory (30). All constructs were verified by sequencing.

Cell culture and transfection
Transfection assays were performed using the two pituitary gonadotrope cell lines {alpha}T3-1 and LßT2, generated by P. Mellon (31, 32) and occasionally, Chinese hamster ovary (CHO) cells. Cells were cultured in DMEM (Sigma, Saint-Quentin Fallavier, France), supplemented with 10% fetal calf serum (FCS) and 100 U/ml penicillin and 100 µg/ml streptomycin sulfate and grown at 37 C in a humidified atmosphere with 5% CO2. Transfections were performed using the LipofectAMINE-Plus assay (Life Technologies, Inc., Gaithersburg, MD), according to the manufacturer’s recommendations. Briefly, 1 x 105 cells were plated in 24-well plates, in triplicate or in duplicate, 1 d before transfection. In each experiment, the total quantity of DNA per well was standardized to 200 ng with pUC19 vector. Ten to 30 ng pTK-Renilla vector (Promega Corp.) per well was cotransfected to serve as an internal standard for transfection efficiency. DNA was combined with 0.6 µl LipofectAMINE and 0.4 µl Plus-reagent in 250 µl OptiMEM medium (Life Technologies, Inc). Transfection mixture was incubated for 15 min at room temperature, and applied to the cells. Following a 6-h transfection, the medium was replaced by DMEM 2% FCS and 10 U/ml penicillin and 10 µg/ml streptomycin sulfate with an appropriate treatment of either the GnRH agonist triptorelin ([D-Trp6] GnRH, Sigma) or cholera toxin (CTX). After 18 h, cells were harvested, lysed and reporter-gene activities were measured as described previously (26).

Nuclear extracts and EMSAs
Nuclear extracts were prepared from {alpha}T3-1, LßT2, and CHO cells. Cells were seeded at 3 x 106 ({alpha}T3-1 and CHO cells) or 6 x 106 (LßT2) cells in 100-mm dishes in DMEM 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate and cultured for 24 h. The medium was then replaced by OptiMEM medium and cells were cultured for an additional 6 h and then a 18 h period in DMEM 2% FCS, 10 U/ml penicillin, and 10 µg/ml streptomycin sulfate, after which cells were harvested and nuclear extracts were prepared as described (33). Wild-type (-75/-49), CRE (-10/+10), consCRE, and Sp1 consensus-synthetic double-stranded oligonucleotides, designed to contain 5' protruding ends, were labeled by filling in the recessed 3' termini with {alpha}[32P]deoxy-CTP (Amersham Biosciences, Orsay, France) using the klenow fragment from Escherichia coli DNA polymerase I then purified on a Sephadex G50 fine column. Nuclear extracts (8 µg) were incubated for 15 min at 4 C in binding buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 1 mM EDTA, 300 µg/ml BSA, and 12% (vol/vol) glycerol] containing 1 µg poly(deoxyinosine-deoxycytidine). Labeled probe and unlabeled competitor were then added and incubated for an additional 30 min at room temperature. For supershift assays, the antibodies against transcription factors Sp1, Sp2, Sp3 (sc-59, sc-643, sc-644, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Kruppel-like factor (sc-1905, Santa Cruz Biotechnology, Inc.), CREB/activating transcription factor 1 (ATF)-1 (sc-186, Santa Cruz Biotechnology, Inc.), cAMP regulatory element modulator (CREM)-1 (sc-440, Santa Cruz Biotechnology, Inc.), ATF-1 (sc-243, Santa Cruz Biotechnology, Inc.), CREB (no. 244, Ref.34 ; and no. 9192, Cell Signaling Technology/Ozyme, Montigny-le-Bretonneux, France) or the control antibodies (polyclonal anti LH and monoclonal anti Myc) were incubated with nuclear extracts for 1 h at 4 C before addition of the labeled probes. The DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel in 1x Tris-borate-EDTA buffer. Gels were dried and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The -73/-49 sequence is essential for constitutive NOS I promoter activity
To discriminate between elements of the -73/+60 NOS I promoter region involved in basal activity, a series of 5' deleted fragments ending at positions -73, -59, -33, -10, +19, +37, and +60 were generated by PCR and inserted upstream of the firefly Luc reporter gene in the pGL3-basic vector. The constructs were then transiently transfected into the gonadotrope-derived cell line LßT2. As shown in Fig. 1Go, the initial deletion that affected the -73/-59 region resulted in a strong decrease in Luc activity (P < 0.001), whereas further deletions were ineffective. Similar results were obtained with the {alpha}T3-1 cells, an alternate gonadotrope-derived cell line. With CHO cells, promoter activity of the -73 construct was notably reduced in agreement with our previous data (26) and consequently, the decrease induced by the 5' deletions was lowered in proportion. This was probably due to the absence or low levels of cognate transcription factors. These data suggested a key role in constitutive promoter activity in the -73/-59 region, hereafter referred to as the NOS I cell-specific (NCS) enhancer. To more precisely define the response elements present in the NCS enhancer, block-replacement mutagenesis were performed within the -81/-51 sequence by generating four adjacent mutations of 7-bp in the context of the -246/+387 promoter (Fig. 2AGo). The mutant and wild-type (-246, -73, and -59) constructs were subsequently transfected into LßT2 cells. As illustrated in Fig. 2BGo, mutations B (mutB), C (mutC), and D (mutD) strongly inhibited the promoter activity by 88–100% (P < 0.001) compared with the wild-type construct, mutC being the most efficient. Similarly, the same mutations resulted in a 71–100% loss of promoter activity (with mutC being again the most effective), after transfection in {alpha}T3-1 cells (results not shown). Unexpectedly, mutation A (mutA) induced a 45% and 56% decrease in Luc activity compared with the wild-type construct (P < 0.001) after transfection in LßT2 and {alpha}T3-1 cells, respectively. Because mutA was located outside but close to the 5' upstream to the NCS enhancer, it most likely acted on promoter activity through conformational modulation of the neighboring DNA motifs. Together, these results suggested the presence of elements important for constitutive promoter activity within the NCS enhancer.



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  		FIG. 1. Localization of elements involved in constitutive NOS I promoter activity using 5' end deletions. A, General organization of the 5' part of the NOS I gene. B, The plasmid constructs indicated in the figure were transiently transfected into LßT2, {alpha}T3-1, and CHO cells. A dashed line indicates the start sites of transcription (+370/+385) and the 5' end of each construct is numbered by reference to exon 1a. Cells were harvested 18 h after transfection and evaluated for Luc reporter gene activity as described in Materials and Methods. Luc activity was normalized for TK- renilla levels, and the values of the different constructs were expressed as fold-induction over Luc activity of the promoterless basic vector. Each bar represents the mean ± SD of four to six separate experiments, each performed in duplicate.

 


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  		FIG. 2. Block replacement mutagenesis analysis of the -73/-59 region of the NOS I promoter. A, NOS I promoter sequence was scanned by block replacement mutagenesis from position -81 to -51 (-246 mutA to -246 mutD) as described in Materials and Methods. B, Constructs containing the wild-type (-73/+387, -59/+387 and the promoterless vector) or -246/+387 mutated promoters were transiently transfected into LßT2 cells and Luc activity was evaluated as described in the legend of Fig. 1Go and Materials and Methods. Each bar represents the mean ± SD of eight separate experiments, each performed in duplicate.

 
A LIM-homeodomain- and an Sp1/Sp3- related elements lie within the NCS enhancer
To analyze the binding capacity of the NCS enhancer, gel-shift experiments were performed using LßT2 and {alpha}T3-1 nuclear extracts. In addition, nuclear extracts isolated from CHO cells were used to evaluate the cell specificity of the shifted complexes. Using a (-75/-49) radiolabeled probe corresponding to the NCS enhancer, i.e. the Wild-NOS described in Fig. 3DGo, several shifted complexes were observed with nuclear extracts from either cell lines (Fig. 3AGo, arrows). Specificity of the interactions was confirmed by homologous competition with an excess of unlabeled probe (50x or 200x, lanes 3, 4, 7, 8, 11, and 12). Comparison between LßT2, {alpha}T3-1 and CHO cells suggested that complexes I–III were related to the gonadotrope-specific activity of the NOS I promoter because they were only present (complexes I and III) or considerably more abundant (complex II) in gonadotrope than in CHO cells (Fig. 3AGo, lanes 2, 6, and 10). To define which motifs within the NCS enhancer were engaged in complex formation, oligonucleotides bearing the equivalent 7-bp block-replacement mutations (mutB, mutC, and mutD in Fig. 3DGo) employed in the previous transfection experiments (see Fig. 2AGo) were then used together with the NCS-labeled probe in competition experiments. In the presence of LßT2 and {alpha}T3-1 nuclear extracts, the probe containing mutB was capable of competing with the labeled NCS probe for formation of either complex in a dose-dependent manner (Fig. 3Go, B and C). In contrast, the probe bearing mutC had no effect on complexes I and III formation notably in {alpha}T3-1 cells, and mutD was totally ineffective on all three complexes. These data indicate that complexes I and III involved the sequences affected by both mutC and mutD that extended from -65 to -51 and that complex II involved the sequence affected by mutD (-57/-51), exclusively. The search for putative transcription factors in the 3' region (-65/-51) of the NCS enhancer revealed the presence of motifs related to consensus binding sites for the zinc finger transcription factor Sp1 (-66/-54), the POU homeodomain transcription factor Oct-1 (-56/-43) and for members of the LIM-homeodomain transcription factor family such as Lhx2 or Lhx3 (-53/-46).



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  		FIG. 3. Identification of multiple DNA-protein complexes in the -75/-49 sequence. EMSAs were performed using LßT2, {alpha}T3-1 and CHO nuclear extracts. 32P-Labeled probe spanning the -75/-49 region of the NOS I promoter was incubated with 8 µg nuclear extracts. Arrows indicate specific bands. A, Lanes 1, 5, and 9; B and C, lane 1 represent results obtained with free labeled probe. A, A 50-fold (lanes 3, 7, and 11) and 200-fold (lanes 4, 8, and 12) excess of unlabeled probe was used as competitor. B and C, Unlabeled mutant probes (mutB, mutC, and mutD) were used as competitors in a 50-fold (lanes 3, 6, and 9) and 200-fold (lanes 4, 7, and 10) molar excess. D, Sense strand sequence of wild-type and mutant probes with mutations highlighted.

 
To evaluate whether these factors were able to bind the NCS enhancer, we performed heterologous competition with unlabeled nucleotides harboring consensus-binding sites for Sp1, Oct-1 and Lhx2/3 (Fig. 4AGo). Concerning the latter probe, we used the pituitary glycoprotein basal element (PGBE), a motif found in the glycoprotein {alpha}-subunit gene promoter that binds Lhx2 and Lhx3 (35). As shown in Fig. 4BGo, competition experiments with the consensus Sp1 probe (ConsSp1) resulted in the abrogation of complex I formation (lanes 11–13), whereas those performed with the PGBE probe resulted in the abrogation of complex II formation (lanes 19–21). In contrast, complex formation was unmodified following the addition of a 1000-fold molar excess of the Oct-1 probe (lanes 23–25). Likewise, mutant Sp1 or mutant PGBE probes (mutSp1 and mutPGBE, respectively) were ineffective (lanes 7–9 and 15–17, respectively).



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  		FIG. 4. LIM-homeodomain and Sp-related transcription factors bind to the -66/-46 sequence. A, Sense strand sequence of the probes used with consensus binding sites are highlighted. B, EMSAs were performed using LßT2 nuclear extracts (8 µg) and the -75/-49 wild-type-labeled probe. Competitions were performed with a 10-, 100-, or 1000-fold molar excess of unlabeled probes. C, Nuclear extracts were incubated with polyclonal Sp1-, Sp2-, Sp3-, and Kruppel-like factor (GKLF) antibodies or with an equal concentration of a rabbit polyclonal LH antibody, before the addition of the Wild-NOS and the consSp1 radiolabeled probes. Exposure time was longer for Wild-NOS than for consSp1.

 
To further characterize the Sp factors that interacted with the NCS enhancer, supershift experiments were performed with antibodies directed against Sp1, Sp2, Sp3, and the Kruppel-like factor (Fig. 4CGo). The labeled NCS (wild-NOS) as well as ConsSp1 probes were tested using nuclear extracts isolated from LßT2 cells. Whatever the probe used, a partially supershifted complex was observed with the Sp1 antibody indicating that the initial complex contained an immunologically related Sp1 factor. Because Sp1 often acts together with Sp3, the presence of both factors within the complex I was evaluated by adding both Sp1 and Sp3 antibodies in the same binding reaction. Under these conditions, and regardless of the probe used, a fully supershifted complex was detected suggesting that Sp3 may be involved in addition to Sp1 (lanes 6 and 14). In contrast, no supershifted complex was obtained with either Sp2 or Kruppel-like factor antibodies.

All of these data were in agreement with the results derived from competition experiments with the mutant NCS enhancer (Fig. 3Go, B and C). Indeed, mutD (-57/-51) that affected the Lhx2/3-related binding site and partly, the Sp1 motif prevented the formation of complexes I–III, whereas mutC, which affected the Sp1 motif, inhibited the formation of complexes I and III. Altogether, these data suggested that a LIM homeodomain-related protein and zinc finger transcription factors, notably Sp1, were capable of binding the -53/-46 and the -66/-54 motifs of the NCS enhancer, respectively.

GnRH and PKA-induced activation of the NOS I promoter are independent of the NCS enhancer
We have previously shown that the NOS I promoter was positively regulated by the hypothalamic decapeptide GnRH as well as through activation of the PKA-dependent signaling pathway. The elements implicated in these regulations were localized within the -73/+60 region and thus might involve the NCS enhancer. To elucidate this question, LßT2 cells were transfected with a series of 5' deleted constructs (-73, -59, -33, -10, +19, +37, and +60) used in previous experiments (see Fig. 1Go). Treatment with a potent GnRH agonist or with the endogenous cAMP generator CTX led to an increased Luc activity in lysates from cells transfected with the -73, -59, and -33 constructs (Fig. 5Go). However, further deletion encompassing the -33/-10 region abrogated both GnRH- and CTX-induced stimulation, indicating that elements of critical importance were located within this region, thereafter referred to as the GnRH response element I (GnRE I). In addition, these results indicated that: 1) the NCS enhancer was not required for GnRH and PKA-dependent regulations; and 2) identical elements were likely involved in GnRH and PKA-dependent regulations. Interestingly, the latter conclusion, together with our earlier published data, showing that the effect of GnRH was not additive with that induced by either CTX or phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (26) suggested that GnRH stimulation of the NOS I promoter might occur through activation of the PKA-dependent signaling pathway. To evaluate this hypothesis, LßT2 cells were transfected with the full-length NOS I promoter construct and cotransfected with a vector expressing the rat PKI (PKA inhibitor protein) cDNA under the control of a cytomegalovirus promoter (pcDNA3PKI). A vector containing the ß-galactosidase gene driven by a cytomegalovirus promoter (pcDNA3ßgal) was used as control. Cells were then stimulated or not with GnRH agonist (3 nM) or CTX (3 nM). Promoter activity was significantly stimulated following treatment with GnRH (2-fold) or CTX (3.2-fold) in cells transfected with the full-length NOS I promoter construct and cotransfected with the pcDNA3ßgal control vector (Fig. 6Go). However, when cells were cotransfected with the vector expressing the PKA inhibitor, the GnRH-induced stimulation was abrogated (P < 0.001). Accordingly, and as expected, the PKI expression vector decreased significantly the CTX stimulation by 69% (P < 0.001).



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  		FIG. 5. Elements involved in the response to GnRH and CTX are situated within the -33/-10 region. Entire or 5'-deleted constructs inserted upstream to the Luc reporter gene were transiently transfected into LßT2 cells. Cells were treated with GnRH (3 nM) or CTX (3 nM). Luc activity was evaluated as described in the legend of Fig. 1Go and Materials and Methods. Each bar represents the mean ± SD of four separate experiments, each performed in duplicate. Asterisks indicate significant differences between treated vs. control cells (*, P < 0.001; ***, P < 0.05).

 


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  		FIG. 6. PKI inhibits the GnRH- and CTX-induced stimulation of NOS I promoter. LßT2 cells were transiently cotransfected with the full-length promoter sequence (-1523/+387) and pcDNA3PKI or pcDNA3ßgal vector. Cells were then treated with GnRH (3 nM) or CTX (3 nM). Luc activity was evaluated as described in the legend of Fig. 1Go and Materials and Methods and expressed as fold-stimulation over NOS I promoter vector activity in control cells. Each bar represents the mean ± SD of six separate experiments, each performed in duplicate or triplicate. Asterisks indicate significant differences (GnRH vs. GnRH + PKI and CTX vs. CTX + PKI) (P < 0.001).

 
A CREB transcription factor binds to the -4/+4 site
From these data, it might be expected that the regulatory sequence involved in GnRH regulation of the NOS I promoter, the GnRE I either contained a cAMP-related response element or acted in combination with an auxiliary motif that itself contained a cAMP-related response element. Indeed, most of the genes regulated by the PKA-dependent signaling pathway often contain at least two elements, one of which being related to the CRE element and other ones being unrelated and commonly involved in cell-specific regulation (36). The -33/-10 (GnRE I) region was thus investigated by scanning mutagenesis introducing four adjacent mutations of seven base pairs. All these mutations induced a marked reduction (by 80–100%) in the response to stimulation suggesting that most of this sequence was required to support the GnRH and PKA-mediated responsiveness. Furthermore, inspection of GnRE I revealed no cAMP-related elements. Therefore, we explored the neighboring DNA sequences and found one putative binding site at position -4/+4 that differed by one base from the consensus CRE sequence (GGACGTCA vs. TGACGTCA, respectively). Gel retardation experiments using LßT2 nuclear extracts and a wild-type (-10/+10) radiolabeled probe (Fig. 7CGo), showed a single predominant complex (Fig. 7AGo, lane 2). Specificity of the complex was attested by the strong competition obtained with the homologous unlabeled probe (lanes 3–5), whereas an excess of mutant competitor probe (mutCRE) did not prevent the formation of the protein-DNA complex (lanes 7–9). Interestingly, a 10- to 1000-fold molar excess of a CRE consensus oligonucleotide (consCRE) decreased the intensity of the shifted band in a dose-dependent manner (lanes 11–13), suggesting that the -4/+4 motif corresponded to a functional CREB binding site. To ascertain this hypothesis, we performed supershift assays with a rabbit polyclonal anti-CREB antibody that was directed against the DNA-binding domain of several members of the CREB-ATF family (CREB-1, ATF-1, and CRE modulator). As shown in Fig. 7BGo, addition of the anti-CREB/ATF antibody completely abrogated the complex produced in presence of the radiolabeled NOS I probe (lanes 6–8), whereas the control antibody, a rabbit polyclonal anti-LH antibody, (lanes 3–5) had no effect. Similarly, the use of the anti-CREB antibody abrogated the complex formed with the radiolabeled CREB/ATF consensus probe (lanes 14–16).



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  		FIG. 7. CREB-/CREM-related factors bind to the -4/+4 sequence. A, EMSAs were performed using LßT2 nuclear extracts. Wild-type labeled probe (-10/+10) was incubated with 8 µg nuclear extracts. Wild-type (Wild-NOS), mutated (mutCRE) and consensus (consCRE) unlabeled probes were used as competitors in 10-, 100-, or 1000-fold molar excess. B, Nuclear extracts were incubated with a rabbit polyclonal anti-CREB-antibody or with an equal concentration of a rabbit polyclonal anti-LH-antibody, before the addition of the Wild-NOS- and the consCRE-radiolabeled probes. C, Nuclear extracts from {alpha}T3-1 were incubated with increasing concentrations (from 0.02–2 µg) of antibodies directed against members of the CREB/ATF family or with an equal concentration of control anti-Myc antibody before the addition the labeled probe. D, Sense strand sequences of wild-NOS, mutCRE, and consCRE probes. CRE site and mutations are highlighted.

 
To further discriminate between various members of the CREB/ATF family, we tested four different antibodies directed against ATF-1, CREM-1, and CREB (Fig. 7CGo). The specific anti-ATF-1 antibody was as inefficient as the unrelated anti-Myc antibody, at the same concentration, at inhibiting complex formation with the CRE-NOS probe, suggesting that ATF-1 was not involved in NOS I gene transcription. In contrast, the partially specific anti-CREM-1 antibody induced a clear supershifted complex in a dose-dependent manner, with the highest concentration (2 µg) being the most efficient. Likewise, the antibodies directed against CREB induced supershifted complexes. The first anti-CREB antibody (no. 244) displayed high binding affinity since a supershifted complex was detected at a 1/100 dilution (0.02 µg). At higher antibody concentration (1/10 dilution) a second high molecular weight complex was visible in addition to the first one, suggesting the formation of a complex containing more than one antibody molecule. The second, specific CREB antibody (no. 9192) similarly induced a supershifted complex, however, with lower affinity. Together, these data indicated that the NOS I probe binds specifically to a CREB factor and also, likely, a transcription factor related to the CREM family. Similar experiments performed with the consensus probe gave identical results, except that this probe displayed a higher affinity than the NOS I probe. Nuclear extracts from either {alpha}T3-1 or LßT2 cells gave similar results.

The CRE site is crucial for NOS I promoter activity
To further analyze the contribution of the CRE-related, motif the wild-type motif (GGACGTCA) was replaced by either an unrelated sequence that included an EcoRI site (GAATTCGG) or the consensus CREB binding site (TGACGTCA). These constructs were then transfected into LßT2 cells, treated or not with GnRH or CTX. Surprisingly, disruption of the CRE (mutCRE) site caused a dramatic decrease in constitutive Luc activity (Fig. 8AGo). Furthermore, neither GnRH nor CTX induced any additional Luc activity. Likewise, the CRE consensus-containing construct displayed a decrease in both basal and GnRH-stimulated promoter activity as compared with the wild-type promoter. Nevertheless, this construct remained able to respond to CTX indicating that the consensus CRE was functional. The CRE-related motif, thereafter referred to as the GnRE II, was thus essential for both constitutive and GnRH-regulated promoter activities. Replacement of the GnRE II by the consensus CRE likely disrupted functional interactions that were essential for promoter activity. These interactions might notably involve factors that bind the NCS, the GnRE I and the GnRE II.



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  		FIG. 8. The CRE (-4/+4) site is required for GnRH responsiveness. A, Constructs containing the full-length promoter sequence with the wild-type (Wild-NOS-Luc, GGACGTCA), mutated (mutCRE-Luc, GGATTCGG) or consensus (consCRE-Luc, TGACGTCA) CRE were transfected into LßT2 cells and treated with GnRH (3 nM) or CTX (3 nM). Results were expressed as fold-induction over Luc activity of the promoterless vector. Each bar represents the mean ± SD of four separate experiments, each performed in duplicate. Asterisks indicate significant differences between treated vs. control cells (P < 0.001). B, Schema depicting the constructs containing the NOS I promoter fragment (-80/+13) upstream of the minimal PRL promoter (-35/+36). Boxes indicate the response elements involved in constitutive (NCS) or regulated expression (GnRE I and II). C, Constructs depicted in B were transiently transfected in {alpha}T3-1 or LßT2 cells and treated or not (open bars) with GnRH (black bars) or CTX (gray bars).

 
Finally, to evaluate whether the promoter domain that contained the NCS (-73/-46) and the GnRE I (-33/-10) and II (-4/+4) was able to act alone and to confer constitutive expression, be regulated by GnRH and the PKA- dependent signaling pathway, the -80/+13 promoter region was placed upstream of the -35/+36 minimal PRL promoter (48). The corresponding construct containing the Luc reporter gene under the control of the chimeric NOS I/PRL promoter was then transfected into {alpha}T3-1 and LßT2 cells. The presence of the -80/+13 NOS I promoter region increased constitutive expression of the minimal promoter in both cell lines (Fig. 8Go, B and C). Furthermore, treatment of these transfected cells with CTX and GnRH led to a strong increase in promoter activity, demonstrating that this restricted domain of the NOS I promoter contains elements sufficient to confer basal activity as well as GnRH and PKA-dependent responsiveness.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The NOS I gene is expressed in gonadotropes of the anterior pituitary gland and its expression is up-regulated by GnRH, the principal regulator of reproductive function in mammals. To understand the mechanisms involved in the specific expression of the NOS I gene and its regulation by GnRH, we have previously identified a particular exon 1, termed 1p, predominantly expressed in the rat pituitary gland. We have subsequently isolated and partially characterized the promoter that directs its transcription (26). This earlier study emphasized the unexpected diversity in the regulation of NOS I gene, which was initially thought to be expressed in a constitutive way and therefore, was studied less intensely than genes encoding for the other NOS isoforms. In particular, little is known about the transcription factors as well as the cognate response elements involved in NOS I gene expression and regulation. In the present study, we show that Sp1 as well as the LIM-homeodomain-related factors bind a cell-specific (NCS) enhancer and are required for constitutive activity of the pituitary-specific NOS I promoter in gonadotropes. Promoter activity also appears to be dependent on the presence of a member of the CREB family. This factor together with those that bind the GnRE I element are also required for the GnRH- and PKA-dependent up-regulation of NOS I promoter activity.

Several of our data argue in favor of an implication of Sp1 transcription factor in the constitutive expression of the rat NOS I gene. Firstly, the distal sequence of the NCS enhancer contains a motif that displays compelling analogies with the canonical sequence that binds this zinc-finger transcription factor and competition experiments with consensus probes prevent the formation of the complex involving this motif. Secondly, in contrast to antibodies directed against Sp2 and Kruppel-like factors, the anti-Sp1 antibody induces a supershifted complex in EMSA experiments. Lastly, this factor is present in the gonadotrope-derived cell line used, as deduced from experiments performed using consensus Sp probes. Similar to other promoters under the control of Sp1, transactivation may occur through direct association with members of the general transcription machinery, notably TFIID. Consistent with the absence of a TATA box in the pituitary NOS I gene promoter, Sp1 may act as a basal transcription factor since it often plays this role in the case of TATA-less promoters that direct the expression of housekeeping as well as tissue-specific or viral genes (37, 38).

It cannot be excluded, however, that Sp3 isoforms may also be involved in the activity of the NOS I promoter because addition of the anti-Sp3 antibody increased the amplitude of the supershifted complex. In this regard, three Sp3 isoforms have been described; all bind the same motif as Sp1 and thus may compete with Sp1 transactivation in the expression of the NOS I gene. The largest 115 K Sp3 isoform contains two activation domains as well as a repression domain in close proximity to the first zinc finger function. Two other isoforms of Sp3, with a molecular mass of approximately 70K, have a deletion of the first activating domain in N-terminal position and represent very weak activators (37). These smaller isoforms have even been described as potent inhibitors of Sp1 as well as of the 115K Sp3 isoform (39, 40). In LßT2 cells, at least one 70K Sp3 isoform is expressed because the consensus Sp probe forms a fast migrating complex that is shifted with the anti-Sp3 antibody (Fig. 4CGo). Thus, the activity of the NOS I gene promoter may be the result of both stimulatory and inhibitory influences mediated by Sp1 and Sp3 transcription factors. Interestingly, a functional Sp1 motif is also required for full constitutive activity of the LHß subunit gene promoter, a marker of the gonadotrope lineage (41, 42), suggesting that Sp1 or closely related factors may be important for the expression of genes specifically expressed in pituitary gonadotropes. In addition, Sp1 and Sp3 have been found to be essential for the constitutive transcription of the human NOS I exon 1c since mutations in these motifs abolished promoter activity (25). A similar conclusion was drawn from transient transfection experiments with constructs containing the rabbit NOS I gene (43).

The PGBE previously identified in the glycoprotein hormone {alpha}-subunit promoter is functional in gonadotrope and thyrotrope cell lines and binds both Lhx2 and Lhx3 LIM-homeodomain factors (35, 44). Lhx3 expression is restricted to the pituitary gland, whereas Lhx2 expression is ubiquitous (45, 46, 47). The PGBE-like element is functional in the NOS I promoter because its mutation (mutD) inhibits promoter activity. In addition, the consensus PGBE probe but not its mutated counterpart is able to abrogate complex formation. Consistent with these data, LIM-homeodomain-related factors have been shown to mediate the tissue-specific expression of several marker genes of the anterior pituitary gland, notably the glycoprotein {alpha}-subunit and the GnRH receptor gene (35, 44, 48). Besides, targeted disruption of the Lhx3 gene prevents the development of the anterior and intermediate lobes of the pituitary gland and affects the determination of pituitary cell lineages, except corticotropes (49). Hence, similar to Sp1, LIM homeodomain proteins play a key role in pituitary gene expression and, as suggested by our present data, most likely in the gonadotrope-specific expression of the NOS I gene.

A recent work published by Dawson’s group led to the identification of a SF-1 motif in exon 2 of the mouse NOS I gene that was crucial for the cell-specific expression after transient transfection in the tumor-derived {alpha}T3-1 cell line (50). The start site of transcription was identified using mRNA isolated from {alpha}T3-1 cells and proved to be different from that identified by our group using mRNA from rat pituitary gland. It is possible that these conflicting data result from the differences in the species (rat vs. mouse) and/or in the origin of the tissue (normal vs. tumoral). Nevertheless, this SF-1 motif is present downstream from the pituitary promoter 1p in the rat NOS I gene and could contribute to tissue-specific expression of NOS I in normal rat gonadotrope cells, just like the factors that we have characterized in the present work.

In this study, we have also identified a CREB-related element that is required not only for constitutive promoter activity, but also as a mediator of GnRH stimulation (Fig. 8Go). This is consistent with our previous results indicating that GnRH activation of the NOS I promoter is probably mediated through a cAMP-dependent signaling pathway (26). Unexpectedly, substitution by the consensus CRE of the GnRE II inhibited basal Luc activity and abrogated GnRH stimulation. As shown in the EMSA, the CREB/CREM factors nevertheless display a higher affinity for the consensus than for the degenerated NOS I CRE element. Furthermore, PKA responsiveness was preserved suggesting that the consensus CRE was functional in this specific promoter context. The absence of GnRH response may, therefore, result from abrogation of functional interactions between the factors that bind the NOS I CRE (GnRE II) and those that bind the GnRE I. This is reminiscent of the inhibin {alpha}-promoter, where weak binding of CREB protein and steroidogenic factor-1 to their natural site, rather than strong binding to their respective consensus site, facilitates synergistic activation by both factors (51).

The mechanisms involved in constitutive and GnRH- regulated activity of the NOS I promoter seems to be intimately connected as depicted in the theoretical model presented in Fig. 9Go. Indeed, mutation of the GnRE II element decreases constitutive promoter activity suggesting functional, and possibly, physical interactions between the cognate transcription factors and the basal transcription machinery. It is hypothesized that coactivators such as CBP/P300 may contribute in these interactions by cooperating with the CREB/CREM-related factors and with factors that interact with the NCS enhancer such as Sp1, Sp3, and LIM-homeodomain-related proteins. Sp1 and Sp3 may themselves interact directly with general transcription factors. If physical interactions between all these factors are truly required this implies that DNA motifs separated by approximately 300 bp must be juxtaposed in close proximity through mechanisms that remain, however, still speculative.



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  		FIG. 9. Illustration of the potential interactions between transcription factors involved in pituitary-specific and NOS I gene regulated expression. Sp1/Sp3 transcription factors and LIM homeodomain-related protein, involved in the pituitary-specific expression of the NOS I gene, bind the NCS region and are thought to interact with the general transcription factors (TAF) and with coactivators such as CBP/P300. The regulatory factors, activated through GnRH stimulation of the PKA-dependent signaling pathway, interact with a CREB-like factor that binds the GnRE II, as well as with CBP/P300, on the one hand, and with as yet unidentified factors that bind the GnRE I. The CREB-like factor may be a determinant for recruiting CBP/P300 because deletion of the GnRE II results in the simultaneous abrogation of both the constitutive and regulated expression of the NOS I gene.

 
As underlined in the introduction, several data from our laboratory, as well as from others, have demonstrated the implication of GnRH in the up-regulation of NOS I in vivo in experimental, pathological, and physiological situations. In male rats, orchidectomy increases hypothalamic GnRH secretion by relaxing feedback regulation by gonadal steroids and also induces a dramatic increase in NOS I gene expression resulting in protein accumulation (27). In females, the level of NOS I is significantly increased on the afternoon of proestrus and decreases thereafter to basal levels during estrus (29). In these female rats as well as in castrated rats, the increase in NOS I is completely prevented by a GnRH antagonist, establishing a predominant role for GnRH in the up-regulation of NOS I. Whereas the modulation of NOS I levels clearly affect cell responsiveness/reactivity in terms of NO/cGMP production (28, 29, 52) the physiological consequences of these second intracellular messengers are, however, far from well understood. Initial studies have reported a reduction in the GnRH-induced release of LH suggesting that NO produced by NOS I may be a signal for the termination of LH release and thus may participate in the characteristic pulsatile secretion of gonadotropins (12). However, in our hands, manipulation of NOS I activity by means of metabolic drugs did not correlate with parallel changes in LH release (Ref.29 and our unpublished data).

An alternate route in the pharmacological approaches is the analysis of the consequence of gene disruption in transgenic mice. The first inactivation of the NOS I gene was achieved by excision of exon 2 and did not reveal any noticeable dysfunction in the reproductive tract (53). However, further studies on maternal behavior have shown that homozygous NOS I-/- lactating females display significant deficits in maternal aggression relative to wild-type mice (54). Such a deficit most likely originates from perturbation at the central level, notably in the hypothalamus and again, without any direct relationship with the pituitary NOS I. However, in these transgenic mice, a residual NOS I activity was present due to the expression of alternatively spliced mRNA encoding for NOS I ß- and {gamma}-isoforms (53, 55). This first approach was recently reinvestigated by generating mice disrupted for the NOS I gene through the excision of exon 6 that contains the catalytic heme-binding domain and therefore, fully abolishes NOS I activity (56). Under these conditions, the mated homozygotes do not produce litters, the transgenic males do not display mating behavior and the females showed decreased ovary weight and decreased corpus luteum. Interestingly, ovarian transplantation from transgenic to wild-type mice leads to a reversal of phenotypic deficits suggesting that alterations do not occur at the gonadal level but rather along the hypothalamo-pituitary axis.

These data do not allow interpretation of the respective contribution of hypothalamic or pituitary NOS I in the observed alterations because the gene disruption strategy employed was not tissue specific; nevertheless, they are coherent with a crucial role for NOS I in the neuroendocrine control of the reproductive function. In this regard, the present study that demonstrates that transcription factors involved in gonadotrope specific gene expression such as Sp1 and LIM homeodomain proteins are also implicated in the constitutive activity of a pituitary-specific NOS I promoter strengthens the idea that NOS I is important in the physiology of gonadotropes. This is further reinforced by the established regulation of NOS I promoter activity by GnRH, the major regulator of gonadotropes, providing molecular support to the previously observed increased expression of NOS I (mRNA, protein, and activity), notably during an important physiological event such as the midcycle surge in the female rat. The involvement of the PKA-dependent signaling pathway in GnRH up-regulation of NOS I is particularly attractive since most of the well-known actions of the neurodecapeptide are mediated through other signaling pathways such as phospholipase C/protein kinase C-, Ca2+-, or MAPK-dependent cascades. Whether cAMP/PKA can mediate the GnRH action in normal rat pituitary gonadotropes is under current investigation.


   Acknowledgments
 
We would like to thank Drs. Wilfried Legoff and John Chapman for the gift of the Sp-family antibodies, Dr. Pamela Mellon for providing the LßT2 cell line and Drs. Danielle Gourdji and Claude Kordon for {alpha}T3-1 cell line generated by Dr. Mellon. We are also grateful to Dr. Jérôme Bertherat for the ATF-1, CREM, and CREB antibodies and Dr. Christian Bleux for the purified anti-LH antibody. Thanks are given to Mrs. Danielle Duchêne for her technical assistance and Mrs. Marie-Claude Chenut for the preparation of this manuscript. We are very grateful to Dr. Lisa Oliver for her expert English correction and Mr. Jean-Pierre Lagarde for DNA sequencing.


   Footnotes
 
This work was supported by grants from the Centre National de la Recherche Scientifique and Pierre et Marie Curie University (Paris). L.K.B. is a recipient of a fellowship from the Ministère de la Recherche et de l’Education Nationale and from the Association pour la Recherche sur le Cancer. A.L. is a recipient of funds from the Association pour la Recherche sur le Cancer, from the Chancellerie des Universités de Paris, and from the Fondation pour la Recherche Médicale.

Abbreviations: ATF-1, Activating transcription factor 1; CHO Chinese hamster ovary; CREB, cAMP response element binding protein; CREM, cAMP regulatory element modulator; CTX, cholera toxin; FCS, fetal calf serum; GnRE, GnRH response element; Luc, luciferase; NCS, NOS I cell-specific; NO, nitric oxide; NOS, NO synthase; NOS I, neuronal NOS; NOS II, inducible NOS; NOS III, endothelial NOS; PGBE, pituitary glycoprotein basal element; PKI, PKA inhibitor protein.

Received December 23, 2002.

Accepted for publication May 13, 2003.


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 Results
 Discussion
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