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Endocrinology Vol. 142, No. 11 4631-4642
Copyright © 2001 by The Endocrine Society

ARTICLES

Isolation and Characterization of a Rat Nitric Oxide Synthase Type I Gene Promoter that Confers Expression and Regulation in Pituitary Gonadotrope Cells

Lydia K. Bachir, Jean-Noël Laverrière and Raymond Counis

Endocrinologie Cellulaire et Moléculaire de la Reproduction, Centre National de la Recherche Scientifique-ESA 7080, Université Pierre et Marie Curie, 75252 Paris, France

Address all correspondence and requests for reprints to: Dr. Raymond Counis, Endocrinologie Cellulaire et Moléculaire de la Reproduction, Centre National de la Recherche Scientifique-ESA 7080, Université Pierre et Marie Curie, 4, place Jussieu, Case 244, 75252 Paris cedex 05, France. E-mail: raymond.counis{at}snv.jussieu.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide synthase type I (NOS I) is expressed and up-regulated in rat pituitary gonadotrophs. Using rapid amplification of cDNA ends-PCR, 2 major transcripts with 5' ends corresponding to exon 1a but truncated of its first 369 or 384 nucleotides, indicative of two pituitary-specific transcription start sites, were identified. By chromosome walking, we isolated 5'-upstream of this truncated exon termed 1p, a novel -1653/+384-bp genomic region. Transient transfections, using the gonadotrope-derived {alpha}T3–1 and LßT2 cell lines and the full-length or 5'-deleted sequences fused to a luciferase reporter gene, demonstrated that cell-specific positive and negative regions were present especially within the -246/-73 region, whereas the +12/+384 region was crucial for transcription. Moreover, in LßT2 cells, the luciferase activity was increased by GnRH, with the full-length sequence being the most efficient and the -73/+60 region corresponding to the essential zone. The latter region was also crucial for cholera toxin-induced activation. Interestingly, GnRH and cAMP effects were not additive, implying a convergent step in the transduction cascade. These data provide evidence for the presence of several elements controlling NOS I expression in gonadotrophs and demonstrate that GnRH, the prime regulator of gonadotrope function, and cAMP may induce the transcription of NOS I in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO) is a short-lived radical that acts as both an intra- and intercellular second messenger on a wide variety of physiological processes, including immune- and endocrine response, neurotransmission, and vascular tone. NO synthases (NOSs) are responsible for NO production by catalyzing the conversion of L-arginine to L-citrulline in a NADPH and calmodulin-dependent reaction. To date, three isoforms of NOS [neuronal (NOS I), macrophage (NOS II), and endothelial (NOS III)] have been identified.

Initially, NOS I expression was considered only constitutive; recently, its regulation by steroids, as well as various peptidic factors, has been demonstrated (1, 2, 3). A hormone-regulated NOS I has seemed to be involved in several aspects of reproductive functions. Using in situ hybridization, an estrogen-stimulated expression of NOS I was shown in the ventromedial nucleus of the hypothalamus, an area central for the reproductive behavior in female rats (4, 5). Similarly, a testosterone up-regulation of NOS I mRNA was demonstrated in NOS I-expressing neurons of the major pelvic ganglion in rats, in relation to the male reproductive function (6). Furthermore, an increasing number of studies have suggested that NOS I plays an important role in gonadotrope function by acting at both the hypothalamic and pituitary levels. Thus, the NO produced by NOS I-containing cells adjacent to the GnRH neurones would be a determinant in the hypothalamus for both {alpha}1-adrenergic and N-methyl-D-aspartate-induced release of GnRH (7, 8, 9, 10, 11, 12, 13). In the rat anterior pituitary, NOS I has seemed to be expressed exclusively in gonadotrope and folliculostellate cells (11). In addition, several studies have described the regulation of NOS mRNA, protein, and/or activity in this tissue, in particular after castration or during parturition (11, 14, 15, 16). We have, ourselves, demonstrated that GnRH enhanced the steady-state levels of NOS I protein and mRNA in gonadotrope cells (17). Moreover, the level and activity of NOS I, as well as NO-induced 3', 5'-cyclic GMP production, was up-regulated in gonadotrope cells during proestrus, when GnRH released from the hypothalamus is at a maximum (18).

GnRH is known to play a critical role in the neurohormonal control of reproduction by stimulating the release and the synthesis by gonadotrope cells of pituitary gonadotropins LH and FSH. These hormones support the production of gonadal steroids and gametogenesis. Altogether, these data raise questions about the functional relevance of NOS I expression in the anterior pituitary and the potential link with GnRH action/signalization and multigenic control (19). In this study, we have attempted to characterize the promoter directing the expression of the NOS I gene in the anterior pituitary gland, with the objective of determining the mechanisms involved in constitutive and regulated expression of NOS I in gonadotrope cells. Of particular interest was the elucidation of whether GnRH up-regulation of NOS I mRNA takes place at the transcriptional level.

In humans, NOS I is encoded by an extremely complex gene, spanning a locus greater than 240 kb as a single copy in the haploid genome (20, 21, 22). The transcription unit is composed of 29 exons, and it generates a number of mRNA transcripts through multiple processing, including alternate promoter usage, exon splicing, and 3' untranslated regions cleavage. Some of these transcripts have been the subject of considerable attention in humans as well as in rodents. In humans, 2 functional promoters directing the expression of the exons termed 5'1 and 5'2 have been identified and characterized to date. In rats and in mice, although the gene has not, as yet, been isolated, several exon 1s have been characterized, showing alternative splicing to exon 2 and/or exon 3 (23, 24, 25). In rats, three exon 1 isoforms (referred to as 1a, 1b, and 1c) have been described, resulting in transcripts expressed with distinct temporal and spatial patterns (23). Based on these data, we have examined the mRNA diversity of NOS I in the rat anterior pituitary. In the present study, we show that two transcripts containing a truncated form of exon 1a, which we have designated exon 1p, are the predominant mRNA variants expressed in the anterior pituitary. Using a PCR-based approach, combined with chromosome walking, we have isolated the exon 1p 5'-flanking sequence. Nucleotide sequence comparisons indicate a high degree of homology with the human promoter that directs the expression of the exon 5'2 containing transcripts and, similarly, with the mouse 5'-flanking sequence of exon 1a (26). Furthermore, using transient transfection assays in gonadotrope-derived cell lines, we have identified negative and positive regulatory domains as well as cAMP- and GnRH-responsive regions within the isolated NOS I promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA extraction and RT-PCR
Anterior pituitaries were dissected from intact male Wistar rats. Total RNA was extracted using Tri-Insta-Pure (Eurogentec, Seraing, Belgium). Reverse transcription was achieved using 5 µg RNA and 200 U Superscript II RNaseH- reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) and 2 pmol primer A (see Table 1Go for primers). Nested PCR was carried out in a 50-µl vol, using 2.5 U Eurobiotaq DNA polymerase and 100 pmol primers: primer A coupled to C, D, or E, respectively, for the first round, and primer B coupled to primer C, D, or E for the second round. The PCR program included an initial denaturation of 2 min at 94 C, followed by 40 or 35 cycles for the first or the nested PCR, respectively, consisting of successive incubations at 94 C for 40 sec, 50 C for 50 sec, and 72 C for 1 min, and a final extension at 72 C for 7 min. Amplicons were electrophoresed in a 2% agarose gel.


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  		Table 1. Sequences and positions of the oligonucleotides used in PCR and 5'-RACE-PCR experiments

 
Rapid amplification of cDNA ends (RACE)-PCR, Southern blot, and sequencing
For RACE-PCR, reverse transcription was carried out as described above, using primer F. Total cDNA was extracted with phenol/chloroform/isoamylalcohol (25/24/1), then precipitated twice with ethanol. The water-dissolved pellet was denatured for 2 min at 100 C and tailed with dATP in a 10-µl vol with 0.1 mM dATP and 30 U terminal deoxynucleotidyl transferase (Eurogentec) for 30 min at 37 C. The enzyme was then heat-denatured for 2 min at 65 C. The polyadenylated cDNA was ethanol-precipitated and amplified, as described above, using primer F coupled to oligo dT12–18 primer for the first round and primer G coupled to the same oligo dT primer for the second round. The amplicons were fractionated in a 1% agarose gel and transferred to nylon membrane. Southern blots were performed using, as probe, a random labeled 32P-NOS I cDNA of 1.2 kb (Alexis corporation, San Diego, CA). Amplicons were purified from agarose gels and cloned into pGEM-T Easy vector (Promega Corp., Lyon, France). Clones were sequenced on an LI-COR automatic sequencer (MWG Biotech, Lincoln, NE), using fluorescent sense and antisense M13 primers, with the chain-termination method.

Promoter isolation and DNA cloning
The unknown sequence at the 5'-end to exon 1a was amplified using the Universal Genome Walker kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Briefly, rat genomic DNA libraries were constructed using different restriction enzymes that leave blunt ends, and then ligated to the genome walker adaptor. The 5'-flanking region was amplified by two nested PCRs, using adaptor primer 1 from the kit coupled to the antisense primer H for the first round, and adaptor primer 2 coupled to primer I for the second round. Amplicons were analyzed in a 1% agarose gel. High-size amplicons were purified and cloned into pGEM-T Easy vector. Clones were sequenced as described above. Sequence comparison and analysis were performed using CLUSTAL V (27) and TFDSITE programs (28), respectively.

Plasmid constructions
To construct vectors containing exon 1p start sites of transcription (+370 and +385), rat genomic DNA was amplified using sense primer J and antisense primer K; the latter incorporated an Nsi I restriction site (Fig. 1Go), using Expend High Fidelity Taq DNA polymerase (Roche Molecular Biochemicals, Meylan, France). After Nsi I and SacI digestion, the amplicon was ligated to the exon 1a-5'-flanking region and part of exon 1a previously cloned into pGEM-T Easy vector (see above), digested with the same enzymes. The construct extending from -1523 to +387 was obtained by amplifying the entire 5'-flanking region with antisense primer L coupled to sense primer M, with the high-fidelity enzyme Deep Vent Taq DNA polymerase (New England Biolabs, Inc., Beverly, MA). The amplicon was then digested with restriction enzymes HindIII and KpnI and ligated to pGL3 Basic vector (Promega Corp.) upstream to the firefly luciferase (Luc) reporter gene. All other plasmids were generated according to the same protocol. Plasmids extending from -841, -246, -73, +60, +203, and +289 to +387, respectively, were amplified with the same antisense primer L coupled to sense primers N, O, P, Q, R, and S, respectively. Plasmid extending from -246 to +12 was generated with antisense primer T and sense primer O.



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  		Figure 1. The three NOS I transcripts that contained either exon 1a, 1b, or 1c, present in the rat anterior pituitary gland. Panel A, Structure of the 5' ends of rat NOS I mRNAs, which illustrate the alternative splicing of exon 1a, 1b, or 1c to exon 2, as reported by Lee et al. (23 ). The position of the antisense (A and B) and sense primers (C, D, and E) used in RT-PCR experiments are indicated (see Table 1Go for primer sequences). Panel B, The detection, by RT-PCR, of exon 1a, 1b, and 1c. Total pituitary RNA were reverse-transcribed using antisense primer A. The resulting cDNAs were then amplified in a seminested PCR reaction using primers A (lanes 1, first PCR) and B (lanes 2, second PCR), coupled to primers C (1a), D (1b), or E (1c). Reaction products were stained with ethidium bromide in agarose gels. Lane M, DNA size markers.

 
Cell culture, transfection, and Luc assay
Transfection assays were performed using the two pituitary gonadotrope cell lines {alpha}T3–1 and LßT2, generated by P. Mellon (29, 30), and the Chinese hamster ovary cell line, CHO-K1. The {alpha}T3–1 cells were cultured in DMEM (Sigma, Saint-Quentin Fallavier, France) supplemented with 10% FCS and 2 mM glutamine, the LßT2 in DMEM with 10% FCS, and the CHO-K1 in F12 with 10% new-born calf serum. Media were supplemented with penicillin and streptomycin. Cells were grown at 37 C in a humidified atmosphere with 5% CO2. All cells were transfected using the lipofectAMINE-Plus assay, according to the manufacturer’s recommendations (Life Technologies, Inc.). Briefly, 105 cells were plated in 24-well plates, in triplicate wells, 24 h before transfection. A total of 200 ng construct, extending from -1523 to +387, or equivalent molar amounts of other plasmids completed with pUC19 vector to 200 ng and 100 ng pCMV-ß-galactosidase expression vector or pTK-Renilla (Promega Corp.) were combined with 0.6 µl lipofectAMINE and 0.42 µl Plus-reagent in 250 µl OptiMEM medium (Life Technologies, Inc.). The mixture was incubated for 15 min, at room temperature, before being added to the cells. After 6 h, the medium was replaced by DMEM 2% FCS and penicillin/streptomycin, in the presence or in the absence of either the GnRH agonist triptorelin ([D-Trp6]GnRH, Sigma), the endogenous cAMP generator cholera toxin (CTX), or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). After 18 h, cells were harvested and lysed, and Luc (firefly and renilla) activities were measured using the dual-Luc reporter assay system (Promega Corp.) (31). ß-galactosidase assay was performed as previously described (32). The ratio of firefly Luc to ß-galactosidase activity, or to renilla Luc activity, served as a measure of normalized Luc activity. Data were analyzed using Tukey-Kramer’s multiple-comparison tests when the F-test was significant (P < 0.001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evidence for the expression of at least three forms of exon 1 in the anterior pituitary
To determine whether the three exon isoforms 1a, 1b, and 1c of the rat NOS I gene previously described in other tissues [i.e. brain, skeletal muscle, and kidney (23)] were expressed in the anterior pituitary, RT-PCR experiments were performed using total rat pituitary RNA with common antisense primers A and B specific for exon 2 combined with isoform-specific sense primers C, D, and E complementary to exons 1a, 1b, and 1c, respectively (Fig. 1AGo). Depending on the position of the primer used, the expected product would be 295, 153, or 232 bp in length after the first PCR and 252, 110, or 189 bp after the second nested PCR, each corresponding to an amplification of exon 1a, 1b, or 1c, respectively (23). Gel electrophoresis (Fig. 1BGo) revealed that the major amplified products obtained after the first and the second PCR (solid arrows) were the expected sizes. The identity of these products was further confirmed by Southern analysis using a radio-labeled NOS I probe that overlapped exon 2 (data not shown).

Identification of the 5' ends of pituitary NOS I mRNAs
RACE-PCR was performed to further characterize the different forms of exon 1 and to define the 5' ends of pituitary NOS I mRNAs. Reverse transcription was initiated with antisense primer F (see Materials and Methods) localized to exon 2, and the resulting single-strand cDNAs were poly-A tailed. A nested PCR was then performed using an oligo-dT sense primer and specific antisense primers F and G. Gel electrophoresis analyses indicated the presence of a major amplified product of approximately 600 bp (Fig. 2AGo). Accordingly, Southern analysis of the amplified products with the NOS I probe revealed a strong hybridization signal corresponding to the 600 bp (Fig. 2BGo, solid arrow). Two additional, weaker signals were also detected, which corresponded to amplified bands of approximately 1000 and 300 bp (open arrows). The prominent 600-bp product was approximately 300 bp shorter than expected from the predicted size of an amplified product containing the entire exon 1a sequence.



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  		Figure 2. The 5' end analysis of pituitary NOS I mRNA. Total RNA from rat anterior pituitary was reverse-transcribed using antisense primer F on exon 2, and the obtained cDNA was then polyadelylated at its 3' end using terminal transferase. Nested PCR was performed using the two antisense primers F and G on exon 2, coupled to oligo-dT as a sense primer. A, Products of the seminested PCR, analyzed by agarose gel electrophoresis and ethidium bromide staining; B, Southern blot analysis of RACE-PCR products. Amplicons were transferred onto nitrocellulose filter and hybridized with a 32P-labeled NOS I probe that overlapped exon 2.

 
The 600-bp product was then gel-purified and cloned into the pGEM-T Easy vector. All of the 14 positive clones selected contained a sequence corresponding to the 3' part of exon 1a spliced to exon 2, which included the additional 30-bp sequence previously described by Lee et al. (23). However, the major 5' part of exon 1a was missing, resulting in a shorter exon truncated by 369 bp (10 clones) or 384 bp (4 clones). These data suggested that the transcription start sites, and thus promoter usage, were different in the anterior pituitary, compared with other tissues described to date. We thus considered the truncated form of exon 1a as a novel isoform, which we have named exon 1p (in reference to its pituitary origin).

Characterization of the 5'-flanking region of exon 1a/1p
To search for the promoter sequence that directs the expression of exon 1p, we used a PCR-based method (chromosome walking). Using two genomic libraries derived from StuI and EcoRV digestion, two products (of approximately 1.9 and 1.1 kb) were amplified with primers H and I (Fig. 3Go). After gel purification, cloning, and sequencing, the sequence of a 1653-bp fragment adjacent to the 5' end of exon 1a was established from four independent clones (Fig. 3Go). Comparative database searches, using the BLAST module, showed that the newly isolated sequence shared an overall high degree of homology (61%) with the promoter of exon 5'2, which contained transcripts of the human NOS I gene (20), with some regions having more than 80% homology (Fig. 4Go). Comparison with the 5'-flanking region of the mouse NOS I gene (26) revealed a higher degree of homology (85% for the complete sequence isolated, whereas the sequences corresponding to A, B, C, and D regions shown in Fig. 4Go displayed 94, 99, 95, and 96%, respectively). Scanning of the GenBank database indicated the presence of numerous potential binding sites for ubiquitous regulatory elements, including sequence matches for cAMP response element (CRE) binding protein (CREB), activating protein (AP)1, AP4, nuclear factor {kappa}B, YY1, ERE (estrogen response element), and barbiturate response elements. This promoter sequence displayed a high G/C content (56%, compared with the rat entire genome), and no TATA box upstream of the start sites of transcription was found. The latter observation was also reported for the promoters of the human NOS I gene identified to date (21, 22) as well as for the region 5' adjacent to the mouse exon 1a.



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  		Figure 3. Nucleotide sequence of the rat NOS I gene 5'-flanking region upstream of exon 1p. Bold characters indicate the sequence of exon 1a that is not transcribed in the rat anterior pituitary gland. This leads to a shorter mRNA containing the novel exon 1p (lowercase letters). The sequence is numbered according to Lee et al. (23 ). Dark triangles show the two new transcription start sites identified in anterior pituitary. The position of sense and antisense primers used for vector construction are indicated by solid arrows, whereas primers used for chromosome walking are indicated by dashed arrows.

 


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  		Figure 4. Alignment of rat and human nucleotide sequences exhibiting more than 80% identity. Identical nucleotides are indicated by asterisks. Gaps were introduced to align the sequences. The black triangle indicates the position of the transcription start site described by Lee et al. (23 ), whereas the open triangle indicates that of the human 5'2 exon (21 ). Rat and human sequences were numbered according to Lee et al. (23 ) and Xie et al. (21 ), respectively.

 
Cell-specific activity of the 5'-flanking region of the NOS I gene
Although the characteristics described above suggested that the NOS I gene belongs to the category of ubiquitously expressed genes, we nevertheless examined the cell-specific influence on the promoter activities of the exon 1p- and 1a 5'-flanking regions. For this, the 633-bp 5'-flanking sequence of exon 1p, extending from -246 to +387 (see Fig. 3Go), was placed upstream to the firefly Luc reporter gene in the pGL3-basic vector. A second construct was designed that contained the sequence -246 to +12 only (Fig. 5Go). These constructs were then tested by transient transfection using the gonadotrope-derived cell lines {alpha}T3–1 and LßT2 (29, 30, 33) and the CHO cell line.



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  		Figure 5. The +12/+387 region immediately upstream of exon 1p was required for promoter activity. The promoterless vector and the constructs that contained the -246/+387 or the -246/+12 region inserted upstream of the Luc reporter gene were transiently transfected into {alpha}T3–1, LßT2, and CHO cells. Cells were harvested 18 h after transfection. Luc activity was calculated as Luc activity/ß-galactosidase activity and then normalized as fold-induction over Luc activity of the promoterless vector (Basic). Each bar represents the mean ± SD for three to seven separate experiments (n), each performed in triplicate. *, P < 0.001, significantly different from Basic-Luc activity.

 
As illustrated in Fig. 5Go, the construct that contained the 1p-flanking sequence (-246/+387) displayed a Luc activity that was 4.0- to 5.5-fold higher than that of the promoterless vector in gonadotrope-derived cell lines {alpha}T3–1 and LßT2, respectively, in comparison with the observed increase in CHO cells, which was much less (1.75-fold that of the promoterless vector). In contrast, the construct containing the -246/+12 region was unable to induce any significant Luc activity in the gonadotrope cell lines. In CHO cells, the Luc activity was below that of the promoterless vector. Similar results were obtained with the Luc gene driven by the entire 5'-flanking region (-1523 to +12) of exon 1a or a region extending from -841 to +12 (not shown). More important, in the latter constructs, the insertion of the +12/+387 region (i.e. immediately upstream adjacent to 1p) restored full promoter activity. These data suggest that the gonadotrope-specific promoter activities are strictly dependent on the presence of the +12/+387 sequence that extends over exon 1a and includes the novel start sites of transcription defined above by RACE-PCR.

Evidence for the presence of gonadotrope cell-specific elements in the -246/-73 region of the NOS I gene
To characterize putative regulatory elements present in the 5'-flanking region of the NOS I gene, a series of constructs that included the totality, or part of, the 1p 5'-flanking region were designed. The longest construct (-1523/+387), encompassed a 1910-bp fragment inserted in front of the Luc reporter gene. Seven 5'-deleted constructs ending at positions -841/-246/-73/+60/+203/+289 were obtained by PCR amplification (Fig. 6Go) using the full-length 5'-flanking sequence as a template. The constructs were subsequently transfected into {alpha}T3–1, LßT2, and CHO cells. As shown in Fig. 6Go, the largest construct (-1523/+387) displayed a significant increase in Luc activity, compared with the promoterless vector, in all cell lines. However, as previously observed with the shorter constructs, the promoter activity in CHO cells was distinctly lower than in the pituitary cell lines (1.52 ± 0.17-fold vs. 2.74 ± 0.24-fold and 2.54 ± 0.19-fold over promoterless vector in {alpha}T3–1 and LßT2 cells, respectively). Deletion of the sequence extending from -1523 to -841 resulted in a significant increase in promoter activity in both {alpha}T3–1 and LßT2 cells. A similar, but less pronounced, increase was also noted in the CHO cell line. Further deletion of approximately 600 bp from -841 to -246 yielded an additional significant increase in promoter activities in LßT2 and {alpha}T3–1 cells only. These data suggested the presence of negative regulatory regions in the distal part of the NOS I promoter. In contrast, deletion of the region extending from -246 to -73 significantly decreased promoter activities in gonadotrope {alpha}T3–1 and LßT2 cells, but was ineffective in CHO cells, as was the deletion from -841 to -246. Further deletions to +60 markedly decreased in {alpha}T3–1 cells, and even abrogated in LßT2 and CHO cell lines, promoter activities. Positive regulatory regions could, therefore, be present in the proximal part of the NOS I promoter. The -246/-73 region includes elements that may be recognized by factors that are specifically expressed in the pituitary cell lineage, whereas cis-acting elements that belong to the -73/+60 region likely bind cognate transcription factors that are expressed in the three cell lines tested.



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  		Figure 6. Localization of sequences required for gonadotrope-specific promoter activity. The promoterless vector (Basic) and the entire or 5'-deleted constructs inserted upstream of the Luc reporter gene were transiently transfected into {alpha}T3–1, LßT2, and CHO cells. Cells were harvested 18 h after transfection. Luc activity was adjusted for ß-galactosidase activity, and the values of the different constructs were expressed as fold-induction over Luc activity of the promoterless vector. Each bar represents the mean ± SD for three to seven separate experiments (n), each performed in triplicate. Different letters indicate significant differences between constructs in the same cell line (P < 0.01).

 
Influence of GnRH on NOS I promoter activity in gonadotrope cells
Because the concentration of NOS I mRNA was enhanced in anterior pituitary gonadotrope cells in response to GnRH (17, 18), we evaluated the effect of a potent GnRH agonist, triptorelin, on transcription. Gonadotrope cells were transfected with the entire or deleted promoter sequences and incubated with 3 nM GnRH agonist. As shown in Fig. 7Go, Luc activity was significantly induced in LßT2 cells using the full-length (-1523/+387) as well as the 5'-deleted (from -1523 to -73) constructs. However, the highest degree of stimulation (about 1.8-fold) was obtained with the full-length construct. GnRH stimulation was abrogated when the sequence extending from -73 to +60 was deleted, indicating that the major GnRH-responsive elements were present in this region. In contrast to LßT2 cells, in {alpha}T3–1 cells, no significant GnRH induction of NOS I promoter was observed, as illustrated in Fig. 7Go, for the complete promoter sequence. All other constructions were also ineffective in these cells (data not shown).



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  		Figure 7. GnRH stimulated the transcriptional activity of the NOS I promoter. LßT2 and {alpha}T3–1 cells were transiently transfected with the full-length or progressive deleted constructs in the presence (shaded bars) or in the absence (open bars) of the GnRH agonist triptorelin (3 nM). No effect of GnRH was detected in {alpha}T3–1, regardless of the type of construct. Therefore, only the data with the full-length promoter were presented. Luc activity was adjusted for TK-renilla activity, and the values were expressed as fold-increase over untreated cells. Each bar represents the mean ± SD for three to six (n) separate experiments, each performed in duplicate. Asterisks indicate significant differences (P < 0.001) between treated and untreated cells.

 
To identify the intracellular signaling pathway by which GnRH might stimulate NOS I promoter activity, we tested different drugs that mimic the receptor-induced activation of the PKC or PKA cascade or intracellular calcium concentration increase. For this, we used 12-O-tetradecanoyl 13-phorbol acetate (TPA), a direct activator of PKC; CTX, a potent cAMP inducer; and BayK8644, a potent agonist of the voltage sensitive L-type calcium channel. In LßT2 cells, despite the well-established preferential coupling of GnRH receptor to the PLC/phosphatidylinositol/Ca2+ transduction pathway, neither TPA (50 nM) nor BayK8644 (1 µM) was able to influence the NOS I promoter activity (data not shown). These drugs had no effect on the induction of NOS I promoter activity in {alpha}T3–1 cells. Nevertheless, TPA significantly stimulated (3.7-fold), in LßT2 cells, the activity of a TPA-responsive construct (31) consisting of an AP1 module linked to the minimal PRL promoter (-35/+36 bp) and fused to the firefly Luc (data not shown). In contrast, treatment with 3 nM CTX increased Luc activity, with a maximum of about 3-fold, in LßT2 cells transfected with the full-length construct, compared with untreated cells (Fig. 8AGo). Progressive deletion of the NOS I promoter, by regions extending from -1523 to -73, did not alter the stimulatory effect of CTX on the Luc activity (presence vs. absence of CTX). This action of CTX was, however, abrogated by a further deletion of the sequence extending from -73 to +60 and beyond. In {alpha}T3–1 cells, CTX was also effective, increasing by 2-fold the Luc activity of the full-length construct (Fig. 8BGo). In the latter cells, as well as in LßT2 cells, deletion of the region -73 to +60 completely abrogated the stimulatory effect of CTX. Response elements for cAMP and, at least some for GnRH, would thus colocalize within the same promoter domain between position -73 and +60, suggesting a convergent step in the transduction cascade for either regulator.



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  		Figure 8. Cholera toxin response elements are located in the -73/+60 region. Luc activity was expressed as fold induction over that of the promoterless vector (Basic). {alpha}T3–1 cells (A) and LßT2 cells (B) were transiently transfected with full-length or 5'-deleted constructs in the presence (shaded bars) or in the absence (open bars) of CTX (3 nM). Luc activity was adjusted for TK-renilla activity, and the values were expressed as fold-increase over untreated cells. Each bar represents the mean ± SD from eight (A) or three (B) separate experiments (n), each performed in triplicate. Asterisks indicate significant differences between treated and untreated cells (P < 0.001).

 
In agreement with this hypothesis, in LßT2 cells, activation of the NOS I promoter by a combined treatment of CTX and GnRH agonist at maximally effective concentrations (3 nM each) yielded a result not significantly different from that obtained after a treatment with the GnRH agonist alone, indicating that the effects of GnRH and CTX were not additive (Fig. 9AGo). Similarly, a combined treatment with the GnRH agonist (3 nM) and IBMX (0.3 mM), a cyclic nucleotide phosphodiesterase inhibitor, enhanced promoter activity to a level equivalent to that induced by IBMX alone. Therefore, GnRH might modulate NOS I promoter activity through nuclear factors that are also the ultimate targets of the cAMP-dependent signaling pathway, such as those related to the CREB/activating transcription factor (ATF) family.



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  		Figure 9. Effects of GnRH, CTX, and IBMX on the transcriptional activity of NOS I promoter. LßT2 cells were transiently transfected with the full-length construct (-1523 to +387) and treated with maximally effective concentrations of (A) CTX (3 nM), GnRH (3 nM), or a combination of both factors; and (B) GnRH (3 nM), IBMX (0.3 mM), or a combination of both. Luc activity was normalized to the activity of TK-renilla Luc expression vector and expressed as fold-stimulation over basic-Luc vector. C, LßT2 cells were transfected with the {Delta}MMTV-Luc(wtCRE) vector and treated with either GnRH or CTX. Controls were treated with the vehicle alone. Luc activity was normalized to the activity of TK-renilla Luc expression vector. Each bar represents the mean ± SD for 13 (A) or 5 (B and C) separate experiments, each performed in duplicate. In each experiment, treated and control cells were compared, and different letters indicate significant differences between treatments (P < 0.001).

 
To test this hypothesis, we analyzed the ability of GnRH to stimulate a cAMP-responsive promoter containing several copies of the canonical cAMP-responsive enhancer (TGACGTCA) placed upstream of the {Delta}MMTV-Luc(wtCRE) promoter (34). This artificial promoter was transfected into LßT2 cells that were treated with either GnRH or CTX. As expected, CTX stimulated Luc activity by 4.1 ± 0.6-fold, compared with untreated cells, suggesting that the cAMP signaling pathway was functional in LßT2 cells. Under GnRH treatment, the Luc activity was also significantly increased, however, to a lesser extent than in CTX-treated cells (2.2 ± 0.4-fold). Together, these findings support the idea that GnRH stimulation may lead to the activation of transcription factors that belong to the CREB/ATF family. Interestingly, as for the NOS I promoter, the stimulatory effect of the GnRH agonist on the {Delta}MMTV-Luc(wtCRE) remained consistently lower than that induced by CTX. This suggests that the mechanisms underlying GnRH action on the NOS I promoter most probably involve signaling factors that differ partially from those revealed here with PKA-specific tools.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NOS I seems the most complex gene yet described, in terms of RNA diversity. Multiple mRNA transcripts are generated through various mechanisms that include the use of alternate promoter, alternative splicing, cassette insertion/deletion, and varied sites for 3'-untranslated regions cleavage and polyadenylation. The isoforms resulting from the use of alternate promoter contribute, in large part, to this structural diversity. In the present study, we demonstrate that the three exon 1 isoforms (a, b, and c) previously described in brain, skeletal muscle, and kidney (23) are expressed in the rat anterior pituitary. More importantly, we show that a novel exon 1, referred to as exon 1p, is the most prominent isoform expressed in this tissue. This conclusion is supported by 5'-RACE-PCR experiments, which indicate that most of the pituitary NOS I mRNAs terminated with a 5' end corresponding to the previously described sequence of exon 1a deleted by the first +369/+384 nucleotides. Thus, at least four NOS I mRNA variants are expressed in the anterior pituitary, arising from alternative splicing of exons 1a, 1b, and 1c and exon 1p to a common exon 2. Because the translation initiation codon is located within exon 2, the N-terminal sequence of proteins encoded by all these mRNA variants should not be altered.

Previous studies have demonstrated that rat NOS I mRNA isoforms are expressed in a tissue- and developmental stage-specific manner (23). Isoform 1b displays the most restricted expression, detected only in E18 embryo and in the small intestine of rats (24). Isoform 1c seems to be present in the kidney, the embryo, and skeletal muscle, although its presence in the latter tissue remains controversial (35). Isoform 1a is the most widely expressed. In rats, the highest expression is found in the brain, followed by kidney, small intestine, adrenal, and heart. An increasing degree of complexity in the rat NOS I gene, in relation to the exon composition, exon splicing, and/or tissue distribution of transcripts, has been documented (24, 35), resulting in at least 14 different transcripts. For instance, in the rat kidney, two exon 1 forms, termed C1 and B1, have been identified, the former corresponding to a truncated form of exon 1a, the latter to an extended form of exon 1a. Similarly, in humans, several alternate mRNA transcripts of the NOS I gene have been identified that differ essentially in the 5' mRNA termini, representing 9 exon 1 forms alternatively spliced to an unique exon 2 (21, 36). Diversity in the 5' end is also found in mice. In NOS I knock-out mice generated by the deletion of exon 2 (37), 2 different transcripts have been observed in which exon 1 forms (5'a or 5'b) are directly spliced to exon 3 (25). Collectively, these data illustrate the amplitude of NOS I mRNA diversity and suggest a high plasticity in promoter usage. The identification of the novel exon 1p isoform in the anterior pituitary gland is thus coherent with this concept.

Based on the finding that transcripts containing exon 1p are the most abundant in the anterior pituitary, we isolated and sequenced the 5'-flanking sequence adjacent to this exon. Comparative analysis reveals that this DNA fragment not only shares strong homology with the human NOS I promoter that directs expression of exon 5'2 in the cerebellum (21) but also displays similar features. The same conclusions can be drawn from a comparison with the mouse 5'-flanking sequence that was published late during the course of our study (26). The 5'-flanking regions of the rat exon 1p, the human exon 5'2, and the mouse exon 1a are pyrimidine-rich (56%, 54%, and 53%, respectively), compared with the rat, human, and mouse genomes. In addition, all three contain TG repeats at similar locations, although the repeats extend over a larger region in the human promoter, compared with that of the rat (Fig. 4Go) and the mouse (not shown). These TG repeats are considered to favor left-hand DNA (Z) formation instead of the usual right-hand DNA (B), which may cause secondary structure formation, allowing response elements localized distal to the transcription start site to be brought into close proximity and, consequently, to alter DNA transcription (20, 38). Furthermore, some potential regulatory elements are positionally conserved, in particular a CRE-like element (GGACGTCA) present at position -4/+4 in the rat promoter, +313/+320 in the human promoter (according to the numbering by Xie et al., Ref. 21), and 2150/2157 in the corresponding mouse region (according to the numbering by Sasaki et al., Ref. 26). Although the analysis of the latter region has revealed structural characteristics similar to those of the human and of the rat sequences, its promoter activity remains to be evaluated in appropriate cell culture models.

Our results, based on transient transfection assays using gonadotrope and nongonadotrope cells, indicated that the DNA elements of crucial importance for basal transcriptional activity are located within the part of the previously described exon 1a sequence upstream of exon 1p, because deletion of the region extending from +12 to +387 abolished promoter activity (Fig. 5Go). Loss of promoter activity is also observed after deletion of the region extending from -73 to +60. Taken together, these data suggest that the +12/+60 region is a determinant for transcriptional activity in all cell lines tested. However, full promoter activity in gonadotrope, but not in CHO cells, requires a larger 5' region that extends farther upstream, because deletion of the -246/-73 fragment results in a significant decrease in Luc activity. Interestingly, the homologous region of the human 5'2 promoter (+50/+234) is similarly required for full transcriptional activity in HeLa cells (21). Furthermore, among the 9 human exon 1 isoforms (36), the 1f isoform seems an extended version (+250/+614) of exon 5'2 (+443/+515). Similarly, the large rat 1a isoform (+1 to +442) could be considered as an extended version of exon 1p (+370/+385 to +442). Thus, the rat 1p promoter seems both structurally and functionally related to the human 5'2 promoter. Interestingly, related isoforms, referred to as exons 1a and 1b, have been also very recently identified by 5'-RACE-PCR in mouse cortical cells (26).

The constitutive expression of the NOS I gene seems thus dependent on two distinct proximal regions extending from -246 to +60 that contain positive regulatory elements. We also show that the distal part of the NOS I promoter extending from -1523 to -841 includes repressor elements that counteract, in gonadotrope cells but apparently not in CHO cells, the influence of the proximal regulatory regions. Stimulation of constitutive promoter activity could, therefore, be controlled by enhancement or repression of the activity of the proximal and the distal regulatory regions, respectively. Inhibition of promoter activity, in turn, might result from opposing mechanisms.

Screening of potential regulatory factors indicates that the promoter sequence also contains response elements that mediate stimulation by GnRH and cAMP, suggesting a hormone-responsive promoter. The most potent activator of the NOS I promoter activity in both {alpha}T3–1 and LßT2 cells is the cAMP-inducer CTX. At a maximally effective concentration, the effects of GnRH are less pronounced and occur specifically in LßT2 cells, although GnRH receptors are functional in {alpha}T3–1 cells (29, 30, 33). Because the latter cells are developmentally less mature than LßT2 cells (which express LHß and, under activin, FSHß in addition to GnRH receptor and the {alpha}-subunit also expressed in {alpha}T3–1), it should be emphasized that some elements of the GnRH-induced transcriptional activation of NOS I are lacking in {alpha}T3–1 cells. The extent of Luc activation by GnRH in LßT2 cells (approximately 2-fold after 18 h in the presence of a maximal GnRH concentration) seems consistent with, nevertheless somewhat lower than, the 2.6-fold increase in steady-state levels of NOS I mRNA observed in the rat pituitary 48 h after injection of a GnRH agonist or the 3.4- to 4.0-fold increase observed 4 d (or longer) after castration (11, 15, 17), which represents a model in which the pituitary is stimulated by high-frequency GnRH pulses. These differences might come from the experimental models (in vivo vs. in vitro, normal rat tissue vs. tumoral mouse cell lines, and other factors) or the conditions used (concentration of GnRH or GnRH agonist, nature and duration of treatments, and other conditions). A detectable effect of GnRH, on the transcriptional activity of the NOS I promoter sequence in LßT2 cells but not in {alpha}T3–1 cells, as discussed above, reinforces the importance of the host cell. Other possibilities include the occurrence of additional transcriptional and/or posttranscriptional regulations. For instance, human exon 1f, which shares identity with rat exon 1a, decreases the translation efficiency of NOS I gene (36). Further investigation is required to determine whether exon 1p might alter the half-life of NOS I mRNA, compared with other exon 1 isoforms. The present study, nevertheless, strongly suggests that the previously established effect of GnRH on NOS I gene expression is, at least in part, mediated by GnRH-induced stimulation of NOS I gene transcription and also that the 1p promoter contains regulatory sequences involved in this process.

In this respect, it is quite surprising that the PKC activator, TPA, is ineffective in inducing the NOS I gene, because the phospholipase C/PKC cascade is considered the preferential signaling pathway for the activated GnRH receptors (39). Indeed, under the same experimental conditions, TPA was shown to be capable of inducing the activation of Luc activity in LßT2 cells, after transfection, using a TPA-responsive control plasmid, confirming the functionality of TPA as well as the PKC pathway in these cells. It is now accepted that GnRH can activate, in addition to the PLC/PKC pathway, directly or indirectly, several other transduction pathways. The possibility that GnRH could signal through the cAMP pathway has been the object of a huge controversy. The most recent data, however, suggest that the GnRH receptor has the potentiality to couple with a G{alpha}s (40, 41) or may indirectly increase cAMP production via the Ca2+/calmodulin kinase-mediated activation of adenylate cyclase I (42). Recent studies from our laboratory indicate that both GnRH and CTX are capable of inducing NOS I in primary cultures of normal rat pituitary cells. In the latter studies, CTX was more efficient than GnRH, thus in total agreement with our present observation (G. Garrel, A. Lozach, L. Bachir, and R. Counis, manuscript in preparation). Whatever the mechanism involved, our data suggest that the GnRH- and the PKA pathway may share common mechanisms in the NOS I promoter activation. Indeed, this is apparent in the effects of substances that increase intracellular levels of cAMP (like the G{alpha}s activator CTX or the phosphodiesterase inhibitor IBMX) which, in addition to mimicking the effects of GnRH, do not act additively with this neurohormone. These data are in agreement with those showing that the {Delta}MMTV-Luc(wtCRE) reference construct is activated in response to both CTX and GnRH in LßT2 cells. Therefore, we could speculate that this interaction may occur through a potential cross-talk between downstream targets of the PKC- and PKA-dependent signaling pathways, as it may happen via the CREB/ATF family of transcription factors (43).

Interestingly, the -73/+60 region (which contains a CRE at position -4/+4, susceptible of interacting with such transcription factors) seems necessary (but not sufficient) for full promoter activation by GnRH. This suggests a complex contribution of transcription factors in interaction with cis-acting regulatory element distantly distributed along the promoter. It is rather difficult, at the moment, to speculate on the nature of the elements involved in the tissue-specific and regulated expression of NOS I in the gonadotrophs because of the great number of such potentially functional sequences revealed in multiple copies throughout the promoter sequence by computer analysis (see the partial list given in Results). This abundance may account for the wide tissue-expression pattern of NOS I and complex promoter usage. Alternatively, the search for elements responsive to transcription factors involved in the gonadotrope-specific expression of various genes (including the GnRH receptor and LHß- and {alpha}-subunits) reveals the presence of consensus, as well as degenerated, potential binding sites for GATA and LIM (-1523/-821 and -246/+60), ets-1 (-1523/-246), and Ptx1 (-1523/+60), whereas no sequence for Egr-1, and only two (imperfect) sequences for SF-1, were noted. Whether and how some of these and other elements and transcription factors are functionally involved in the expression of NOS I in gonadotrophs is under current investigation.


   Acknowledgments
 
The authors express their warmest thanks to Dr. Pamela Mellon, at the University of California, San Diego, for kindly providing the LßT2 cells and to Drs. Danielle Gourdji and Claude Kordon for generous provision of the {alpha}T3–1 cells generated by Dr. Mellon. We are grateful to Dr. Alexandre Appert for providing us with the rat genomic libraries used for chromosome walking and to Dr. Dietmar Spengler for the generous gift of the {Delta}MMTV-Luc(wtCRE) plasmid. The expert technical assistance of Mrs. Danielle Duchene, as well as the contribution of Dr. Lisa Oliver and Mrs. Marie-Claude Chenut for the English correction and preparation of this manuscript, respectively, are wholeheartedly acknowledged. We are indebted to M. Yves Brossas for his help in automated DNA sequencing and to Dr. Yves Courtois for giving us free access to LI-COR DNA sequencer.


   Footnotes
 
This work was supported by grants from the Centre National de la Recherche Scientifique and Pierre et Marie Curie University (Paris). L. K. Bachir is a recipient of a fellowship from the Ministère de la Recherche et de l’Education Nationale.

The nucleotide sequence of the NOS I promoter region described in this paper will appear in the EMBL nucleotide sequence database under accession number AJ305233.

Abbreviations: AP, Activating protein; ATF, activating transcription factor; CHO, Chinese hamster ovary; CRE, cAMP response element; CREB, CRE binding protein; CTX, cholera toxin; IBMX, 3-isobutyl-1-methylxanthine; Luc, luciferase; NO, nitric oxide; NOS, NO synthase; RACE, rapid amplification of cDNA ends; TPA, 12-O-tetradecanoyl 13-phorbol acetate.

Received March 7, 2001.

Accepted for publication July 9, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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