The Orphan Nuclear Receptor, Steroidogenic Factor 1, Regulates Neuronal Nitric Oxide Synthase Gene Expression in Pituitary Gonadotropes
Xueying Wei,
Masayuki Sasaki,
Hui Huang,
Valina L. Dawson and
Ted M. Dawson
Institute for Cell Engineering (V.L.D., T.M.D.), Departments of Neurology (M.S., H.H., V.L.D., T.M.D.), Neuroscience (V.L.D., T.M.D.), and Physiology (V.L.D.), and the Program in Cellular and Molecular Medicine (X.W., V.L.D., T.M.D.), Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
Address all correspondence and requests for reprints to: Ted M. Dawson, Institute for Cell Engineering, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Carnegie 2-214, Baltimore, Maryland 21287. E-mail: .
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ABSTRACT
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Steroidogenic factor 1 (SF-1), an essential nuclear receptor, plays key roles in steroidogenic cell function within the adrenal cortex and gonads. It also contributes to reproductive function at all three levels of the hypothalamic-pituitary-gonadal axis. SF-1 regulates genes in the steroidogenic pathway, such as LHß, FSHß, and steroid hydroxylase. Abundant evidence suggests that nitric oxide (NO) has an important role in the control of reproduction due to its ability to control GnRH secretion from the hypothalamus and the preovulatory LH surge in pituitary gonadotropes. Recently, we cloned and characterized the promoter of mouse neuronal NO synthase (nNOS). nNOS is localized at all three levels of the hypothalamic-pituitary-gonadal axis to generate NO. We find that its major promoter resides at exon 2 in the pituitary gonadotrope
T31 cell line and that there is a nuclear hormone receptor binding site in this region, to which SF-1 can bind and regulate nNOS transcription. Mutation of the nuclear hormone receptor binding site dramatically decreases basal promoter activity and abolishes SF-1 responsiveness. A dominant negative of SF-1, in which the transactivation (AF-2) domain of SF-1 was deleted, inhibits nNOS exon 2 promoter activity. Dosage-sensitive reversal- adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1), which colocalizes and interferes with SF-1 actions in multiple cell lineages, negatively modulates SF-1 regulation of nNOS transcription. These findings demonstrate that mouse nNOS gene expression is regulated by the SF-1 gene family in pituitary gonadotropes. nNOS, a member of the cytochrome p450 gene family, could be one of the downstream effector genes, which mediates SF-1s reproductive function and developmental patterning.
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INTRODUCTION
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NITRIC OXIDE (NO) is an important biological messenger (1, 2). NO is synthesized by three different isoforms, neuronal NO synthase (NOS) (nNOS, type I), endothelial NOS (eNOS, type III) and inducible NOS (iNOS, type II) (1). Through the action and production of NO by these different isoforms, NO subserves many important and different physiological functions (1, 2). NO exerts an important influence on neuroendocrine function. nNOS is enriched in both the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic- pituitary-gonadal (HPG) axis. nNOS is enriched in the paraventricular, supraoptic, and ventral medial nuclei of the hypothalamus (3, 4). In the pituitary gland, nNOS is enriched in the posterior pituitary (5). The anterior pituitary expresses nNOS, and it is localized to gonadotropes and folliculo-stellate cells (6). In the adrenal gland, NO is localized to adrenal chromaffin cells (5). nNOS is enriched within both female and male reproductive tracts, where it regulates the fertility of both male and female mice (3, 7). NO may be important for the secretion and regulation of a variety of neural hormones including CRH, ACTH, GnRH, LH, and FSH (3, 4, 7, 8, 9, 10, 11, 12, 13).
The expression of the various neural hormones and neural modulators within the HPA and HPG are tightly regulated and controlled in a temporal and developmental manner (3, 4, 8, 9, 10, 12). The nuclear receptor, steroidogenic factor-1 (SF-1), plays a prominent role in regulating the expression of a variety of important HPA and HPG genes (14, 15, 16, 17, 18). Due to the potential importance of nNOS in regulating hypothalamic-pituitary function, we sought to characterize the molecular mechanisms by which nNOS expression is regulated. We show in the pituitary gonadotrope cell line
T31 (19) that nNOS expression is driven primarily by the nNOS exon 2 promoter and that SF-1 plays a prominent role in regulating the expression of nNOS.
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RESULTS
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nNOS Exon 2 Is The Major Transcript in Pituitary Gonadotropes
The regulation of the nNOS gene is extremely complicated. In humans, the genomic structure spans more than 240 kb, where nine separate alternative first exons splice to a common exon 2 to yield an identical protein (20). These alternative first exons contain alternative promoters, which control the diversity of nNOS expression in many different organs in a spatial and temporal manner (21). Characterization of the mouse nNOS genomic structure revealed that nNOS is primarily regulated by at least three promoters contained within exon 1a, 1b, and 1c, which splice to a common exon 2 (21). Although exon 2 contains the translation initiation start methionine, it also contains 5'-flanking sequences that function as a potent promoter, depending upon the transcription initiation start site (21). Accordingly, we sought to determine which promoter within the 5'-flanking sequences of exon 1a, 1b, 1c, or exon 2 regulates the expression of nNOS in the mouse pituitary gonadotrope cell line
T31 (Fig. 1
). A sensitive RT-PCR assay was used to measure the level of each nNOS mRNA in
T31 cells (22). To verify the specificity of the amplified nNOS cDNA, a nNOS-specific primer recognizing all amplified transcripts was used as a probe. Only the exon 2 transcript is detected in
T31 cells (Fig. 1A
). To confirm that exon 2 is the primary nNOS transcript in
T31 cells, we used a series of reporter plasmids containing exon 1a (pEx1a), exon 1a + 1b (pEx1a +1b), exon 1b (pEx1b), exon 1c (pEx1c), and exon 2 (pEx2) fused to the ß- galactosidase (ß-gal) reporter gene. These reporter constructs as previously described (21), contain the alternate promoters that regulate nNOS expression. Each reporter construct was transfected into
T31 cells via lipofectamine transfection, and then 2 d later cells were harvested to assess reporter activity. Consistent with the RT-PCR analysis the promoters and elements within exons 1a, exon 1a + 1b, exon 1b, and exon 1c that reside 5' to the transcription start site have minimal promoter activity, whereas exon 2 has greater than 10-fold higher reporter activity in
T31 cells (Fig. 1B
). These results, taken together, indicate that nNOS exon 2 is the primary transcript in mouse
T31 cells.

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Figure 1. nNOS exon 2 Is the Primary Transcript in Mouse T31 Cells
A, RT-PCR with primers specific for exon 1a, 1b, 1c, and exon 2 coupled with a common antisense primer was used to measure the level of each nNOS mRNA in T31 cells. To verify the specificity of the amplified nNOS cDNA, an nNOS specific primer recognizing all amplified transcripts was used as a probe in Southern blotting. Only the exon 2 transcript is detected in T31 cells. B, For the reporter gene assay, we used a series of reporter plasmids containing exon 1a (pEx1a), exon 1a + 1b (pEx1a +1b), exon 1b (pEx1b), exon 1c (pEx1c), and exon 2 (pEx2) fused to the ß-gal reporter gene. Depicted on the left are the nNOS promoter-ß-galactosidase reporter gene constructs. The numbers on the promoters of exon 1a, 1b, and 1c indicate the positions relative to the transcription initiation sites of the respective exons. For exon 2, the number indicates the position relative to the translation initiation site. The details of the construction procedure are described in Materials and Methods. A RSV-luciferase expression vector (5 ng) was used as an internal control to quantify transfection efficiency. Each reporter construct (0.8 µg) was transiently transfected into T31 cells via lipofectamine, and then 48 h later cells were harvested to assess reporter activity. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. C, To characterize the 5' terminus of nNOS mRNA in T31 cells, 5'-RACE was performed. Two gene-specific primers, nos. 1 and 2, were designed to amplify the 5' end of mouse nNOS according to the sequence around the translation initiation site of nNOS within exon 2. Ten micrograms total RNA extracted from T31 cells were treated with calf intestinal phosphatase and tobacco acid pyrophosphatase enzyme to leave full-length mRNA decapped. A RNA adapter was then ligated to the 5' end. After reverse transcription with a random primer, cDNAs were amplified through two rounds of PCR using as primers nNOS gene-specific primer no. 1 and 5'-RACE outer primer in the first round and an nNOS gene-specific primer no. 2 and 5'-RACE inner primer in the second round. PCR products were analyzed by 3% agarose gel electrophoresis, and ethidium bromide-stained DNAs were visualized under UV. Lane 1 represents molecular weight marker. This experiment was replicated with similar results.
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To establish that exon 2 contains the transcription initiation site, rapid amplification of 5'-cDNA ends (5'-RACE) was carried out using primers located in exon 2. This approach allowed isolation of full-length nNOS mRNA sequences upstream of exon 2. Two different cDNA species are identified from
T31 cells (Fig. 1C
). Sequence analysis of the PCR products shows transcription starting from either -71 or -82 of translation start codon, which is about 30 bp downstream of two CRE sites on the exon 2 promoter. Thus, the ß-gal reporter construct designated pEx2 contain sequences upstream of the -71 or -82 transcription initiation start site that function as the nNOS promoter in
T31 cells.
nNOS Exon 2 Contains a Nuclear Hormone Receptor (NHR) Regulatory Sequence
The NHR, SF-1, regulates a variety of important transcripts within the HPA and HPG (Fig. 2A
). As such, we wondered whether nNOS expression could be regulated in a similar manner by SF-1. Analysis of the mouse nNOS genomic sequence reveals the presence of a consensus NHR DNA (23) recognition site, 5'-PyCAAGGPyPyPu-3', located at -206 to -198 upstream of ATG translation start site 5'-206-CAACCTTG-198-3'. The NHR site is conserved in rat, mouse, and rabbit nNOS exon 2 and relatively conserved in human nNOS (Fig. 2B
). To determine the importance of the NHR site within the nNOS exon 2 5'-untranslated region, in context substitution mutants of the entire exon 2 promoter were generated that were expected to disrupt transactivating factor binding to the exon 2 promoter (Fig. 2C
). Mutation of the NHR DNA recognition site dramatically reduces nNOS pEx2 ß-gal reporter gene activity.

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Figure 2. nNOS Exon 2 Contains an NHR Regulatory Sequence
A, A consensus SF-1 binding site is located in the 5' flanking regions of a variety of genes within the HPG axis including the -subunit gene of LH, FSH, thyrotropin, hCG, and LHß, GnRH receptor and Müllerian-inhibiting substance gene, as well as cytochrome p450 genes such as 21-hydroxylase and aromatase. Numbers indicate the position of the 5' end of the sequences relative to the transcription start site. B, Analysis of the mouse nNOS genomic sequence reveals the presence of a consensus NHR DNA recognition site, 5'-PyCAAGGPyPyPu-3', located at -206 to -198 of ATG translation start site 5'-206CAACCTTG-198-3'. The NHR site is conserved in mouse, rat, and rabbit nNOS exon 2. Numbers represent the position of the 5' end of the sequences relative to the ATG translation start site. The monomeric NHR site is potentially a SF-1 binding site. C, To test if the NHR site is required for the nNOS exon 2 promoter activity, T31 cells were transiently transfected with ß-gal reporter construct pEx2 (0.8 µg) or an in-context-substitution mutant pEx2/NHRm (0.8 µg) with pEx2 NHR site CAACCTTG changed to CAAAATTG along with RSV-luciferase (5 ng). Forty-eight hours after transfection, cells were harvested. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three experiments performed in triplicate.
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SF-1 Interacts with the nNOS NHR Site
To identify the nature of factors in nuclear extracts that interact with the NHR site in nNOS exon 2, EMSAs were performed with a 28-bp fragment of 32P-labeled DNA corresponding to the nNOS promoter sequence that includes the NHR site (Fig. 3
). When the DNA probe was incubated with nuclear extracts prepared from
T31 cells, one major complex is identified (Fig. 3
). The binding of the protein to the NHR site is specific, in as much as the formation of the complex is completely inhibited by excess of unlabeled sequence, and not by an excess of sequence in which the NHR element was mutated (24). To determine whether SF-1 is a component of the protein complex that binds to the NHR site, specific polyclonal antibodies to SF-1 were added to the protein/DNA binding fraction mixture, and the effect on complex formation from nuclear extracts prepared from
T31 cells was assessed (25). We find that a polycolonal anti-SF-1 antibody, disrupts the protein/DNA complex. In the presence of another anti-SF-1 antibody the DNA protein complex is disrupted (Fig. 3
). Taken together, these results suggest that SF-1 is a component of the NHR/protein complex and that SF-1 may be a critical regulator of nNOS expression.

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Figure 3. SF-1 Binds to the NHR Site Contained within the Exon 2 Promoter of nNOS
Binding reactions were performed using T31 nuclear extracts and a 32P-labeled 28-bp probe spanning region -213 to -186 of the nNOS exon 2 promoter. EMSA showed a prominent DNA/protein complex (lane 1), which is completely inhibited by 125-fold molar excess of unlabeled sequence (lane 2), but not by an excess of sequence in which the NHR element is mutated (lane 3). To determine the nature of the protein in the complex, specific polyclonal antibodies to SF-1 (anti-SF1 no. 1) were added, which disrupts the complex (lane 4). In the presence of another anti-SF-1 antibody (anti-SF-1 no. 2), the DNA/protein complex is also disrupted (lane 5). As a control, preimmune rabbit IgG cannot dissociate the complex (lane 6). This experiment was replicated with similar results.
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To further investigate the importance of SF-1 as a regulator of nNOS transcription, the effects of overexpression of SF-1 (26, 27) and a constitutively active SF-1 (VP16-SF-1) (28, 29) was examined. Both SF-1 and VP16-SF-1 induce nNOS ß-gal reporter gene activity (Fig. 4A
). An intact NHR is required for nNOS-promoter induction by SF-1 or VP16-SF-1 because a nNOS exon 2ß-gal reporter gene containing a mutation in the NHR site is not responsive (Fig. 4A
). Because SF-1 is constitutively expressed at high levels in
T31 cells, we examined the effects of forced overexpression of SF-1 in SF-1-deficient NIH3T3 cells (Fig. 4B
). SF-1 dramatically induces nNOS ß-gal reporter gene activity in SF-1-deficient NIH3T3 cells (Fig. 4B
). An intact NHR is required for nNOS-promoter induction by SF-1 because a nNOS exon 2 ß-gal reporter gene containing a mutation in the NHR site is not responsive (Fig. 4B
).

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Figure 4. An Intact NHR Site within the nNOS Exon 2 Promoter Confers SF-1 Responsiveness in Pituitary Gonadotropes
A, T31 cells were transiently transfected with the ß-gal reporter construct pEx2 (0.8 µg) or pEx2/NHRm (0.8 µg). Cells were cotransfected with control vector PCI (0.2 µg), SF-1 overexpression vector PCMV5-SF-1 (0.2 µg), or constitutively active VP16-SF-1 (0.2 µg) along with RSV-luciferase (5 ng). Forty-eight hours after transfection, cells were harvested. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing pEx2 with control vector to pEx2/NHRm with control vector, and comparing pEx2 cotransfected with control vector to pEx2 cotransfected with PCMV5-SF-1 or VP16-SF-1. B, Due to the minimal effect of SF-1 on pEx2 promoter activity in T31 cells and to verify that SF-1 up-regulates exon 2 promoter activity through the NHR site, an SF-1-deficient cell line was used. NIH3T3 cells were transiently transfected with ß-gal reporter construct pEx2 (0.8 µg) or pEx2/NHRm (0.8 µg). Cells were cotransfected with control vector PCI (0.2 µg), or SF-1 overexpression vector PCMV5-SF-1 (0.2 µg) along with RSV-luciferase (5 ng). Forty-eight hours after transfection, cells were harvested. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing pEx2 with control vector to pEx2/NHRm with control vector, and comparing pEx2 cotransfected with control vector to pEx2 cotransfected with PCMV5-SF-1.
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To ascertain whether SF-1 is an important mediator of nNOS transcription, a dominant negative to SF-1 (SF-1 del 443462) was created by deleting amino acids 443462. Deletion of amino acids 443462 removes the transactivation (AF-2) domain, which is required for transcriptional activation by SF-1 (27, 30, 31). SF-1 del 443462 dose dependently reduces nNOS exon 2-driven ß-gal expression (Fig. 5
). SF-1 del 443462 still inhibits nNOS exon 2 ß-gal reporter gene containing a mutation in the NHR site (data not shown), thus SF-1 del 443462-mediated inhibition of nNOS expression may not require an intact SF-1 binding site. The finding that SF-1 enhances nNOS exon 2 promoter activity, as well as the finding that an NHR mutation in the nNOS exon 2 promoter and a dominant inhibitory form of SF-1 reduce nNOS transcription, suggests that SF-1 binds to the nNOS NHR element and regulates nNOS transcription.

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Figure 5. A Dominant-Negative SF-1 (SF-1 del 443462) Dose Dependently Inhibits nNOS pEx2 Promoter Activity
A, AF-2 domain, residing at the carboxyl terminal of SF-1, is responsible for transcriptional activation of SF-1 (30 ). SF-1 del 443462 is an expression plasmid that removes the AF-2 domain corresponding to amino acids 443 to 462. B, pEx2 (0.8 µg) and increasing amounts of SF-1 del 443462 (0, 0.1, 0.2, 0.5, 1, 2 µg) were cotransfected into T31 cells along with RSV-luciferase (5 ng). The total amount of the transfected PCI expression vector was kept constant in each transfection. Forty-eight hours after transfection, cells were harvested for reporter activity. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing pEx2 to pEx2 cotransfected with increasing amounts of SF-1 del 443462.
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To test whether SF-1 is sufficient to regulate nNOS transcription, we employed the GAL4 system using cytomegalovirus (CMV) promoter driven expression of a GAL4 DNA binding domain fusion proteins. We replaced the NHR site within the nNOS exon 2 ß-gal reporter gene with 1 GAL4 binding site (pEx2/NHRGAL4). Consistent with our mutation analysis, introduction of the GAL4 binding site significantly reduces basal nNOS ß-gal reporter gene activity (Fig. 6
). In the presence of GAL4-SF-1, basal promoter activity is restored. A dominant-negative form of SF-1 fused to GAL4 (GAL4-SF-1 del 443462) further reduces ß-gal reporter gene activity. Taken together, these experiments indicate that SF-1 bound to nNOS exon 2 promoter controls basal nNOS transcription.

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Figure 6. SF-1 Bound to nNOS Exon 2 Promoter Controls Basal nNOS Transcription
A, NHR site within the nNOS exon 2 ß-gal reporter gene was replaced with 1 GAL4 binding site (pEx2/NHRGAL4). B, GAL4-SF-1 lacks the first 132 amino acids of SF-1 and has the DNA binding domain and Ftz-f1 box of SF-1 replaced with the GAL4 DNA binding domain (30 32 ). SF-1-GAL4 del 443462 contains first 19 residues of SF-1 and the GAL4 DNA binding domain but has amino acids 20132, 443462 of SF-1 deleted (30 ). The first 19 amino acids are not essential for SF-1 transactivation (30 ). C, pEx2 (0.8 µg) and pEx2/NHRGAL4 (0.8 µg) were transiently transfected into T31 cells. pEx2/NHRGAL4 was also cotransfected with pCMV-GAL4 (0.2 µg), GAL4-SF-1 (0.2 µg), or SF-1-GAL4 del 443462 (0.2 µg) along with RSV-luciferase (5 ng). Forty-eight hours after transfection, cells were harvested for reporter activity. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing pEx2 to pEx2/NHRGAL4; **, P < 0.05 when comparing pEx2/NHRGAL4 cotransfected with GAL4 compared with pEx2/NHRGAL4 cotransfected with GAL4-SF-1 or pEx2/NHRGAL4 cotransfected with SF-1-GAL4 del 443462.
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DAX-1 (dosage-sensitive reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) is a negative regulator of SF-1 function (32, 33, 34, 35, 36, 37). Accordingly, we monitored whether DAX-1 could regulate nNOS transcription (Fig. 7
). DAX-1 dose dependently inhibits nNOS exon 2 reporter gene activity (Fig. 7A
). A DAX-1 mutant (DAX-1 del 92470), which lacks the SF-1 interaction domain, fails to reduce nNOS exon 2 reporter gene activity (Fig. 7B
). To test whether an intact NHR site is needed for DAX-1-mediated inhibition, we examined the effects of DAX-1 on pEx2/NHRm. We find that DAX-1 still inhibits pEX2/NHRm (data not shown); thus, DAX-1 mediated transrepression of nNOS expression may not require an intact SF-1 binding site. Further support that DAX-1 inhibits nNOS transcription through an interaction with SF-1 and not through direct DNA binding is the observation that DAX-1 inhibits ß-gal reporter activity from pEx2/NHRGAL4 reporter construct in the presence of GAL4-SF-1 (Fig. 7C
).

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Figure 7. DAX-1 Negatively Modulates SF-1 Transactivation of nNOS Exon 2 Promoter Activity
DAX-1 colocalizes with SF-1 in multiple cell lineages including pituitary gonadotropes and regulates SF-1 function in the endocrine system (32 33 34 35 ). We tested whether DAX-1 could regulate nNOS transcription in T31 cells. A, 0.8 µg pEx2 and increasing amounts of DAX-1 (0, 0.1, 0.2, 0.5, 1, 2 µg) were cotransfected into T31 cells along with RSV-luciferase (5 ng). The total amount of the transfected PBKCMV expression vector was kept constant in each transfection. Forty-eight hours after transfection, cells were harvested for reporter activity. B, To test the effect of an inactive DAX-1 truncation mutant (DAX-1 del 92470) on SF-1 transactivation of nNOS, 0.8 µg pEx2 and 0.5 µg DAX-1 or 0.5 µg DAX-1 del 92470 were cotransfected into T31 cells along with RSV-luciferase (5 ng). The total amount of the transfected PBKCMV expression vector was kept constant in each transfection. Forty-eight hours after transfection, cells were harvested for reporter activity. C, To see if DAX-1 inhibition of SF-1 mediated nNOS transactivation is independent of DNA binding, 0.8 µg pEx2/NHRGAL4 and 0.2 µg GAL4-SF-1 were cotransfected with increasing amounts of DAX-1 (0, 10, 20, 50, 100 ng) into T31 cells along with RSV-luciferase (5 ng). The total amount of the transfected PBKCMV expression vector was kept constant in each transfection. Forty-eight hours after transfection, cells were harvested for reporter activity. Levels of ß-gal activity were internally standardized by levels of RSV-luciferase activity. Promoter activity of each transfection was normalized to the average of pEx2 promoter activity. Results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing reporter construct to reporter construct cotransfected with various amounts of DAX-1 or DAX-1 del 92470.
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To ascertain whether SF-1 is a critical regulator of endogenous nNOS gene expression in
T31 cells, we assessed nNOS levels via Western blot analysis after
T31 cells were transfected with either the dominant-negative SF-1 construct or the constitutively active SF-1 (28, 29) construct (Fig. 8
). The dominant negative SF-1 construct significantly reduces nNOS expression by approximately 40%, whereas the constitutively active SF-1 construct increases nNOS expression by approximately 40% (Fig. 8
).

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Figure 8. Overexpression of Dominant-Negative or Constitutively Active SF-1 Affects Endogenous nNOS Gene Expression in T31 Cells
Two micrograms of PCI, SF-1 del 443462, or constitutively active VP16-SF-1 were transfected into T31 cells via the lipofectamine method. Forty-eight hours later, cells were harvested and Western blots were performed to detect endogenous nNOS protein expression levels. The amount of nNOS protein was quantitated by densitometry. Values of nNOS protein level were standardized to ß-tubulin levels of the same sample. nNOS protein expression levels after PCI treatment was used to normalize nNOS protein levels for comparison with SF-1 del 443462 or VP16-SF-1 treatment (PCI = 100%). Results are the mean ± SEM from at least four independent experiments. Statistical significance was determined by a Students t test at *, P < 0.05 when comparing PCI treatment to SF-1 del 443462 or VP16-SF-1 treatment.
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DISCUSSION
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Our results demonstrate that nNOS is an SF-1-regulated gene. nNOS contains a consensus NHR DNA recognition site that is conserved in human, rat, and mouse. Mutation of the NHR site within the nNOS exon 2 promoter substantially reduces nNOS exon 2 ß-gal reporter gene activity. SF-1 binds specifically to the NHR region of the mouse nNOS exon 2 gene promoter and through this binding regulates nNOS exon 2 promoter activity. Our experiments indicate that the nNOS NHR site contained within exon 2 contributes significantly to nNOS transcription. First, the nNOS NHR site is a consensus NHR sequence that is conserved in rodents and humans, suggesting that the nNOS NHR might function as a binding site for SF-1 or SF-1-related family members. Second, EMSA using an nNOS exon 2 fragment (-213 to -186) shows a DNA protein complex that is competed with cold probe, whereas a mutant probe fails to compete with the DNA protein complex, indicating that the complex contained within nNOS exon 2 contains an NHR binding protein. Third, two separate antibodies to SF-1, but not control serum, disrupts the complex formed with the nNOS exon 2 probe, indicating that SF-1 is part of the complex. Fourth, expression of a SF-1 interfering mutant (SF-1 del 443462) markedly attenuates transactivation of the nNOS exon 2 ß-gal reporter gene. Fifth, SF-1 is sufficient to activate the nNOS exon 2 reporter transcription in the SF-1-deficient cell line NIH3T3 cells, and this activation requires an intact NHR site. Mutation of the NHR site dramatically decreases nNOS exon 2 promoter activity in the SF-1 expressing
T31 cells. Sixth, DAX-1, a negative regulator of SF-1 function, potently inhibits nNOS exon 2 ß-gal reporter gene activity. And finally, overexpression of a dominant-negative SF-1 or overexpression of the constitutively active SF-1 construct (VP16-SF-1) down-regulates or increases nNOS expression, respectively, in
T31 cells. Furthermore, SF-1 is sufficient to regulate nNOS transcription as indicated by use of the GAL4 transactivation system. These observations, taken together, strongly suggest that the nNOS NHR contributes to the regulation of nNOS transcription and that a component of the NHR regulation is mediated via SF-1.
Within the HPG and HPA, SF-1 functions at multiple levels to regulate the expression of a variety of important genes including LHß, GnRH receptor, cytochrome p450 steroid hydroxylases, steroidogenic acute regulatory protein, ACTH receptor, oxytocin, and others (14). nNOS is structurally related to the cytochrome p450 gene family (1). Members of the cytochrome p450 gene family regulates steroid hormone synthesis in conjunction with hydroxysteroid dehydrogenases (38). The expression of these enzymes is regulated in a temporal and tissue-specific manner, and the expression is mainly regulated at the transcriptional level (38). Analysis of the promoter regions of the steroidogenic p450 gene family reveals a crucial NHR element, which binds to SF-1. SF-1 acts at the adrenal cortex, multiple levels of the reproductive axis and the hypothalamus, pituitary, and gonads. Based upon our results, SF-1 may play a critical role in the coordinated expression of nNOS. nNOS joins the list of SF-1-regulated proteins within the HPG. Due to the importance of nNOS in regulating neuroendocrine function, our data suggest that SF-1 may play a critical role in the temporal and tissue-specific expression of nNOS within the HPA and HPG.
Quantitative RT-PCR followed by Southern-blotting with an nNOS specific promoter indicates that exon 2 is the primary transcript for nNOS in
T31 cells. ß-gal reporter gene activity assays also showed that in
T31 cells exon 2 contains the primary promoter for regulation of nNOS expression in
T31. Furthermore, 5'-RACE reveals that the transcription initiation start site is contained with nNOS exon 2. This is consistent with previous findings that in mouse cortical neurons exon 2 is the main inducible transcript regulated by calcium influx through L-type voltage-sensitive channels (21). In cortical neurons, nNOS expression is regulated by two cAMP/Ca2+ regulatory elements (CRE) (21). The CRE binding protein (CREB) binds to the two CREs within nNOS to regulate the expression in nNOS in response to calcium (21). It is likely that, in
T31 cells, the CRE sites are also important for expression. NHR transactivation is often coordinated through an interaction with the CREB binding protein (30, 39, 40, 41). It is likely that SF-1-regulated expression of nNOS is coordinated through interactions with proteins that bind to the CRE/CREB/CREB binding protein complex contained within the nNOS exon 2 promoter.
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MATERIALS AND METHODS
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Materials
T31 cells were generously provided by Pamela Mellon (University of California San Diego, La Jolla, CA). PCMV5-SF-1, the vector containing the mouse SF-1 cDNA, was given by Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX). PBKCMV-DAX-1, with the full-length human DAX-1 cDNA, GAL4-SF-1, SF-1-GAL4 del 443462, were gifts from J. Larry Jameson (Northwestern University Medical School, Chicago, IL).
Plasmid Construction
The construction of pßgal-301, pEx2, pEx1a, pEx1a + 1b, pEx1b, and pEx1c have been previously described (21). Briefly, to facilitate construction of reporter gene constructs we introduced unique recognition sites for EcoRI and XhoI upstream of ß-gal cDNA. The multiple cloning site of pSL301 (Invitrogen Life Technologies, Carlsbad, CA) was digested by NcoI, made blunt, and excised by HindIII digestion. This multiple cloning site fragment was subcloned into SmaI-HindIII site of pßgal-Basic (CLONTECH Laboratories, Inc., Palo Alto, CA) and designated pßgal-301.pBam9, which contains a 9-kb BamHI fragment of the mouse genomic DNA that includes mouse exon 2 (kindly provided by Dr. Paul L. Huang, Massachusetts General Hospital, Boston, MA). Using pBam9 as a template, a DNA fragment containing partial exon 2 and its 5' flanking region (-859 to -9 of the translation initiation site) was generated by PCR using a sense primer (5'-TAC GGA ATT C1AT ATC TGC CCA GTA-3') and an antisense primer (5'-TGT GTC CTC GAG2 AAG ACC TCC AAG-3') introducing EcoRI site (underline 1) in its 5' end and XhoI (underline 2) site in the 3' end, respectively. After digestion of the PCR product with these enzymes, the fragment of exon 2 and its 5' flanking region was subcloned into EcoRI-XhoI site of pßgal-301 and designated pEx2. To isolate genomic DNA containing mouse nNOS exon 1, a mouse genomic DNA library constructed in a BAC system (Bacterial Artificial Chromosomes, Research Genetics, Inc., Huntsville, AL) was screened with a probe isolated from pBam9. A positive 4.5-kb fragment containing exon 1a was subcloned into BamHI-EcoRI site of pBluescript II KS(-) (Stratagene, La Jolla, CA) and was designated pEB4.5. Using pEB4.5 as template, a DNA fragment of exon 1b and 300 bp of its 5' flanking region was produced by PCR with a sense primer (5'-TTC AGA ATT C1AT AGG ATA AAG CAG-3') and antisense primer (5'-CCT GCT CTC GAG2 CCC GCG CAG TTC-3'). After enzyme digestion, these fragments were subcloned into EcoRI-XhoI site of pßgal-301 and designated pEx1b. Using pBam9 as a template, the DNA fragment of exon 1c and 600 bp of its 5' flanking region was created with a sense primer (5'-CCA AAT GAA TTC1 GTT ACA TAA TAA TAA CCA GA-3') and antisense primer (5'-TGA TGT AGG GGC TCC TAC CTG GGT AAC3 TAG CT-3') containing a KpnI site (underline 3). After digestion by EcoRI and KpnI, the fragment was subcloned into the EcoRI-KpnI site of pßgal-301 and designated pEx1c. A DNA fragment containing exon 1a and its 5' flanking sequence, 3.5 kb was excised by EcoRI and EagI from pEB4.5, subcloned into EcoRI-EagI site of pßgal-301 and the same site of pEx1b, and designated pEx1a and pEx1a + 1b, respectively.
To introduce point mutations in putative cis-regulatory elements, site-directed mutagenesis was employed using a PCR overlap protocol (42). Briefly, the PCR was carried out using an outer sense primer and an inner antisense primer. Another PCR was performed using the combination of an inner sense primer and the outer antisense primer. DNA fragments from both PCRs had overlapping ends and were linked together in a subsequent fusion reaction in which the overlapping 3' ends functions as a primer for the 3' extension of the opposite strand. This combined fragment was further amplified in the second PCR using the outer sense and antisense primer. The resultant fragment has EcoRI and XhoI site in its ends and were inserted into the EcoRI-XhoI site of pßgal-301. The pEx2/NHRm was created by introducing a 2-bp mutation into the pEx2 construct with a pair of mutagenic primers (sense strand: 5'-ACG TCT GCC TGG TCA AAA TTG ACT TCC TTA GAG AT-3').
The SF-1 del 443462 has amino acids 443462 of SF-1 protein deleted and was made by adding a termination codon and XbaI site and subcloning the EcoRI/XbaI fragment into the PCI vector (Promega Corp., Madison, WI). The VP16-SF-1 was constructed by subcloning PCR products containing the VP16 activation domain sequence into SF-1 del 443462. DAX-1 del 92470 was created by introducing a termination codon and XbaI site through PCR and subcloning the products back to the same expression vector.
To construct pEx2/NHRGAL4, the same PCR-based technique was employed. First, using as inner primer pair (sense primer: -206 to -180 TCT AGA1 CAA CCT TGA GAT CT2T CCT TAG AGA ATA AG, antisense primer: -231 to -197 AGA TCT2 CAA GGT TGT CTA GA1C CAG GCA GAC GTC AG) and pEx2 as template, XbaI site (underline 1) and BglII site (underline 2) were introduced into pEx2. This site-direct mutated pEx2 had the same promoter activity in
T31 cells as pEx2 (data not shown). The XbaI-BglII region containing NHR site in the mutated pEx2 was replaced with a synthesized double-stranded oligonucleotide containing a copy of the consensus GAL4 binding sequence (underline 3) with XbaI and BglII at its 5' and 3' end, respectively (sense strand: 5'-CTA GAG CAG GTG CGG AGT ACT GTC CTC CG3A AGA CAG AGG A-3'), antisense strand: 5'-GAT CTC CTC TGT CTT CGG AGG ACA GTA CTC CG3C ACC TGC T-3').
To eliminate the problem of nonspecific mutation generated through PCR, a thermostable DNA polymerase harboring a proofreading activity (pfu DNA polymerase, Stratagene) was employed in all PCR for construction of the reporter genes. All constructs were verified by direct sequencing.
Cell Culture and Transient Transfection
The
T31 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml streptomycin and penicillin (Life Technologies, Inc.) at 37 C in a humidified atmosphere containing 5% CO2 and 95% air. The NIH3T3 cells were cultured using DMEM with 10% FBS, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, and 100 U/ml streptomycin and penicillin under the same conditions.
For transient expression studies, the cells were plated in 35-mm culture wells and cultured for 1 d. The following day, transient transfections were performed by a lipofection method (LIPOFECTAMINE PLUS Reagent, Life Technologies, Inc.). For the purpose of normalizing transfection efficiency between each well, a luciferase expression vector with a Rous sarcoma virus (RSV) promoter was cotransfected. The reporter constructs (0.8 µg/well) and the luciferase expression vector (5 ng/well) with or without the amount of overexpression vectors (0.2 µg/well or as indicated) were treated with 6 µl/well PLUS Reagent and 4 µl/well LIPOFECTAMINE reagent in 0.1 ml/well of reduced serum DMEM for 15 min sequentially. The cells were washed twice and finally replaced with 1 ml media containing the DNA-PLUS-liposome complex. After incubation for 35 h, 1 ml of the growth media containing 20% of FBS was added and the cells were further incubated overnight. The following day, the transfection medium was removed and new culture media was applied. The cells were harvested 48 h following transfection in 200-µl cell lysis buffer (Lysis Solution, Galacto-Star, TROPIX, Bedford, MA). The ß-gal activity in the whole cell lysate was measured with a luminometer (Monolight 2000, Analytical Luminescence Laboratory, Ann Arbor, MI) using a chemiluminescence-based detection system (Reaction Buffer, Galacto-Star, TROPIX) after the manufacturers protocol. Luciferase activity was determined using a chemiluminescence-based detection system (Luciferase Assay System, Promega Corp.). The reporter activity was normalized by dividing the observed ß-gal activity by the luciferase activity of the same sample. The reporter activity was further normalized by dividing the sample values by the average of pEx2 promoter activity (assuming pEx2 = 100%). The data are presented as mean ± SEM and represent multiple independent experiments with each individual experiment performed in triplicate. Statistical significance was calculated by ANOVA and the Students t test, and significance was established as P < 0.05.
EMSA
For preparation of nuclear extracts,
T31 cells were harvested with ice-cold hypoosmotic buffer [10 mM HEPES, pH 7.8; 10 mM KCl; 2 mM MgCl2; 0.1 mM EDTA; 10 µg/ml aprotinin; 0.5 µg/ml leupeptin; 3 mM phenylmethylsulfonyl fluoride; and 3 mM dithiothreitol (DTT)]. After incubation in the ice-cold hypoosmotic buffer, Nonidet P-40 was added to a final concentration of 0.6% to disrupt the cell membrane. The nuclei were collected by centrifugation for 5 min and resuspended in a high salt buffer (50 mM HEPES, pH 7.4; 50 mM KCl; 300 mM NaCl; 0.1 mM EDTA; 10% glycerol; 3 mM DTT; and 3 mM phenylmethylsulfonyl fluoride). To facilitate solubilizing DNA binding proteins, the solution was gently shaken for 30 min at 4 C. Soluble proteins in the high salt buffer were separated by 10 min of centrifugation from insoluble matter and stored at -80 C for further use.
A double-stranded oligonucleotide as shown (see Fig. 3
) was used as a probe and end-labeled with T4 polynucleotide kinase in the presence of [
-32P]ATP and purified through G-50 Sephadex columns (NICK Columns, Amersham Pharmacia Biotech, Arlington Heights, IL). Ten micrograms of nuclear extract were subjected to 30 µl of binding reaction (43) containing 15 mM Tris-HCl (pH 7.8), 50 mM KCl, 1 mM EDTA, 1 mM DTT, 4% Ficoll 400, 4 mg/ml BSA, 2 µg of poly(deoxyinosine-deoxycytidine) and, as competitors, excess unlabeled wide-type or mutated oligonucleotides, polyclonal anti-SF-1 antibody (anti-SF-1 no. 1; Upstate Biotechnology, Inc.), anti-SF-1 antiserum (anti-SF-1 no. 2; provided by Dr. Ken-ichirou Morohashi, National Institute for Basic Biology, Okazaki, Japan), or rabbit preimmune IgG (Sigma). After incubation for 30 min on ice, 25,000 cpm of labeled probe was added and further incubated for 30 min on ice. DNA-protein complexes were separated from free probe on a native 5% polyacrylamide gel. After running at 4 C, the gel was dried and subjected to autoradiography.
RT-PCR Assays
T31 cellular RNA was isolated using guanidinium isothiocyanate/phenol/chloroform (44). Total RNA (10 µg) was reverse transcribed by the addition of 40 U Moloney murine leukemia virus reverse transcriptase at 37 C for 1 h in the presence of 300 ng oligo(deoxythymidine) primers, 25 mM deoxynucleoside triphosphates (ProSTAR First-stand RT-PCR Kit, Stratagene). PCRs (22) included specific sense primers for exon 1a, 1b, 1c, exon 2, and a common nNOS antisense primer. The primers were: exon 1a sense strand 5'-AAA TAA ATT CCC CCT CAC GAC C-3', product 800 bp; exon 1b sense strand 5'-GGG GTG AAC AGC GTG ATC CAC-3', product 500 bp; exon 1c sense strand 5'-AGG TGG GAG GAG AGC CAA TGG-3', product 500 bp; exon 2 sense strand 5'-CCT GTG GGA GTC GTC TTG GC-3', product 370 bp; common antisense strand 5'-GTG GTC TCC AGG TGT GTA GTA AAG C-3'. Cycle conditions were 94 C for 3 min, 94 C for 1 min, 54 C for 1 min, 72 C for 1.5 min. Amplification was carried out for 28 cycles.
Southern Blot Analysis
RT-PCR products were separated on 1.5% agarose gel. Standard Southern blotting procedures were followed with DNA fixed on Hybond-N+ membrane (Amersham Pharmacia Biotech) by UV cross-linking. nNOS-specific primer 5'-CAA GCC AAG ACG ACT CCC ACA GGA-3' was used as a probe and end-labeled with T4 polynucleotide kinase in the presence of [
-32P]ATP. After prehybridization of the membrane with 5x saline sodium phosphate/EDTA (SSPE), 5x Denhardts solution, 0.5% sodium dodecyl sulfate (SDS), and denatured 0.5 mg sonicated Herring sperm DNA (Life Technologies, Inc.) at 65 C for 1 h, the labeled nNOS-specific probe was denatured by heating to 100 C for 5 min and added to the prehybridization solution. After hybridization for at least 12 h at 65 C, the membrane was washed using high stringency conditions, twice in 2x SSPE, 0.1% SDS at room temperature for 10 min, one time in 1x SSPE, 0.1% SDS at 65 C for 15 min, and one time in 0.1x SSPE, 0.1% SDS at 65 C for 10 min. After washing, the membrane was wrapped in Saran Wrap and autoradiographed.
5'-RACE
To characterize the 5' terminus of nNOS mRNA from
T31 cells, 5'-RACE was carried out using the FirstChoice RLM-RACE kit (Ambion, Inc., Austin, TX). Total cellular RNA was isolated using guanidinium/phenol/chloroform (44). Ten micrograms of total RNA were treated with calf intestinal phosphatase to remove free 5'-phosphates from degraded mRNA, tRNA, rRNA, and DNA. Tobacco acid pyrophosphatase was then used to incubate the RNA to remove the cap structure from full-length mRNA, leaving a 5'-monophosphate. A 45-base RNA adapter oligonucleotide was ligated to the 5' end of mRNA using T4 RNA ligase. RNA was then reverse transcribed with Moloney murine leukemia virus reverse transcriptase by priming with random decamers. The first strand cDNA was amplified in 50 µl of the first-round PCR containing 20 pmol of both the nNOS gene-specific primer no. 1 (5'-GGT ATC TGT GTC CTT CAG AAG ACC-3') and 5'-RACE outer primer. The temperature and cycling conditions were as follows: 1 cycle of 94 C for 3 min, 35 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min and 1 cycle of 7 min of elongation time at 72 C. One microliter of first PCR product was subjected to a second round of amplification in 50 µl of reaction mixture containing 20 pmol of both the nNOS gene specific primer no. 2 (5'-CAA GCC AAG ACG ACT CCC ACA GGA-3') and 5'-RACE inner primer using the same temperature and cycling conditions as used in the first round. The amplified DNA fragments were analyzed in an agarose gel electrophoresis and the bands were purified from the gel, subcloned to a PCR cloning vector (PCR 2.1, Invitrogen Life Technologies) and subjected to DNA sequence analysis.
Western Blot Analysis
T31 cells were transfected with 2 µg control vector PCI, SF-1 del 443462, VP16-SF-1 using the lipofection method (LIPOFECTAMINE PLUS Reagent, Life Technologies, Inc.) with 10 µl/well PLUS Reagent and 10 µl/well LIPOFACTAMINE Reagent. Forty-eight hours after transfection, culture plates were rinsed twice with cold PBS (pH 7.4), and cells were harvested in RIPA buffer (20 mM Tris-HCl, pH 7.4; 0.1% SDS; 1% Triton X-100; 1% sodium deoxycholate). nNOS protein detection was performed using standard procedures (45). Briefly, the cells were sonicated on ice using a Branson sonifier and cell extracts were stored at -70 C. Total protein concentration was determined using the Bradford method (Pierce Chemical Co., Rockford, IL). Proteins in the cell lysates were resolved by 7.5% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a nitrocellulose membrane in Tris-glycine-methanol buffer. The membrane was blocked for 1 h at room temperature in a blocking solution mixture of 3% nonfat dry milk, 0.1% Tween-20, and Tris-buffered saline (pH 8.0). The membrane was then incubated for at least 1 h at room temperature with primary mouse anti-nNOS monoclonal antibody (Transduction Laboratories, Lexington, KY) at a 1:2500 dilution. ß- Tubulin was used as a control to quantify the amount of protein loaded in each lane and was detected with mouse anti- ß-tubulin monoclonal antibody (Sigma) at a 1:5000 dilution. The membrane was washed and incubated for 1 h at room temperature in a 1:1500 dilution of goat antimouse peroxidase-labeled IgG (Pierce Chemical Co.) for nNOS or 1:4000 dilution of the antibody for ß-tubulin. The blot was washed and then processed for analysis using an enhanced chemiluminescence detection kit as described by the manufacturer (Pierce Chemical Co.). The Western blots were quantitated by densitometric scanning. Values of nNOS protein expression levels were normalized to that of ß-tubulin of the same sample.
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ACKNOWLEDGMENTS
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We thank Weza Cotman and Paulette Sackett for manuscript preparation. We thank Drs. Pamela Mellon, Keith Parker, and J. Larry Jameson for cell lines and constructs used in this report. We thank Dr. K. Morohashi for the SF-1 antiserum.
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FOOTNOTES
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This work was supported by U.S. Public Health Service Grant NS-37090.
Abbreviations: CMV, Cytomegalovirus; CRE, cAMP/Ca2+ regulatory elements; CREB, CRE binding protein; DAX-1, dosage-sensitive reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; DTT, dithiothreitol; eNOS, endothelial NOS; FBS, fetal bovine serum; ß-gal, ß-galactosidase; HPA, hypothalamic-pituitary-adrenal; HPG, hypothalamic-pituitary-gonadal; iNOS, inducible NOS; NHR, nuclear hormone receptor; NO, nitric oxide; NOS, NO synthase; nNOS, neuronal NOS; SF-1, steroidogenic factor 1; RACE, rapid amplification of 5' cDNA ends; RSV, Rous sarcoma virus; SDS, sodium dodecyl sulfate; SSPE, saline sodium phosphate/EDTA; VP16-SF-1, constitutively active SF-1.
Received for publication October 15, 2001.
Accepted for publication August 8, 2002.
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