Transcription Factors Oct-1 and C/EBPß (CCAAT/Enhancer-Binding Protein-ß) Are Involved in the Glutamate/Nitric Oxide/cyclic-Guanosine 5'-Monophosphate-Mediated Repression of Gonadotropin-Releasing Hormone Gene Expression

Denise D. Belsham1 and Pamela L. Mellon

Departments of Reproductive Medicine and Neurosciences The Center for Molecular Genetics University of California, San Diego La Jolla, California 92093


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1–7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPß. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1–7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPß protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPß are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/cGMP signal transduction pathway.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The signal transduction pathway utilizing nitric oxide (NO) as a second messenger in the brain has been well characterized (1, 2). NO mediates the actions of the excitatory amino acid glutamate in the cerebellum and hippocampus, which may be linked to the phenomena of long term potentiation and memory (3, 4, 5). NO has also been postulated to be an important modulator of reproductive function. Acting on the specific hypothalamic neurons responsible for the synthesis and secretion of GnRH, NO has been shown to stimulate GnRH secretion (6, 7, 8). Furthermore, NO serves as the downstream signaling intermediate for induction of GnRH secretion by the glutamate receptor agonist N-methyl-D-aspartic acid (NMDA) (9). Since in vivo, GnRH is found in only a small number of neurons scattered from the preoptic to the anterior hypothalamus (10), the GnRH-secreting hypothalamic cell line, GT1–7, has been invaluable in facilitating investigations of GnRH gene expression and function. The GT1–7 cells were developed using targeted oncogenesis by introduction of a hybrid gene containing the 5'-flanking region of the rat GnRH gene linked to the coding region of the potent oncogene, SV40 T-antigen, into transgenic mice (11). The cells are morphologically and physiologically neuronal (11, 12, 13) and secrete GnRH in a pulsatile manner, very similar to GnRH neurons within the hypothalamus in vivo (14, 15, 16). NMDA and NO also stimulate GnRH secretion from GT1 cells (6, 7, 8, 9). In fact, inhibitors of NO synthases block pulsatile release of GnRH by GT1–7 cells (17), implicating NO synthesis as an obligate intermediate in the pulsatile secretion of GnRH from the hypothalamus. Thus, NO may serve as a potential modulator of the synchronization of GnRH release, thereby implying a broader role in the physiological control of the hypothalamic-pituitary-gonadal axis.

Previous attempts to study the molecular mechanisms involved in the expression of genes within discrete populations of neurons, such as the GnRH-secreting neurons of the hypothalamus, have been difficult. Regulation of GnRH gene expression by second messengers has been the focus of a number of studies in the GT1–7 cells. GnRH gene expression is repressed by phorbol ester through the down-regulation of protein kinase C (13, 18, 19, 20) and by glucocorticoids (21, 22) and PRL (23). We have shown that GnRH gene expression is repressed by NMDA, acting through a NO, cGMP signal transduction pathway that results in activation of cGMP-dependent protein kinase. Further, we have found that repression of GnRH gene expression is through a linear, obligate pathway involving NO and requiring calcium (24). Upon binding the NMDA receptor, glutamate (or NMDA) causes an influx of extracellular calcium, which binds calmodulin and activates nitric oxide synthase, thus producing NO. NO then binds guanylyl cyclase, thereby increasing cyclic GMP (cGMP) levels and activating cGMP-dependent protein kinase. We demonstrated that this pathway was acting at the transcriptional level since in transfections, hybrid genes containing 3 kb of the 5'-regulatory region of the rat GnRH gene linked to a reporter gene were also down-regulated after treatment of the GT1–7 cells with NMDA, sodium nitroprusside (SNP, a NO donor), or 8Br-cGMP. The region necessary for repression of GnRH gene expression was localized to 300 bp of DNA within the 5'-regulatory region known to contain a neuron-specific enhancer that directs GnRH gene expression to the GT1–7 neuron (24).

The GnRH neuron-specific enhancer is known to bind a number of nuclear proteins from the GT1–7 cells (25). Some of these protein-binding regions are required for basal GnRH gene activity. One of two GATA transcription factor consensus sequences within the neuron-specific enhancer binds GATA-4 and is essential for basal GnRH gene expression (26). Further, two POU homeodomain transcription factor consensus sequences, AT-a and AT-b, bind the transcription factor Oct-1, which is also essential for GnRH gene expression (27).

The mechanisms by which the glutamate/NO/cGMP pathway regulates transcription are not yet known. Developmental control of gene expression has generally been shown to be due to the interactions of multiple activator proteins bound to specific cis-regulatory regions that together specify appropriate transcription (28). In contrast, signal transduction pathways more often act by posttranslational modification of an individual transcription factor. Regulatory proteins occur in families that have common DNA recognition properties (29). Members of the POU-homeodomain family bind an octamer motif (30, 31, 32) and regulate transcription through differential expression of the family members (30) or perhaps through interactions with other transcription factors (33, 34, 35, 36). Similarly, members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, which are basic region-leucine zipper proteins, are differentially expressed (37, 38) and are capable of protein-protein interactions with members of this family or other transcription factor families (38, 39, 40, 41, 42).

In this study, we show that two regions, AT-b (-1695 to -1702) and -1676 (-1676 to -1684), within the 300-bp neuron-specific enhancer of the GnRH gene are critical for repression of GnRH by NO. These two sequences bind GT1–7 nuclear proteins, individually or as a continuous sequence encompassing both sites, and contain DNA-consensus sequences for the POU-homeodomain and C/EBP families of transcription factors. We find that two of the proteins bound to these regions are Oct-1 and C/EBPß. In addition, treatment of GT1–7 cells with an NO donor increases the intensity of the DNA/protein complex formed on the oligonucleotides representing the AT-b element, although overall Oct-1 or C/EBPß protein content in the GT1–7 cells does not appear to be altered. Instead, Oct-1 in nuclear extracts from GT1–7 cells treated with an NO donor demonstrates increased relative binding affinity for the AT-b site indicating a potential mechanism for regulation. That this mechanism is likely to involve increased phosphorylation of the Oct-1 complex after activation of the glutamate/NO/cGMP signaling pathway is supported by our finding that dephosphorylation reduces the binding of the Oct-1 complex in NO-treated nuclear extracts. Thus, the repression of GnRH gene expression by NO occurs at the transcriptional level in the GT1–7 neuronal cells, and the downstream effector molecules necessary for the effects of NO include the known transcription factors, Oct-1 and C/EBPß.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two Elements within the GnRH Neuron-Specific Enhancer Confer Repression by the Glutamate/NO/cGMP Pathway
We have previously identified a neuron-specific enhancer 1.6 kb upstream of the GnRH gene essential for GnRH gene expression in GT1–7 cells (25). This 300-bp neuron-specific enhancer (-1863 to -1571) contains several protein binding regions as determined by DNase I footprinting assays and electrophoretic mobility shift assay (EMSA) and also confers repression by the glutamate/NO/cGMP pathway (24). To study basal enhancer activity during the characterization of the 300-bp region, block replacement mutants were previously prepared within the major footprints located in the enhancer and placed upstream of the truncated (-173) GnRH promoter and the chloramphenicol acetyl transferase (CAT) reporter gene (25). We used these reporter plasmids, as well as some point mutations in the two well characterized AT-rich elements (27), to determine which region(s) of the GnRH enhancer confers NO repression of GnRH gene expression by transfecting them into GT1–7 neurons and treating with SNP, a NO donor, and ionomycin, a calcium ionophore. Treatment with SNP and ionomycin repressed reporter gene activity from most of the mutants, reproducing the repression observed with the intact GnRH enhancer (EN on -173 vs. EN on -173 with SNP+ Ca++; lanes 1 and 2, Fig. 1Go). Although repression was slightly less effective for most of the mutations (averaging ~65% compared with 45% for wild type), only two elements abolished repression upon treatment with NO, the block mutation from -1684 to -1676 and m AT-b, a double-point mutation in one of the AT-rich elements (Fig. 1Go).



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Figure 1. Two Elements within the 300-bp GnRH Enhancer (-1863 to -1571) Confer Repression by NO

GT1–7 cells were transiently transfected with 15 µg of one of the CAT expression vectors containing block replacement or point mutations within the enhancer and then treated with SNP and ionomycin in Opti-MEM or maintained in Opti-MEM alone. Treatments were for 4 h after previous transfection of the DNA for 12 h. Bars represent the percent of CAT activity remaining after SNP treatment as compared with control activity of each mutant enhancer. EN on -173 represents the wild-type enhancer on the GnRH promoter and is normalized to 100%. With SNP + Ca++, the activity of EN on -173 is repressed to approximately 40%. All block replacement mutants contain at least 7 of 9 bp changes within the region indicated (25 ) and the AT-a, AT-a flank, and AT-b are double-point mutations (see Fig. 2Go and Ref. 27). RSV-luciferase was included as an internal control for transfection efficiency. Each value is an average of at least three independent measurements in duplicate or triplicate ± SEM normalized to the internal control.

 
Specific Proteins Bind the Elements Necessary for Repression by the Glutamate/NO/cGMP Signal Transduction Pathway
The two regions found to confer repression of GnRH gene expression by NO both bind proteins, as determined by deoxyribonuclease I (DNaseI) footprinting (25). To further study the proteins bound to these regions, EMSAs were performed with oligonucleotide probes representing the AT-b and -1684/-1676 regions (termed -1676 region) (Fig. 2AGo). The two elements are adjacent within the enhancer (Fig. 2Go), but these oligonucleotides do not overlap.



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Figure 2. Sequences of the GnRH Enhancer at the AT-b and -1676 Regions and the Oligonucleotides used for EMSA Studies

A, The boxed sequences represent the AT-b and -1676 elements DNase I footprinted by GT1–7 nuclear proteins (25 ). The wild-type oligonucleotides used as probes in EMSA analysis are represented under the appropriate regions. Some oligonucleotides have a GATC linker sequence added to each 5'-end, which is not part of the GnRH enhancer sequence. B, The mutations within the sequence of the GnRH enhancer and the mutant oligonucleotides are represented. The mutated bases within the sequences are changed accordingly in bold and indicated by an X. C, The sequences of the consensus C/EBP and Sp1 oligonucleotides are also shown.

 
Nuclear extracts were prepared from GT1–7 cells untreated or treated for 1 h with NMDA, NO, or cGMP (all with ionomycin) or ionomycin alone, since in our initial study we found that calcium was required for the repression of GnRH gene expression (24). The DNA-protein complexes formed on the AT-b region are represented by three differentially migrating bands, as are the complexes formed on the -1676 region (Fig. 3Go). The slowest migrating complex formed on each of the two regions appears to be unique, while the two lower complexes comigrate, although the sequences of the two oligonucleotides are quite different. Interestingly, the intensity of the upper band formed with the AT-b oligonucleotide was consistently higher with the NMDA/NO/cGMP-treated GT1–7 nuclear extract, but not with untreated or treatment with ionomycin alone (Fig. 3Go, lanes 1–5; Fig. 4Go, lanes 1–2; Fig. 5AGo, lanes 1 and 6; Fig. 6Go, lanes 1 and 3). The upper band with the -1676 oligonucleotide is also increased by the treatments in this figure, but this increase is not as distinct or reproducible (Fig. 3Go, lanes 6–10; Fig. 4Go, lanes 5 and 6; Fig. 5BGo, lanes 1 and 6; Fig. 6Go, lanes 6 and 9).



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Figure 3. GnRH Enhancer AT-b and -1676 Oligonucleotide Probes Form Unique Complexes with GT1–7 Cell Nuclear Proteins, Some of Which Are Induced by Treatment of the GT1–7 Cells with NMDA, SNP, or 8-Br-cGMP

GT1–7 cells were treated with NMDA, SNP, or 8-Br-cGMP (all with calcium ionophore ionomycin), with ionomycin alone, or not treated (none), as indicated. EMSAs were performed with 3 µg of treated or control GT1–7 nuclear extract using the AT-b (lanes 1–5) or -1676 (lanes 6–10) oligonucleotides as probes, which are described in the legend of Fig. 2Go. The arrows indicate the specific complexes formed on the AT-b or -1676 probes.

 


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Figure 4. AT-b and -1676 Mutant Oligonucleotides Fail to Bind Most of the GT1–7 Nuclear Protein Complexes

GT1–7 cells were treated with SNP and ionomycin. Nuclear protein extracts were prepared from treated and untreated cells as described. EMSAs were performed using 3 µg of GT1–7 nuclear extract ± SNP+Ca++ treatment and the wild-type (AT-b) or mutant AT-b (m AT-b) oligonucleotides (lanes 1–4) or wild-type (1676) or mutant (m 1676) -1676 oligonucleotides (lanes 5–8) as probes. Figure 2Go describes the specific mutations found within the oligonucleotides. The arrows indicate the complexes formed on the probes.

 


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Figure 5. AT-b, -1676, Consensus C/EBP, and Consensus Sp1 Oligonucleotides Compete for Binding of the Protein Complexes Formed on the AT-b, -1676, and Consensus Sp1 Probes

EMSAs were performed with a labeled oligonucleotide containing the AT-b (panel A), -1676 (panel B), or consensus Sp1 sequences (panel C) and 3 µg of GT1–7 nuclear extract ± SNP + Ca++ treatment as indicated. All competitor lanes represent 100-fold (AT-b or -1676) or 10-fold (consensus Sp1 or consensus C/EBP) molar excess of unlabeled oligonucleotide. Formation of the upper complex on the AT-b probe was eliminated by the AT-b competitor alone, while the middle complex on the AT-b probe was competed by the unlabeled -1676 or the consensus Sp1 competitors. The upper complex on the -1676 probe was eliminated by the unlabeled -1676 oligonucleotide or by the consensus C/EBP competitor, while the middle complex was competed by both AT-b and consensus Sp1 oligonucleotides. The single complex formed on the consensus Sp1 probe was competed by the unlabeled consensus Sp1 or -1676 oligonucleotides. None of the excess unlabeled oligonucleotides were able to eliminate the lower complex formed on the AT-b or -1676 probes. Arrows indicate the specific complexes formed on the probes.

 


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Figure 6. Oct-1 and C/EBPß Antibodies Supershift the Upper Complexes Formed on the AT-b and -1676 Oligonucleotide Probes, Respectively

EMSAs were performed with labeled oligonucleotides representing the AT-b (lanes 1–5) or -1676 (lanes 6–12) sequences and 3 µg of nuclear extract from GT1–7 cells ± SNP + Ca++ treatment as indicated. Specific antibodies toward Oct-1 (lanes 2 and 4), C/EBPß (lanes 7 and 10), or Sp1 (lanes 8 and 11) were used, while an equivalent amount of purified normal rabbit IgG was used as a negative control (lanes 5 and 12). The uppermost arrow indicates the supershifted complex with either probe.

 
Oligonucleotides were also prepared containing the same mutations used to map the regions necessary for NO-mediated repression of GnRH gene expression (Fig. 2BGo). The bottom strand of the AT-b region contains an octamer motif (ATTAAAAT, matches 6 of 8 bases) that may contain a putative POU-homeodomain transcription factor binding site (43), while the upper strand of the -1676 region contains a putative C/EBP consensus sequence (TGAAGCAAT, matches 9 of 9 bases) (44). The mutant oligonucleotides incorporate base changes that specifically eliminate the POU homeodomain and C/EBP consensus binding sequences. Using these oligonucleotides as probes for EMSA analysis, we found that no DNA/protein complexes were formed on the AT-b double point mutant, while only the middle complex remained with the -1676 block replacement mutant oligonucleotide (Fig. 4Go). These results confirm that the AT-b and -1676 regions bind proteins specifically and if mutated, with the same base changes as the block replacement mutants, protein binding is not tolerated with the exception of the middle band on the -1676 probe.

To determine the specificity of protein binding, competition studies were performed with each of the two regions. The upper and middle complexes formed on the AT-b oligonucleotide are dramatically reduced when a 100-fold molar excess of unlabeled AT-b oligonucleotide is added to the EMSA reaction mixture, indicating that these two complexes represent a specific interaction between protein and the AT-b sequence (Fig. 5AGo, lane 2). Similarly, the upper and middle complexes formed on the -1676 oligonucleotide are competed by inclusion of a 100-fold molar excess of unlabeled -1676 oligonucleotide (Fig. 5BGo, lane 2). The fastest migrating complex is not competed by any of the competitors used with either of the oligonucleotides, indicating that it is a nonspecific DNA/protein complex. Further, the upper complex formed with the -1676 oligonucleotide is also eliminated by a 10-fold molar excess of C/EBP consensus oligonucleotide (Fig. 2CGo), indicating that the protein(s) bound at this complex are also capable of binding the C/EBP consensus sequence (Fig. 5BGo, lane 5). When the C/EBP consensus sequence is used as a labeled probe, the single band migrates to the same position as the -1676 upper complex (data not shown).

The middle complex on AT-b is completely abolished with a 100-fold molar excess of the AT-boligonucleotide, while it is also dramatically decreased by a 100-fold molar excess of the -1676 and 10-fold excess of Sp1 consensus oligonucleotides (Fig. 5AGo, lanes 2–4). Conversely, the middle complex formed on the -1676 oligonucleotide is competed by a 100-fold molar excess of cold -1676, AT-b, and 10-fold Sp1 consensus oligonucleotides (Fig. 5BGo, lanes 2–4). The Sp1 consensus oligonucleotide was originally used as an unrelated DNA sequence control probe since there is no discernible homology to either element (Fig. 2CGo), until it was noted that the complex formed with GT1–7 nuclear extract migrated to the same level as the middle complexes of the AT-b and -1676 oligonucleotides. When using the Sp1 consensus sequence as the labeled oligonucleotide, one complex is formed and migrates at the same position as the middle band in both the AT-b and -1676 oligonucleotides (Fig. 5CGo). Furthermore, a 10-fold molar excess of unlabeled Sp1 consensus sequence and 100-fold -1676 oligonucleotide abolish the appearance of this specific complex, but the complex is not competed by 10-fold molar excess of C/EBP consensus sequence (Fig. 5CGo, lanes 2–4). This indicates that the middle complex may be formed by the binding of similar protein(s) on both the AT-b and the -1676 probes. This middle protein complex is not seen when using the unrelated GATA binding motif of the human preproendothelin 1 (PPET) gene as a labeled control probe (data not shown) (45).

The Transcription Factor, Oct-1, Binds the AT-b Element, While the CCAAT/Enhancer Binding Protein (C/EBP) ß Binds to the -1676 Element of the GnRH Neuron-Specific Enhancer
The identity of the proteins bound to the two elements required for NO repression was determined by antibody supershift assays. The consensus binding sites within the regions indicated which antibodies might be used to determine the identity of the member(s) of the two transcription factor families binding to these regions. Previous studies in our laboratory indicated that Oct-1, a widely expressed POU-homeodomain protein, bound to both the AT-b and AT-a regions (27). AT-b and AT-a are similar elements within the GnRH enhancer containing a POU-homeodomain consensus octamer sequence. When the Oct-1 antibody was used with the SNP-treated GT1–7 nuclear extract, a supershifted complex appeared, similar to that seen with the nuclear extract from untreated GT1–7 cells (Fig. 6Go, lanes 2 and 4). Changing the EMSA conditions did not completely supershift the upper complex, indicating that another protein may be present in the complex that cannot supershift to the larger complex.

Analysis of the C/EBP consensus sequence present within the -1676 region of the GnRH enhancer revealed a 100% match to a C/EBPß binding site. For this reason, we chose the C/EBPß antibody to use in the supershift analysis of this complex. A supershifted complex was seen with nuclear extract preparations from both SNP-treated and untreated GT1–7 cells (Fig. 6Go, lanes 7 and 10). Consistently, with different nuclear extracts and incubation conditions, the untreated cell nuclear extract preparation yielded a stronger supershift with the C/EBPß antibody, indicating that the protein complexes formed on the -1676 region with the nuclear extract from NO-treated and untreated GT1–7 cells may differ.

Finally, a Sp1 antibody did not supershift the protein complexes formed with any of the GT1–7 nuclear extracts (Fig. 6Go, lanes 8 and 11), although we did see a supershift using HeLa cell nuclear extract (data not shown). These results imply that although a protein capable of binding the Sp1 consensus oligonucleotide may be present in the middle complex, Sp1 itself does not appear to be present in this complex, or may not be capable of forming a supershift when it is part of the middle complex, due to hindrance at the antigenic site. However, no Sp1 supershift was produced using the Sp1 consensus oligonucleotide as a probe with the GT1–7 nuclear extract (data not shown), suggesting that Sp1 may not be present in the GT1–7 cell nuclear extract. Rabbit IgG was used as a nonspecific antibody control and showed no supershifts with any of the nuclear extracts (Fig. 6Go, lanes 5 and 12).

Oct-1 and C/EBPß Bind Simultaneously to the Sequence Encompassing Their Adjacent Sites
A 34-bp oligonucleotide (AT-1676) that includes both the AT-b and -1676 binding sites was used to determine whether the Oct-1 and C/EBPß proteins can bind to the adjacent sites simultaneously (Fig. 2AGo). EMSA analysis with the AT-1676 oligonucleotide produced a complex that migrated more slowly than the three complexes previously detected binding at either the AT-b or -1676 sites alone (Fig. 7Go). All four complexes were eliminated by competition with 100-fold molar excess of the AT-1676 oligonucleotide (Fig. 7BGo, lane 3). Interestingly, the intensity of the uppermost complex formed on the AT-1676 oligonucleotide appears to be increased with the SNP-treated nuclear extract (Figs. 7AGo and 7BGo, lane 2; representing nuclear extracts from two separate experiments) when compared with the control nuclear extract (Figs. 7AGo and 7BGo, lane 1).



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Figure 7. Oct-1 and C/EBPß Bind Simultaneously to the Adjacent AT-b and -1676 Elements

EMSAs were performed with a labeled oligonucleotide representing the enhancer sequence encompassing both the AT-b and -1676 sequences (AT-1676, panel A, lanes 1 and 2 and panel B, lanes 1–8), or oligonucleotides with the AT-b site mutated (AT-1676MAT, panel B, lanes 9 and 10) or the -1676 site mutated (AT-1676M76, panel B, lanes 11 and 12), illustrated in Fig. 2Go, with 3 µg of nuclear extract from GT1–7 cells ± SNP + Ca++ treatment. GT1–7 nuclear extracts, from separate experiments (A and B) representing control (A and B, lane 1) or SNP-treated (panel A, lane 2, and panel B, lanes 2–12) cells, were subjected to EMSA analysis. All competitor lanes represent 100-fold molar excess of the unlabeled oligonucleotides, AT-1676 (panel B, lane 3), AT-b (panel B, lane 4), or -1676 (panel B, lane 5). Specific antibodies against Oct-1 (panel B, lane 6) or C/EBPß (panel B, lane 7) were used to perform a supershift analysis of the composite AT-1676 oligonucleotide, while an equivalent amount of purified normal rabbit IgG was used as a negative control (panel B, lane 8). Mutation of the AT-b site within the AT-1676 oligonucleotide was used alone (panel B, lane 9) or with 100-fold molar excess of the unlabeled -1676 oligonucleotide, and mutation of the -1676 site within the AT-1676 oligonucleotide was used alone (panel B, lane 11) or with 100-fold molar excess of the unlabeled AT-b oligonucleotide (panel B, lane 12), in EMSA analysis. The uppermost band, indicated with an arrow, represents the binding of both the Oct-1 and C/EBPß protein complexes.

 
The proteins bound at the uppermost complex were studied by EMSA analysis, using the oligonucleotides representing the individual binding sites and the antibodies against both Oct-1 and C/EBPß. Both the AT-b and -1676 oligonucleotides, at 100-fold molar excess, eliminated the uppermost complex (Fig. 7BGo, lanes 4 and 5). The Oct-1 and C/EBPß antibodies supershifted the uppermost complex (Fig. 7BGo, lanes 6 and 7, respectively), but IgG alone had no effect (Fig. 7BGo, lane 8). The C/EBPß supershift was weak and required a longer exposure of the EMSA gel to be visualized (data not shown). Mutation of either the AT-b or the -1676 site within the AT-1676 oligonucleotide (Fig. 2BGo) abolished the appearance of the uppermost complex, although binding at the remaining nonmutated element remained [Fig. 7BGo, lanes 9 (AT-1676MAT) and 11 (AT-1676M76)]. Competition analysis with 100-fold molar excess of the AT-b or -1676 oligonucleotide shows that the lower three complexes are the same as those described for the individual oligonucleotide probes (Fig. 7BGo, lanes 10 and 12). These results indicate that the protein complexes are also able to bind the AT-1676 oligonucleotide individually.

Neither Oct-1 nor C/EBPß Protein Is Induced in the Nuclear Protein Extracts from SNP-Treated GT1–7 Cells
The upper complex binding the AT-b oligonucleotide appears to be increased in nuclear extract treated with NMDA, SNP, or cGMP (with calcium) and similarly the upper complex binding the combined AT-1676 oligonucleotide appears induced, while induction of the binding on the -1676 probe is variable. Therefore, we determined whether the concentrations of these proteins were increased in the SNP-treated nuclear extract by Western blot analysis. Utilizing the same antibodies as for the antibody supershift analysis, we determined that the nuclear extract from the GT1–7 cells treated with SNP and ionomycin did not contain more Oct-1 or C/EBPß protein than did the untreated GT1–7 nuclear extract (Fig. 8Go). Using Western blot analysis, Oct-1 and C/EBPß antibodies produced bands of the appropriate sizes (37). This finding indicates that although the band intensity is increased in the upper bands with both the AT-b and -1676 oligonucleotides, the increase in band intensity cannot be accounted for by an increased amount of Oct-1 or C/EBPß in the nuclear extract from GT1–7 cells treated with SNP.



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Figure 8. Neither Oct-1 nor C/EBPß Protein Levels Are Increased in Nuclear Extracts after NO Treatment of GT1–7 Cells

Nuclear extracts from GT1–7 cells ± SNP + Ca++ treatment were run on a 12% polyacrylamide gel and Western blotted onto Immobilon-P membranes. Specific antibodies toward Oct-1 and C/EBPß, the same as those used in EMSA supershift analysis, were used as the primary antibody. The secondary antibody was horseradish-peroxidase. The bands were visualized using enhanced chemiluminescence. A specific band of approximately 100 kDa is seen with the Oct-1 antibody (lanes 1 and 2), while a major band of approximately 46 kDa and a 29-kDa minor band are seen with the C/EBPß antibody (lanes 3 and 4).

 
Oct-1 in Nuclear Extracts from GT1–7 Cells Treated with SNP Has Increased Relative Binding Affinity for the AT-b Region in the GnRH Neuron-Specific Enhancer Due to Increased Phosphorylation
Since neither Oct-1 nor C/EBPß protein levels are induced in SNP-treated GT1–7 nuclear extracts by Western blot analysis, it is possible that the binding affinity of the protein(s) for the oligonucleotide has changed. To test this hypothesis, a competition analysis was performed with increasing amount of unlabeled AT-b oligonucleotide. The amounts of oligonucleotide competitor ranged from 30 pg to 10 ng DNA. The relative intensity of the uppermost band (which represents the Oct-1 protein complex), was plotted vs. the amount of competitor using a logarithmic scale. In three separate nuclear extracts prepared from GT1–7 cells treated with SNP and ionomycin, the IC50 for the AT-b oligonucleotide changes by approximately 4- to 10-fold (Fig. 9Go, representative graph) as determined by the 50% displacement values. Because the increase in the upper complex formed on the -1676 oligonucleotide was much less dramatic in EMSA analysis, this same study was not possible using this oligo-nucleotide.



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Figure 9. Competitive Titration of Oct-1 from GT1–7 Cells Treated with SNP Shows Increased Relative Binding Affinity for the AT-b Element

EMSA was performed with labeled oligonucleotide probe representing the AT-b element comparing 3 µg of nuclear extract from GT1–7 cells ± SNP + Ca++ treatment. Changes in relative binding affinity were determined under conditions of increasing concentrations of unlabeled AT-b unlabeled oligonucleotide (represented as picograms DNA added to the EMSA reaction). The dried gels were exposed and Oct-1 band intensity (the uppermost complex on the AT-b probe in EMSA, as seen in Figs. 3–5GoGoGo) was determined by phosphorimager analysis. The relative band intensity was plotted on a log scale vs. increasing concentrations of AT-b oligonucleotide competitor. Dotted lines indicate the competitor DNA concentration at which 50% of the Oct-1 complex is displaced. The above graph is representative of three individual experiments using three separate nuclear protein extract preparations from treated and untreated GT1–7 cells.

 
To determine whether activation of the cGMP-dependent protein kinase signaling pathway resulted in phosphorylation of proteins in the Oct-1 complex, we observed the DNA binding of Oct-1 after phosphatase treatment of the nuclear extracts. Dephosphorylation of the SNP-treated and control nuclear extracts with increasing concentrations of calf intestinal alkaline phosphatase (CIP) was performed to determine whether phosphorylation of nuclear proteins might be responsible for the increased binding of the Oct-1 complex to the AT-b site. After preincubation with CIP, but before the EMSA analysis, extracts were treated with a phosphatase inhibitor cocktail to avoid dephosphorylation of the labeled oligonucleotide probe. The efficiency of the phosphatase inhibitor cocktail was determined by coincubation with CIP before EMSA analysis. EMSAs using SNP-treated nuclear extract demonstrated a decrease in DNA-binding activity to the AT-b element upon increasing concentration of CIP, while binding to the AT-b oligonucleotide by the CIP-treated control extract remained stable (Fig. 10Go). Thus, it is possible that upon NO activation of the signaling pathway involving cGMP-dependent protein kinase, the Oct-1 complex undergoes increased phosphorylation resulting in increased DNA binding activity at the AT-b element.



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Figure 10. Evidence that Phosphorylation Regulates the DNA Binding Ability of the Oct-1 Complex at the AT-b Element from GT1–7 Cells Treated with SNP

EMSA was performed with labeled oligonucleotide probe representing the AT-b element comparing 5 µg of nuclear extract from GT1–7 cells ± SNP + Ca++, treated with increasing concentrations of CIP. Nuclear extracts were preincubated with CIP, followed by incubation of the nuclear extracts with a phosphatase inhibitor cocktail (50 mM sodium fluoride and 1 mM sodium orthovanadate) to avoid dephosphorylation of the labeled probe. The efficiency of the phosphatase inhibitor mix was determined by coincubation with CIP (lane 6).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular mechanisms involved in the physiological action of NO on gene expression are not yet understood. Remarkably, only two neuron-specific genes have been shown to be regulated by NO: microtubule-associated protein 2 in hippocampal granule cells (46) and GnRH in the GT1–7 hypothalamic neurons (24). In this study we have defined two specific regions within the 5'-flanking region of the GnRH gene that are critical for repression of GnRH gene expression, and we find that the transcription factors binding these regions are members of the POU-homeodomain and basic region-leucine zipper transcription factor families.

We have localized two elements within the well characterized 300-bp neuron-specific enhancer of the GnRH gene (25, 26, 47) that are each required for the repression of GnRH gene expression through the glutamate/NO/cGMP pathway. These regions lie adjacent within the enhancer (the entire responsive region encompasses only 34 bp, -1704/-1671), implying that protein-protein interactions may be necessary for NO-mediated repression of gene regulation. Two specific complexes are formed on each of the two important elements. One of these protein complexes appears to be common to both the AT-b and -1676 oligonucleotides, despite the fact that the only similarities between the two sequences are the GATC linkers at the 5'-ends of the oligonucleotides (Fig. 2Go). The 34-bp sequence encompassing both the AT-b and -1676 regions does not contain a GC-rich Sp1 consensus sequence (42, 48), nevertheless a protein(s) capable of binding to both of the oligonucleotides representing this region is also capable of binding a GC-rich Sp1 consensus oligonucleotide in EMSA. This complex is not Sp1 itself, however, since Sp1 cannot be identified in GT1–7 nuclear extracts. Interestingly, this complex is present in the EMSA analysis utilizing the composite oligonucleotide AT-1676, which does not contain any GATC linkers. Further studies will be undertaken to determine the identity of the proteins in this complex. While one of the mutations used in the functional analysis (m AT-b) did not bind this common middle complex (although it contains the same GATC overhang as its wild-type counterpart, AT-b), the block mutation at -1676 did not eliminate this complex in EMSA even though it does prevent NO repression. Thus, the binding of this protein is not likely to be involved in NO repression since it remains bound in the presence of the -1676 block mutation that eliminates repression.

The upper complex binding specifically to the AT-b oligonucleotide is supershifted by antibodies specific for Oct-1. Oct-1 was previously found to bind both the AT-a and AT-b regions of the GnRH neuron-specific enhancer, but only the AT-a region was thought to be important for GnRH enhancer activity since a block replacement mutant in the AT-b region did not affect GnRH gene expression (27). In this study, we prepared a mutation within the AT-b region which specifically altered the critical two bases within the octamer consensus region required for binding POU-homeodomain proteins such as Oct-1 (Fig. 2Go). This specific mutation prevented NO repression. Oct-1 is expressed in several cell types (47, 49) and is known to interact with a number of other DNA-binding proteins (33, 34, 35, 36, 50, 51), basal transcription factors (52, 53), or with tissue-specific coactivators (54, 55). Therefore, it has been postulated that Oct-1 may confer tissue-specific gene expression through its interaction with other transcription factors specifically expressed in a particular cell type or by complex interactions with coactivators. The ability of Oct-1 to participate in NO repression may also be due to its interaction with such proteins.

The upper complex binding specifically to the -1676 oligonucleotide is supershifted by antibodies specific for C/EBPß. Although published C/EBP consensus sequences differ slightly (37, 42, 44), a comparison of our binding site at -1676 shows a 100% match to a consensus sequence generated by comparison of a number of C/EBP binding sites (44, 56). The block replacement mutant -1684/-1676 changed the bases within the C/EBP consensus sequence required to bind C/EBP proteins resulting in the disappearance of the upper protein complex with the -1676 oligonucleotide in EMSA (Fig. 4Go). C/EBPß is a member of the basic region-leucine zipper family that controls transcription of a number of genes through protein-protein interactions at the gene level. In particular, C/EBPß can not only bind to its own family members such as C/EBP proteins (38), fos, and jun (57), but also forms complexes with other transcription factors such as nuclear factor-{kappa}B (40, 41), estrogen receptor (41), and an Sp1 factor (42). Furthermore, similar to Oct-1, C/EBPß may also alter transcription by complex interactions with coactivators and basal transcription factors such as TFIIB and p300 (58, 59).

C/EBPß has been previously shown to be induced by stimulation of the glutamate pathway in rat cortical astrocytes in culture. This induction is calcium and calmodulin dependent and is maximal (1.4-fold) after a 1-h treatment (60). However, the role of NO in this signaling was not investigated.

The NO-stimulated repression of GnRH by Oct-1 and C/EBPß may involve complex interactions between these two proteins, with other transcription factors, the basal transcriptional machinery, and/or coactivators. Our data indicate that the two protein complexes bind the 34-bp region of the enhancer simultaneously. The distance from the center of the consensus site for Oct-1 to the center of the C/EBP consensus site is 20 bp. Oct-1 is known to bind to the major groove (61, 62), and indications are that C/EBP also binds to the major groove (63). Therefore, the two proteins bind to the same side of the DNA helix just one turn apart. In fact, the requirement for both binding sites to be intact for repression to occur implies that they may coordinate to bind a third protein together that is involved in the response.

Our studies indicate that the binding of the GT1–7 nuclear protein complex to the AT-b region, and perhaps to the C/EBP region, is increased in extracts harvested after treatment of the GT1–7 cells with SNP and ionomycin. Using Western blot analysis, we did not detect an increase in the total protein levels of either Oct-1 or C/EBPß using specific antibodies for these two proteins. Since the concentration of protein is not changed within NO-stimulated cells, then the affinity of the Oct-1 protein (and perhaps C/EBPß) for DNA might be changed through a posttranslational modification, as has been seen in other systems (64). Studies of GT1–7 nuclear extracts in EMSA analysis, with or without SNP treatment, indicate that this is the case for Oct-1. By increasing the amount of cold oligonucleotide in EMSA competition studies (65), we demonstrate that the relative binding affinity of Oct-1 to the AT-b region is increased 4- to 10-fold after treatment with NO. Furthermore, dephosphorylation of the nuclear extract shows that this increase is related to the phosphorylation state of the proteins.

We chose to study the regions responsible for repression of GnRH gene expression after treatment of the GT1–7 cells with NO. Nevertheless, it appears that the protein binding at the AT-b and -1676 regions are common to all three components of the glutamate/NO/cGMP signal transduction pathway. After treatment with NMDA, SNP, or 8Br-cGMP (all in the presence of the calcium ionophore ionomycin), we detect the same protein complexes, and the Oct-1 complex appears to be increased by each of the three treatments (see Fig. 3Go).

One of the known effects of the elevation of cGMP is activation of cGMP-dependent protein kinase (66). Thus, it is possible that posttranslational modification of the proteins bound to these two regions may be due to direct phosphorylation by cGMP-dependent protein kinase, since repression of GnRH gene expression requires the action of this kinase (24). Presently, very few substrates for this kinase have been reported, and none of the known substrates to date are transcription factors (66). However, a few as-yet-unidentified specific substrates of cGMP-dependent protein kinase have been found in rat brain (67). There are a number of examples in which the direct phosphorylation of a protein alters its ability to bind DNA (68). Both Oct-1 (68) and C/EBP family members (69) exhibit changes in DNA-binding affinity, depending upon their phosphorylation state. Dephosphorylation of C/EBP{delta} severely inhibits its DNA binding activity on the serum amyloid A promoter in liver (70). Interestingly it has also been postulated that the DNA specificity of Oct-1 can be regulated by the action of different kinases (71). As cells enter mitosis, Oct-1 is hyperphosphorylated by protein kinase A, which results in the inhibition of Oct-1 DNA binding activity (72). We can speculate that depending upon the cell type and the available transcription factors, Oct-1 phosphorylation may result in a differential effect on DNA-binding activity. For instance, in GT1–7 cells, cGMP-dependent protein kinase activation may result in increased phosphorylation of Oct-1, causing the enhanced binding affinity of the Oct-1 complex to the AT-b element. Alternatively, it is possible that another protein, required as a corepressor, is recruited to the enhancer after a change in phosphorylation due to activation of the signaling cascade. Whether cGMP-dependent protein kinase or another protein kinase phosphorylates either Oct-1 or C/EBPß in response to the activation of the glutamate, NO, cGMP signal transduction pathway in the GT1–7 hypothalamic neurons is yet another question that remains to be answered. Although at present we cannot say how an apparent increase in DNA-binding activity might cause repression, this study demonstrates that transcriptional repression of the GnRH gene by the NMDA/NO/cGMP pathway depends on binding of the transcription factors Oct-1 and C/EBPß and indicates a likely mechanism by which NO exerts its effect on gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Reagents
GT1–7 cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS, 4.5 mg/ml glucose, and penicillin/streptomycin and maintained in an atmosphere with 5% CO2 as described (11). Medium was changed to Opti MEM (Life Technologies, Inc.), a serum-free medium, before the treatments. NMDA (used at 500 µM), SNP (used at 50 µM), 8-bromo-cGMP (8-Br-cGMP; used at 250 µM), and ionomycin, (used at 0.5 µM), were obtained from Sigma (St. Louis, MO). Oligonucleotides were prepared by Operon Technologies, Inc. (Los Angeles, CA). Oct-1, C/EBPß, and Sp1 antibodies and consensus oligonucleotides were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Plasmid Constructions and Transfections
The plasmids containing the GnRH neuron-specific enhancer upstream of the GnRH promoter (termed EN on -173) were created by inserting the enhancer (-1863 to -1571 of the GnRH gene) in reverse orientation into the polylinker upstream of the promoter in -173 GnRH-CAT as was previously described (25). The block replacement mutants were constructed as previously described (25) or by designing specific mutations using two homologous PCR primers each containing a 9-bp BamHI linker site (-1606/-1598, -1646/-1638, -1659/-1651, -1684/-1676) or specific 2-bp changes (m AT-b, m AT-a, and m AT-a flank). Briefly, two PCR products were generated using flanking primers within the vector encompassing the GnRH neuron-specific enhancer in combination with one of the homologous mutation-containing primers, isolated, purified, and then used as template to create a single PCR product with only the flanking vector primers. This PCR product containing the GnRH enhancer block replacement mutation was then cloned into the -173 GnRH-CAT plasmid using the upstream polylinker. All plasmids were sequenced by the dideoxy-chain termination method (73) in the presence of {alpha}[32P]dATP, 3000 Ci/mmol (New England Nuclear, Boston, MA) and Sequenase (United States Biochemical Corp., Cleveland, OH). Transfections were performed using the calcium phosphate precipitate method (74) containing 15 µg of plasmid DNA and 5 µg of the internal control plasmid RSV (Rous sarcoma virus)-luciferase (75). The cells were incubated for 12–14 h with DNA, followed by three PBS rinses, and then treated with SNP and ionomycin in Opti MEM for 4–6 h before harvesting. Protein extracts were prepared by freeze-thawing as described previously (76), and protein concentrations were determined using the Coomasie blue procedure (77). CAT (78) and luciferase assays (75) were done as previously described.

Nuclear Extract Preparation and EMSA Analysis
Nuclear extracts were prepared following the method of Lee et al. (79). After protein concentration was determined using the Coomasie blue procedure (77), the extracts were stored frozen at -75 C. EMSA and supershift assays were performed as previously described (27), except EMSA reaction mixes were incubated 10 min at room temperature with all reagents except the probe and then another 15 min or 30 min (AT-1676 oligonucleotides) at room temperature after addition of 20,000 cpm of the labeled oligonucleotide. The oligonucleotide sequences shown in Fig. 2Go had a GATC linker added to each 5'-end that were filled in using Klenow fragment both for labeled probes, in the presence of {alpha}[32P]dATP, 3000 Ci/mmol (New England Nuclear, Boston, MA) or, in the case of competitor oligonucleotides, with cold deoxynucleoside triphosphates. Consensus C/EBP or Sp1 oligonucleotides were radiolabeled by incubation of T4 polynucleotide kinase with {gamma}[32P]dATP (6000 Ci/mmol; New England Nuclear, Boston, MA). All labeled probes were gel purified by 6% polyacrylamide gel electrophoresis, crushed, soaked overnight, and phenol-chloroform extracted, and the amount of radioactivity was determined. Probes were diluted in 25 mM KCl. Competition studies were done by adding a specified amount (10- to 100-fold, as indicated) of unlabeled oligonucleotide to the reaction mix 15 min before the addition of labeled oligonucleotide. Supershift assays were performed exactly as the EMSA analysis except 1 µl of Oct-1, C/EBPß, or Sp1 antibody (obtained from Santa Cruz Biotechnology, Inc.) was added after the initial reaction and incubated for 45 min at room temperature before loading onto the gel. The reactions were electrophoresed on a 30-min prerun 5% polyacrylamide gel in 0.25 x TBE for 3.5 h at 175 V. After electrophoresis, the gels were dried and exposed to Kodak XAR-5 film for autoradiography.

For the Oct-1 DNA binding analysis, the same nuclear protein extracts prepared for the EMSA analysis described above were used. Three nuclear extract preparations from treatments of GT1–7 cells in separate experiments were used for the relative binding affinity analysis. EMSA analysis was performed exactly as previously described except increasing concentrations of unlabeled AT-b oligonucleotide were incubated with the reaction mixture 15 min before the labeled AT-b probe (20,000 cpm) was added. Concentrations of unlabeled AT-b oligonucleotide ranged from 30 pg to 10 ng DNA. The polyacrylamide gels were dried and exposed to an intensifying screen. Quantification of the uppermost complex on the AT-b probe, corresponding to Oct-1 binding, was done using a phosphor imaging system and the ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Relative band intensity was plotted against the amount of oligonucleotide competitor on a logarithmic scale. Change in relative binding affinity was determined by comparing the concentration of unlabeled oligonucleotide required to displace 50% of the Oct-1 protein bound to the AT-b probe. Loading efficiency was controlled by normalization to the lower nonspecific band on the EMSA gels.

Phosphorylation analysis was performed using a previously described method (70), with modifications. Dephosphorylated nuclear extracts were prepared by incubating 5 µg of SNP-treated and untreated nuclear extracts with increasing concentrations of calf intestinal alkaline phosphatase (0.01–0.5 U, CIP, Roche Molecular Biochemicals, Indianapolis, IN) at room temperature for 30 min. In some experiments, in addition to the phosphatase, nuclear extracts were incubated in the presence of a combination of phosphatase inhibitors (50 mM sodium fluoride and 1 mM sodium orthovanadate). The nuclear extracts were then subjected to EMSA analysis, as described above, after incubation with the phosphatase inhibitor cocktail for 10 min at room temperature to avoid dephosphorylation of the labeled probe.

Western Blotting
GT1–7 nuclear extract prepared as described above was also used for Western analysis. Proteins (50 µg) were separated on an 12% SDS-PAGE gel and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The membranes were incubated with antisera to Oct-1 and C/EBPß (Santa Cruz Biotechnology, Inc.) in 1 x PBS with 5% nonfat dry milk and 0.001% sodium azide overnight at room temperature with gentle shaking. Membranes were washed sequentially with PBS, 5% non-fat dry milk, and 0.03% Tween-20 for 4 x 30 min without shaking. Membranes were then incubated with horseradish peroxidase-labeled secondary antisera from rabbit for 3–6 h at room temperature and then washed with PBS containing 0.1% Tween-20 as described above. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) as described by the manufacturer.


    ACKNOWLEDGMENTS
 
We thank Teri Banks and Megan Houseweart for assistance in cloning some block replacement mutant plasmids and Simon Lee and Jocelyn Jovenal for excellent technical support. We are grateful to Melody Clark, Mark Lawson, Shelley Nelson, and Jennifer Taylor for critical reading of the manuscript. We also thank the members of the Mellon laboratory for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Pamela L. Mellon, Reproductive Medicine, 0674, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093.

D.D.B. was supported by a fellowship from the Medical Research Council of Canada. This research was supported by a grant from NIH to P.L.M. (DK-44838).

1 Current address: Department of Physiology/Division of Reproductive Science, University of Toronto, Toronto Hospital Research Institute, 200 Elizabeth Street,Toronto, Ontario, Canada M5G 2C4. Back

Received for publication November 5, 1998. Revision received October 18, 1999. Accepted for publication November 8, 1999.


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