Pituitary Homeobox 1 Activates the Rat FSHß (rFSHß) Gene through Both Direct and Indirect Interactions with the rFSHß Gene Promoter

Marjorie M. Zakaria, Kyeong-Hoon Jeong, Charlemagne Lacza and Ursula B. Kaiser

Endocrine-Hypertension Division (M.M.Z., K.-H.J., C.L., U.B.K.), Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Endocrine Division (M.M.Z.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Ursula B. Kaiser, Endocrine-Hypertension Division, Brigham & Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: ukaiser{at}partners.org.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular mechanisms underlying gonadotrope-specific and hormonal regulation of FSHß gene expression remain largely unknown. We have studied the role of pituitary homeobox 1 (Ptx1), a transcription factor important for regulation of many pituitary-specific genes, in the regulation of rat FSHß (rFSHß) gene transcription. We demonstrate that Ptx1 activates the rFSHß gene promoter both basally and in synergy with GnRH. The effect of Ptx1 was localized to -140/-50, a region also important for basal activity of the promoter. Two putative Ptx1 binding sites (P1 and P2) homologous to consensus Ptx1 binding elements were identified in this region. We demonstrate specific binding of Ptx1 to the P2 but not to the P1 site. Furthermore, functional studies indicate that the P2 but not the P1 site mediates activation of the promoter by Ptx1. Residual activation of the promoter by Ptx1 was observed independent of the P2 site. However, no additional Ptx1 binding sites were identified in this region, indicating that the residual activation observed is likely independent of direct Ptx1 binding to the promoter. These results identify a functional Ptx1 binding site in the rFSHß gene promoter and suggest the presence of an additional activating pathway that is independent of direct binding of Ptx1 to the promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH PLAYS AN important role in gonadal development and gametogenesis. The FSHß-subunit is synthesized by pituitary gonadotropes and is the rate-limiting step in overall FSH production. Studies of null and gain-of-function mutations of FSHß in mice illustrate the importance of FSHß. FSHß-deficient females are infertile due to a block in folliculogenesis; FSHß-deficient males are fertile but have small testes and reduced sperm number and motility (1). The phenotype of these mice was successfully rescued by introducing an FSHß transgene into the FSHß-deficient background (2). Transgenic females overexpressing FSHß are also infertile and exhibit a phenotype similar to human ovarian hyperstimulation syndromes with highly hemorrhagic and cystic ovaries (3). Transgenic males are infertile as well, despite having normal testicular development (3). These studies illustrate the physiological importance of FSHß for normal reproductive development and function. Therefore, studying the mechanisms regulating FSHß gene expression is essential to understanding regulation of FSH synthesis.

Although recent studies have provided insight into cis elements and cognate trans-factors important for regulation of {alpha}-gonadotropin subunit ({alpha}-GSU) and LHß gene expression, relatively little is known about the molecular mechanisms regulating FSHß gene expression. Two functional activating protein-1 (AP-1) sites have been identified in the promoter region of the ovine FSHß (oFSHß) gene (4, 5). These sites are important for GnRH activation of the oFSHß gene promoter in the presence of activin in primary pituitary cultures (6). Progesterone-response element-like sequences have been localized in the promoter of the rat FSHß (rFSHß) gene and have been shown to directly mediate progesterone regulation of the gene (7, 8, 9). Transcriptional inhibition of the oFSHß gene promoter by 17ß-estradiol has been mapped to -105/-72 of the promoter but does not seem to involve direct binding of the estrogen receptor to that region (10). Recently, bone morphogenetic proteins (BMP), specifically BMP-6 and BMP-7, were shown to stimulate activity of the oFSHß gene promoter in primary pituitary cultures and in LßT2 cells (11).

Pituitary homeobox 1 (Ptx1) is a member of the Ptx subfamily of bicoid-related homeodomain proteins. The homeobox domain of Ptx acts as a DNA-binding domain, binding to sequences containing the consensus Ptx binding site TAA(T/G)CC (12). Ptx1 is one of the earliest markers of pituitary organogenesis, and is located upstream of a cascade of regulatory proteins involved in pituitary development (13). It is expressed throughout anterior pituitary development in embryonic life, and remains present in all adult pituitary cell lines. Ptx1 plays an important role in gonadotrope development, as evidenced by the Ptx1 knockout model. Mice homozygous for a Ptx1 gene deletion have normal early pituitary organogenesis, but exhibit defects in late anterior pituitary development. In situ hybridization and immunohistochemical analysis indicated a marked reduction in FSHß, LHß, and TSHß gene expression with some reduction in {alpha}GSU expression (14). The results suggest a reduction in the number of gonadotropes and thyrotropes, as well as in the levels of FSHß, LHß, and TSHß transcripts within the individual cells (14). These data support an important in vivo role for Ptx1 in gonadotrope differentiation and in expression of the gonadotropin subunits.

We hypothesized that Ptx1 is directly involved in FSHß gene regulation. In the present study, we demonstrate that Ptx1 can activate transcription of the rFSHß gene promoter, both basally and in synergy with GnRH. The effect of Ptx1 was localized to -140/-50 bp of the rFSHß gene promoter. Sequence analysis of this region revealed two putative Ptx1 binding sites (P1 and P2), homologous to consensus Ptx1 binding elements (13). The P2 site was shown to bind Ptx1 and to be important for mediating Ptx1 activation of the rFSHß gene. Our results also suggest the presence of a secondary activating pathway that is independent of direct binding of Ptx1 to the rFSHß gene promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ptx1 Expression Levels in LßT2 and GGH3-1' Cells
Ptx1, a pituitary specific transcription factor, is differentially expressed among various pituitary cell lineages (13). Pituitary cells expressing the {alpha}GSU in the adult mouse express the highest level of Ptx1 when compared with other pituitary endocrine cells (15, 16). Studies of immortalized pituitary-derived cell lines have also shown higher levels of Ptx1 mRNA in the gonadotrope-derived {alpha}T3-1 cell line compared with the somatolactotrope-derived GH3 cell line (13). Endogenous Ptx1 was detected by RT-PCR in LßT2 cells (17), a gonadotrope-derived cell line that more closely resembles mature gonadotropes than {alpha}T3-1 cells (18, 19). We planned to use two pituitary-derived cell lines, GGH3-1' and LßT2, in our studies of Ptx1 activation of the rFSHß gene promoter. Therefore, a Northern blot comparing relative levels of Ptx1 expression in these cell lines was performed (Fig. 1Go). GH3 and {alpha}T3-1 cells were also included, and CV-1 cells, a monkey kidney fibroblast cell line, were used as a negative control (13). As expected, Ptx1 mRNA was detected in all pituitary-derived cell lines, but not in CV-1 cells. The highest level of expression was observed in gonadotrope-derived cells. Interestingly, Ptx1 mRNA levels were even higher in LßT2 cells than in {alpha}T3-1 cells. So, in addition to exhibiting features of mature gonadotropes, LßT2 cells express high levels of Ptx1. This cell line was used in our subsequent experiments to study the effects of endogenous Ptx1 on the rFSHß gene promoter. Ptx1 mRNA levels in GH3 and GGH3-1' cells were much lower, and these cells were used for studies of the effects of Ptx1 overexpression on the rFSHß gene promoter.



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Figure 1. LßT2 Cells Express High Levels of Ptx1 Compared with GGH3-1' Cells

Northern blot analysis comparing Ptx1 mRNA levels between different pituitary cell lines including GH3, GGH3-1', {alpha}T3-1, and LßT2 cells. CV-1 cells were used as a negative control for Ptx1 expression.

 
Ptx1 and GnRH Synergistically Activate the rFSHß Gene Promoter
Regulation of the rFSHß gene promoter activity by Ptx1 was studied by overexpression of Ptx1 in GGH3-1' cells. These cells were generated previously by stably transfecting the rat somatolactotrope GH3 cells with the rat GnRH receptor (GnRHR) cDNA (20, 21, 22). GGH3-1' cells have been shown to express the GnRHR, and support expression and GnRH regulation of the gonadotropin subunit gene promoters, including the rFSHß gene promoter (23). GGH3-1' cells were cotransfected with -2000/+698 rFSHßLuc along with either the Ptx1-pcDNA3 expression vector or the empty pcDNA3 vector as a control, and Rous sarcoma virus (RSV)-ß-galactosidase. Forty-eight hours after transfection, cells were treated with 100 nM GnRH agonist (GnRHAg) or with vehicle for 4 h. Cells were then harvested and luciferase activity was measured and corrected for ß-galactosidase activity. Activity of -2000/+698 rFSHßLuc was increased by either GnRHAg (4.9 ± 0.5-fold) or Ptx1 (16.5 ± 2.4-fold) alone (Fig. 2Go). Synergism between GnRHAg and Ptx1 was observed when used in combination, as indicated by a 48 ± 6.0-fold increase in promoter activity. These results indicate that GnRH can activate rFSHß gene transcription and are consistent with our previous findings (23). In addition, Ptx1 is also able to activate rFSHß gene transcription in GGH3-1' cells, consistent with previous reports of Ptx1 activation of the bovine FSHß promoter in CV-1 cells (13). Interestingly, the synergism observed between GnRH and Ptx1 suggests an interaction or cross-talk between these two factors in activation of the rFSHß gene promoter.



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Figure 2. Ptx1 and GnRH Activate the rFSHß Gene Promoter Independently and Synergistically

GGH3-1' cells were transiently cotransfected with -2000/+698 rFSHßLuc, Ptx1-pcDNA3 or control pcDNA3, and RSV-ß-galactosidase expression vector. Forty-eight hours after transfection, cells were treated with either vehicle or 100 nM GnRHAg for 4 h and a luciferase assay was performed. Luciferase activity was normalized to ß-galactosidase activity and results are depicted as fold increase in relative luciferase activity compared with controls in the absence of Ptx1 overexpression and without GnRHAg. Results are shown as mean ± SEM of six separate experiments, each performed in triplicate. Letters (a, b, c, d) indicate statistically significant differences (P < 0.05) between the groups.

 
Ptx1 Responsiveness of the rFSHß Gene Promoter Maps to -140/-50
Mapping studies to localize and identify Ptx1-responsive elements in the rFSHß gene promoter were performed in GGH3-1' cells. Cells were cotransfected with 5'-deletion constructs of the rFSHß gene promoter fused to a luciferase reporter gene, and with either Ptx1-pcDNA3 or control empty vector. Ptx1-mediated activation of the rFSHß gene promoter was observed for all 5'-deletion constructs tested except for -50/+15 rFSHßLuc (Fig. 3Go). Activation by Ptx1 was significantly reduced from 36 ± 10.0-fold to 2.2 ± 0.8-fold by 5'-deletion from -140 to -50 (P < 0.05). Activation of -50/+15 rFSHßLuc by Ptx1 was no greater than that for the control promoterless luciferase vector, pXP2. Thus, Ptx1 activation of the rFSHß gene promoter is localized to DNA sequences between -140 and -50.



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Figure 3. Activation by Ptx1 Is Localized to -140/-50 of the rFSHß Gene Promoter in GGH3-1' Cells

Transient transfections in GGH3-1' cells were performed using 5'-deletion constructs of the rFSHß gene promoter with Ptx1-pcDNA3 or pcDNA3, and RSV-ß-galactosidase. Luciferase activity was normalized to ß-galactosidase activity. Results are shown as fold activation by Ptx1 over control for each FSHßLuc reporter construct. The data are shown as mean ± SEM of three separate experiments, each performed in triplicate. Asterisks indicate statistically significant difference (P < 0.05) compared with pXP2.

 
Sequences between -140 and -50 Are Important for Basal rFSHß Gene Promoter Activity in LßT2 Cells
Transient transfection studies were next performed in LßT2 cells to identify promoter sequences important for basal activity of the rFSHß gene promoter. LßT2 cells were transfected with 5'-deletion constructs of the rFSHß gene promoter fused to a luciferase reporter gene and RSV-ß-galactosidase. Forty-eight hours after transfection, cells were harvested and luciferase activity was measured and corrected for ß-galactosidase activity. Basal rFSHß gene promoter activity greater than that of the promoterless pXP2 plasmid was observed for all constructs except for -50/+15 rFSHßLuc (Fig. 4Go). Basal luciferase activity of the -256/+15 rFSHßLuc construct reached but did not achieve statistical significance (P = 0.08) over pXP2. Furthermore, 5'deletion from -140 to -50 significantly reduced basal activity of the promoter (P < 0.05). These results indicate that the same -140/-50 region that mediates Ptx1 responsiveness of the rFSHß gene promoter in GGH3-1' cells is also important for basal activity of the promoter in the Ptx1-expressing LßT2 cell line.



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Figure 4. Basal Activity of the rFSHß Gene Promoter Is Reduced by Deletion of -140/-50 in LßT2 Cells

Transfections of the 5'-deletion constructs of the rFSHß promoter and RSV-ß-galactosidase were performed in LßT2 cells. Luciferase activity was normalized to ß-galactosidase activity. Results are shown as mean ± SEM of three separate experiments, each performed in triplicate. Relative luciferase activity of the rFSHß deletion constructs was compared with the relative luciferase activity of the control pXP2 vector. Different letters (a, b) indicate statistically significant difference (P < 0.05) between groups.

 
Identification of Putative Ptx1 Binding Sites in the rFSHß Gene Promoter
Sequence analysis of the DNA region between -140 and -50 revealed the presence of two DNA sites with significant homology to the consensus Ptx1 binding site (12, 13, 24, 25, 26). A more distal putative element [CAAGCC] located in reverse orientation at -134/-129 will be referred to as P1 (Fig. 5Go). A more proximal putative element [AAATCC] located at -54/-48 will be referred to as P2 (Fig. 5Go). P1 and P2 each share 83% homology with the consensus Ptx1 binding site. Mutations were introduced in each site as indicated in Fig. 5Go, and the mutants will be referred to as P1M and P2M.



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Figure 5. Two Putative Ptx1 Binding Sites Identified between -140/-50 of the rFSHß Gene Promoter

Sequence analysis of the -140/-50 region of the rFSHß gene promoter indicates the presence of two DNA sequences with 83% homology to the consensus Ptx1 binding element. The more 5'-site is referred to as P1, and the more 3'-site as P2. The arrows indicate the orientation of P1 and P2. Point mutations were introduced in each site. The mutant sites are referred to as P1M and P2M. The underlined nucleotides indicate the point mutations introduced in P1 and P2.

 
Ptx1 Binds Specifically to the P2 Site
Ptx1 can activate its target genes by binding directly to DNA response elements in the promoters of these genes. We hypothesized a similar mechanism of action for Ptx1 on the rFSHß gene promoter. EMSAs were performed using oligonucleotide probes corresponding to P2, P2M, P1, and P1M sequences and nuclear protein extracts derived from LßT2 cells (Fig. 6Go). A bacterially produced glutathione S-transferase (GST)-Ptx1 fusion protein was used as a positive control. The LH and LHM probes encoding the intact and mutated Ptx1 binding site of the mouse LHß gene promoter were also used as controls, and incubated with LßT2 nuclear protein extracts. A single protein-DNA complex is observed when nuclear extracts are incubated with P2 (Fig. 6AGo, lane 2) but not with P2M (lane 7). Binding to P2 is inhibited by a 500-fold excess of unlabeled P2 (lane 3) but not of P2M (lane 4). A partial supershift of the protein-DNA complex is observed with the addition of a Ptx1-specific antibody (lane 5). The GST-Ptx1 protein also binds to the P2 probe, and a supershift of the complex is observed with the Ptx1-specific antibody (lanes 8 and 9). Binding of the LßT2 nuclear protein extracts to the LH probe generated two protein-DNA complexes (lane 11), one of which is partially supershifted by the Ptx1 specific antibody (lane 12). This complex is also eliminated by mutation of the Ptx1 binding site in the LHM probe (lane 13). On the other hand, a weak band is observed when LßT2 nuclear protein extracts are incubated with the P1 probe (Fig. 6BGo, lane 1). This band remains intact despite the addition of a 500-fold excess of unlabeled P1 or P1M probes (lanes 3 and 4). No supershift by the Ptx1-specific antibody is observed (lane 5). Binding of the GST-Ptx1 protein to P1 is observed (lane 8) but is much weaker than GST-Ptx1 protein binding to the P2 probe (Fig. 6AGo). The complex of GST-Ptx1 on P1 is supershifted with the Ptx1-specific antibody (Fig. 6BGo, lane 9).



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Figure 6. Ptx1 Binds Specifically to the P2 Site

EMSAs were performed using nuclear protein extract (N.E.) obtained from LßT2 cells and incubated with (A) P2 or P2M probes, and (B) P1 or P1M probes. Incubation with a 500-fold excess of unlabeled (A) P2 or P2M and (B) P1 or P1M were used for competition. A GST-Ptx1 fusion protein was used as positive control with either the P2 or the P1 probe. A Ptx1-specific antibody was used for supershift studies in each EMSA. Nuclear protein extracts were also incubated with the mouse LH and LHM probe for control. The large arrows indicate the specific protein-DNA complex and the small arrows the complex supershifted with the Ptx1-specific antibody. The asterisk indicates the DNA-protein complex corresponding to Ptx1 binding to the LH and LHM probes. F. P., Free probe.

 
In summary, these results indicate specific binding of a protein(s) in the LßT2 nuclear extracts to the P2 oligonucleotide but not to the P1 oligonucleotide. Antibody supershift assays identify Ptx1 (or an antigenically related protein) as a component of the DNA-protein complex formed by LßT2 nuclear extracts on P2. Studies using the LH and LHM probes confirm the presence of Ptx1 (or an antigenically related protein) in LßT2 nuclear extracts, and confirm specific binding of Ptx1 to cognate DNA elements. In addition, use of the bacterially engineered GST-Ptx1 fusion protein confirms that P2 is a Ptx1 recognition site. Binding of LßT2 nuclear extract proteins to the P1 oligonucleotide, on the other hand, is nonspecific. It is not competed by excess unlabeled P1 oligonucleotide and is not supershifted by a Ptx1-specific antibody. Binding to P1 is observed with the GST-Ptx1 protein but is weak when compared with binding to P2.

The P2 Site Mediates Activation of the rFSHß Gene by Ptx1
Transfection studies in GGH3-1' cells were performed next to assess the functional significance of the P1 and P2 sites in mediating activation of the rFSHß gene promoter by Ptx1. Point mutations were introduced singly and in combination in the P1 and P2 sites of the -140/+15 rFSHßLuc construct. These point mutations are identical with P1M and P2M (Fig. 5Go). GGH3-1' cells were cotransfected with the wild-type and mutant -140/+15 rFSHßLuc constructs and with Ptx1-pcDNA3 or control empty vector. Ptx1 induced a 72 ± 3-fold increase in wild-type -140/+15 rFSHßLuc activity (Fig. 7Go). Activation by Ptx1 was significantly reduced by mutation of the P2 (17 ± 3-fold; P < 0.05) but not the P1 (74 ± 17-fold) site. No further reduction in activation by Ptx1 was observed when the P1 and P2 sites were mutated in combination (16 ± 2-fold). The introduction of a mutation in P2, which eliminated Ptx1 binding to this site, greatly reduced activation of rFSHß transcription by Ptx1. Nonetheless, a residual 16-fold activation by Ptx1 was observed with the P2 mutant and the P1/P2 double mutant constructs. This residual activation is significantly greater than that observed with the control promoterless luciferase vector, pXP2, (P < 0.05), suggesting an additional mechanism of action of Ptx1 on rFSHß gene activation that is independent of the P2 site.



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Figure 7. Mutation of the P2 But Not the P1 Site Reduces Ptx1 Activation of the rFSHß Gene Promoter in GGH3-1' Cells

Mutations of the P1 and P2 sites were introduced singly and in combination in -140/+15 FSHßLuc. Transfection studies were performed in GGH3-1' cells using wild-type or mutant -140/+15 FSHßLuc constructs with Ptx1-pcDNA3 or control pcDNA3 and RSV-ß-galactosidase. Results are expressed as fold activation by Ptx1 over control for each reporter construct. The data are shown as mean ± SEM of three separate experiments, each performed in triplicate. Different letters indicate statistically significant differences (P < 0.05) between groups.

 
The P2 Site Is Important for Basal and GnRH-Stimulated Activation of the rFSHß Gene
Transfections were also performed in the LßT2 cell line, to assess the functional significance of P1 and P2 in basal and GnRH-stimulated activity of the rFSHß gene promoter in a gonadotrope-derived cell line. The wild-type and mutant -140/+15 rFSHßLuc constructs described previously were transiently transfected in LßT2 cells (Fig. 8Go). The promoterless vector pXP2 was used as a control. Forty-eight hours after transfection, cells were treated with or without 100 nM GnRHAg for 4 h, after which cells were harvested and luciferase and ß-galactosidase assays were performed. The results indicate a statistically significant reduction (P < 0.05) in basal rFSHß gene promoter activity when the P2 site was mutated, either singly or in combination with P1. Interestingly, mutation of the P1 site increased basal luciferase activity of the rFSHß gene promoter compared with wild type (P < 0.05) but not in the presence of the P2 mutation. In addition, GnRH-stimulated luciferase activity was significantly reduced by mutation of P2 but not P1 (Fig. 8Go). No further reduction of GnRH-stimulated activity was observed when both the P1 and P2 mutations were present in combination. Surprisingly, mutation of the P1 site appeared to increase GnRH-stimulated luciferase activity, both in the context of the wild-type promoter and in the presence of the P2 mutation. Of note, both basal and GnRH-stimulated activity of -140/+15 rFSHßLuc were reduced by mutation of P2, but the fold activation by GnRH was retained in all constructs tested. Thus, the P2 site appears to contribute to both basal and GnRH-stimulated rFSHß gene promoter activity in LßT2 cells, but does not affect the fold response to GnRH.



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Figure 8. The P2 Site Is Important for Basal and GnRH-Stimulated Activation of the rFSHß Gene Promoter in LßT2 Cells

Transfections were performed in LßT2 cells using the wild-type and mutant -140/+15 FSHßLuc constructs and RSV-ß-galactosidase. Forty-eight hours after transfection, cells were treated with or without 100 nM GnRHAg for 4 h, after which cells were harvested. Luciferase activity was normalized to ß-galactosidase activity, and results are expressed as relative luciferase activity. This is a representative of three separate experiments and results are depicted as mean ± SEM of triplicates within the representative experiment. Basal luciferase activity was compared between all constructs. a, b, c, Statistical significance (P < 0.05). GnRH-stimulated luciferase activity was compared between all constructs. w, x, y, z, Statistical significance (P < 0.05).

 
Ptx1 Binding Sites in -140/-31 of the rFSHß Gene Promoter
The presence of a residual Ptx1 effect on -140/+15 rFSHßLuc despite mutation of the P1 and the P2 sites suggested additional Ptx1 mechanisms of action. 5'-deletion of sequences between -140 and -50 of the rFSHß gene promoter completely eliminated the ability of Ptx1 to activate the rFSHß gene, and reduced the Ptx1 effect to that of the promoterless control vector (Fig. 3Go). In contrast, whereas mutation of P2 in the context of -140/-50 rFSHßLuc markedly reduced activation by Ptx1, a 16-fold activation of the mutated P2 and P1/P2 rFSHß promoter remained and was significantly greater than the effect of Ptx1 on pXP2 (Fig. 7Go). These disparate results suggested that Ptx1 exerts additional effects through other sequences between -140/-50. One possible mechanism is the presence of additional Ptx1 DNA-binding sites that were not recognized by the homology analysis. An EMSA study was performed using overlapping oligonucleotide probes spanning the region between positions -140 and -31 of the rFSHß gene promoter to detect possible additional Ptx1 DNA-binding sites (Fig. 9Go). GST-Ptx1 protein was shown to bind primarily to probe V (-50/-31), which contains the P2 site. Additional weaker binding to probe I (-140/-111), which contains the P1 site, was also observed, consistent with the findings in our earlier EMSA studies (Fig. 6Go). No significant binding of GST-Ptx1 to probes II, III, and IV was observed. Thus, no additional sites for direct Ptx1 binding to this region of the rFSHß gene promoter were detected by EMSA.



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Figure 9. Ptx1 Binds Primarily to the P2 and Less Strongly to the P1 Site.

EMSAs were performed using five overlapping oligonucleotide probes spanning the region between -140 and -31 of the rFSHß gene promoter, with GST or GST-Ptx1 fusion proteins. The probes used include probe I (-140/-111), probe II (-120/-91), probe III (-100/-71), probe IV (-80/-51), and probe V (-60/-31). The arrow indicates the protein-DNA complex. F.P., Free probe.

 
Ptx1 Binds Primarily to the P2 Site in the Context of -140/+15 of the rFSHß Gene
Further protein-DNA binding studies were performed using the -140/+15 region of the rFSHß gene to identify all important Ptx1 binding sites, and to define more precisely the base pairs contacted by Ptx1 in the context of this full region. Methylation and uracil interference assays were performed using sequences corresponding to -140/+15 of the rFHSß gene as a probe, end-labeled on either the sense or the antisense strand, and the GST-Ptx1 fusion protein. Methylation interference assays identified only one Ptx1 binding site on the antisense strand (Fig. 10AGo). Sequence alignment attributed the interference pattern to guanine (G) nucleotides at positions -50 and -49 of the antisense strand, corresponding to the site previously identified and referred to as P2 in EMSA studies (Fig. 10AGo). No additional Ptx1 binding elements, including P1, were identified by methylation interference. Uracil interference assays were performed to further study the sense and antisense -140/+15 rFSHß strands (Fig. 10Go, B and C). Using this approach, again only one Ptx1 binding site was recognized. Sequence analysis indicated that the observed interference pattern occurred at the thymidine (T) nucleotide at position -51 on the sense strand (Fig. 10BGo), again corresponding to the P2 site. There was no other evidence of interference with Ptx1 binding, including the DNA region corresponding to the P1 site on the antisense strand. Taken together, these results identify P2 as the major Ptx1 binding site in the -140/+15 region of the rFHSß gene, with no additional sites of direct Ptx1 binding identified.



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Figure 10. The P2 Site Is Identified as the Only Ptx1 Binding Site by Methylation and Uracil Interference Assays

The -140/+15 fragment of the rFSHß gene promoter was used as a probe in (A) methylation and (B and C) uracil interference assays. In each experiment, the free probe (F.P.) was compared with the Ptx1-bound probe (B.P.). Bidirectional sequence analysis of -140/+15 rFSHß was run in parallel to localize areas of binding interference (data not shown). The area of binding interference is indicated by an arrow and a corresponding DNA sequence that is boxed. The locations of the P1 and P2 sites are indicated. A, Methylation interference assay was performed using -140/+15 rFSH ß 3'-end-labeled on the antisense strand, and incubated in the presence or absence of GST-Ptx1. The area of binding interference is indicated by the arrow and corresponds to the two guanine nucleotides of P2 (boxed sequence). B and C, Uracil interference assay was performed using -140/+15 rFSHß 5'-end-labeled on the (B) sense and (C) antisense strands, and incubated in the presence or absence of GST-Ptx1. The area of binding interference on the sense strand corresponds to the thymidine oligonucleotide of the P2 site as indicated by the arrow and boxed P2 sequence. C, There is no evidence of binding interference on either P1 or P2 on the antisense strand.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our studies were performed in pituitary-derived cell lines as they represent physiologically appropriate models for studying the role of a pituitary-specific transcription factor. The cell lines used include GGH3-1' cells, a modified rat somatolactotrope cell line that stably expresses the rat GnRHR (20, 21, 22), and LßT2 cells, a murine gonadotrope-derived cell line (27). GGH3-1' cells were previously shown to support expression of the gonadotropin subunit genes including FSHß, basally and in response to GnRH (23). LßT2 cells exhibit many features of mature gonadotropes, with expression of all of the gonadotropin subunit genes, including FSHß (18, 28), expression of the GnRHR, and the ability to respond to GnRH. In addition, we have shown that both cell lines express Ptx1 with very low levels in GGH3-1' cells, and higher levels in LßT2 cells. These characteristics made GGH3-1' cells useful for Ptx1 overexpression studies. Similar overexpression studies performed in parallel in LßT2 cells also indicated activation of the rFSHß gene promoter by Ptx1, although to a lesser extent when compared with our studies in GGH3-1' cells (data not shown). These findings likely reflect the presence of higher levels of endogenous Ptx1 in LßT2 cells, making it difficult to achieve high degrees of further activation of the FSHß promoter by Ptx1 overexpression. LßT2 cells, on the other hand, were used to study the physiological relevance of the P1 and P2 sites of the rFSHß gene promoter under basal and GnRH-stimulated conditions.

Mapping studies performed in GGH3-1' cells localized activation of the rFSHß gene by Ptx1 to -140/-50 of the promoter. The same DNA region was shown to be important for basal stimulation of the rFSHß gene promoter in LßT2 cells. Sequence analysis identified two putative Ptx1 binding sites in this region: P1 at -134/-129 and P2 at -54/-48. Both sites share 83% homology with consensus Ptx1 binding sites. Of these two sites, only the more proximal P2 site was shown to be functionally active in both cell models. Structural studies including EMSA, uracil, and methylation interference assays demonstrated direct binding of GST-Ptx1 to the P2 site. Binding of GST-Ptx1 to the P1 site, only observed in EMSA studies, was weak when compared with binding to the P2 site. Binding of GST-Ptx1 to the P1 site was not observed in either uracil or methylation interference assays, suggesting that it is either weak or nonexistent in the context of the -140/+15 region of the rFSHß gene. Functional studies further support a role for the P2 but not the P1 site in regulation of rFSHß gene expression. Mutations of the P2 site that inhibit binding of Ptx1 result in a significant reduction in activation of the rFSHß gene promoter by Ptx1. These mutations also reduced basal and GnRH-stimulated expression of the rFSHß gene promoter in LßT2 cells. On the other hand, inactivating mutations of the P1 site did not interfere with either activation of the rFSHß gene promoter by Ptx1, or with basal and GnRH-stimulated activity of the promoter. In contrast, mutation of the P1 site appeared to increase the activity of the rFSHß gene promoter at times, although this effect was variable depending on the promoter and cell line context. The physiological significance of this effect is not known. Taken together, these results demonstrate that only the P2 and not the P1 site is consistently functionally relevant for rFSHß gene promoter activity in both cell lines. This is in agreement with previous studies showing that only one of several putative Ptx1 binding elements is important for Ptx1 activation of target genes, including LHß, {alpha}GSU, (13) and proopiomelanocortin (12).

In addition to the P2-mediated activation of the rFSHß gene promoter by Ptx1, we observed a P2-independent effect of Ptx1 on the promoter. A residual 16-fold activation of the rFSHß gene promoter by Ptx1 was observed despite introducing inactivating mutations in P2, either singly or in combination with P1 mutations. This residual activation was significantly greater than the activity of pXP2, the promoterless luciferase vector. These results suggest a secondary P2-independent effect of Ptx1 on the rFSHß gene promoter. This effect does not appear to involve direct binding of Ptx1 to the promoter, as no binding to sites other than to P2 was identified in -140/+15 of the rFSHß promoter. 5'-deletion from -140 to -50 fully eliminated the Ptx1 effect, whereas mutation of P2 only led to a partial reduction. Therefore, additional sequences between -140 and -50 are important for the P2-independent effect, but do not involve direct binding of Ptx1 to the rFSHß gene promoter. Residual activation of the rFSHß gene promoter may involve protein-protein interaction between Ptx1 and other transcription factors, as has been described for the LHß gene promoter. Transcriptional cooperation was described between Ptx1 and steroidogenic factor-1, and Ptx1 and early growth response protein (Egr)-1 on the LHß gene promoter (29). The former requires binding of steroidogenic factor-1 but not of Ptx1 to cognate sequences in the promoter, whereas the latter requires binding to only one of the Egr-1 or the Ptx1 sites (29). Similarly, protein-protein interaction between Ptx1 and a yet unidentified transcription factor may account for the P2-independent activation of the rFSHß gene promoter by Ptx1.

The P2 site is important for both basal and GnRH-stimulated expression of the -140/+15 rFSHß gene promoter, as indicated by studies in LßT2 cells. Inactivating mutations of P2, while reducing both effects, did not change the fold activation by GnRH. These results suggest that Ptx1 does not directly mediate the GnRH response. However, Ptx1 was shown to synergize with GnRH in activating the rFSHß gene promoter in GGH3-1' cells. This synergism may not involve direct binding of Ptx1 to the promoter but may involve interaction between Ptx1 and a GnRH-responsive transcription factor, such as described with Egr-1 on the LHß promoter (30).

Nuclear extracts from LßT2 cells were used in EMSA studies demonstrating specific binding to the P2 but not the P1 site. The protein(s) bound to the P2 site was recognized in supershift assays with a Ptx1-specific antibody, indicating that the complex contains Ptx1, or a protein antigenically related to Ptx1. Oligonucleotide probes encoding the intact or mutant Ptx1-binding site of the mouse LHß gene were used as positive controls with LßT2 nuclear extracts. The results obtained further confirmed the presence of Ptx1 protein in the nuclear extracts and specific binding to the Ptx1 binding site. Specific binding to the P2 site was also confirmed using a GST-Ptx1 fusion protein. Although binding of GST-Ptx1 was also observed on the P1 probe, it was weak when compared with binding to the P2 probe, a finding consistent with the lack of a functional role for P1. These results demonstrate that endogenous Ptx1 protein in LßT2 cells binds specifically to the P2 site of the rFSHß gene promoter. It remains possible that proteins other than Ptx1 may also bind to Ptx1 recognition sites and play an additional role in activating the rFSHß gene promoter. This mechanism of action has been suggested by in vivo and in vitro studies of LHß gene regulation. Transgenic mice bearing a DNA-binding inhibiting mutation of the proximal Ptx1 binding site of the LHß gene promoter fused to a CAT reporter gene (31) showed no basal pituitary CAT activity and no CAT response to GnRH. These results indicate that the proximal Ptx1 binding site of the LHß gene promoter is important for both basal expression and GnRH responsiveness of the LHß gene promoter. In comparison, the Ptx1 knockout mouse model showed only a reduction but not complete absence of LHß and FSHß gene expression. Absence of the Ptx1 binding site had a more profound effect on LHß gene expression than absence of the Ptx1 protein, suggesting that proteins other than Ptx1 may regulate this gene via the Ptx1 binding site. Supporting evidence for this observation stems from studies of cell-specific LHß gene expression. These studies demonstrate the presence of an LßT2-specific protein that binds to Ptx1 recognition elements in the LHß gene promoter but does not comigrate with known members of the Ptx and Otx families (32). Similarly, additional LßT2 proteins other than Ptx1 may bind to the P2 site and regulate rFSHß gene expression.

In summary, we confirm that Ptx1 is expressed in gonadotrope cells, as shown by high levels of Ptx1 mRNA in the gonadotrope-derived LßT2 cell line. We also report that Ptx1 can activate the rFSHß gene promoter singly and in synergy with GnRH. The primary mechanism of action of Ptx1 on the rFSHß gene promoter involves binding of Ptx1 to the P2 site, which shares 83% homology with consensus Ptx1 recognition elements. Studies in LßT2 cells identify the same P2 site as functionally important for basal and GnRH-stimulated activity of the rFSHß gene promoter, suggesting that these effects are mediated by binding of endogenous Ptx1 to this site. A residual P2-independent activation of the rFSHß gene promoter by Ptx1 is observed, suggesting the presence of an additional pathway that does not involve direct binding of Ptx1 to the promoter. Future studies will include further investigation of this secondary pathway and identification of transcription factors interacting with Ptx1 to mediate basal and GnRH-stimulated activation of the rFSHß gene promoter.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The GnRH agonist des-Gly10,[d-Ala6]-LHRH ethylamide (GnRHAg) was purchased from Sigma (St. Louis, MO). A polyclonal Ptx1 antibody was generated in rabbits against a peptide corresponding to amino acids 24–52 of mouse Ptx1, conjugated to keyhole limpet hemocyanin as a hapten carrier (Covance Research, Richmond, CA). The amino acid region between 24–52 is unique to Ptx1 based on homology analysis, and is highly hydrophilic and antigenic.

Reporter Plasmids and Expression Vectors
rFSHßLuc constructs contain the indicated regions of the rFSHß gene fused upstream of the luciferase reporter gene in pXP2 (23, 33). -2000/+698 rFSHßLuc has been described previously (23); -472/+15 rFSHßLuc was generated by restriction digestion of -2000/+698 rFSHßLuc with BstNI; -50/+15 rFSHßLuc by digestion with NcoI/BstNI. The appropriate fragments were then subcloned into pXP2. -256/+15 rFSHßLuc and -140/+15 rFSHßLuc were generated by PCR amplification, using forward and reverse complementary primers to the respective DNA sequences of the rFSHß promoter. Primer sequences incorporated restriction enzyme sites BamHI and HindIII (Table 1Go). PCR products were digested with BamHI and HindIII, and subcloned into pXP2. All constructs were confirmed by restriction digest patterns and bidirectional sequence analysis. rFSHßLuc mutant constructs were generated using site-directed mutagenesis. Two point mutations were introduced in each of the putative Ptx1 binding sites (P1 and P2) in the -140/+15 rFSHßluc construct, singly and in combination. These mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer’s instructions. Oligonucleotide primers used to introduce the P1 and P2 mutations are described in Table 1Go.


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Table 1. Oligonucleotides Used in This Study

 
An expression vector expressing ß-galactosidase driven by the RSV-ß-galactosidase was used as an internal standard and control (34).

The mouse Ptx1 cDNA was generated by RT-PCR and subcloned into pcDNA3/Amp (Invitrogen, Carlsbad, CA). A reverse transcription reaction was performed using total RNA from {alpha}T3-1 gonadotrope cell line and an oligo(deoxythymidine) primer, and the reverse transcription-generated first strand poly A+ cDNA products were subjected to a PCR using Ptx1 cDNA-specific primers. PCR primers were designed based on the Ptx1 cDNA sequence (GenBank accession no. U71206) to span the Ptx1 open reading frame region, from +401 to +1435 of the cDNA, and a HindIII or EcoRI restriction enzyme site was inserted into 5'-end of each primer for the purpose of subcloning. Bidirectional sequence analyses were performed using the dideoxynucleotide chain termination method to ensure the correct Ptx1 cDNA sequence.

Generation of GST-Ptx1 Fusion Protein
A GST/Ptx1 plasmid was generated by insertion of the Ptx1 cDNA downstream of the GST coding sequence in the pGEX-4T-2 expression vector (Amersham Pharmacia Biotech, Piscataway, NJ) in the BamHI/NotI sites. This plasmid was introduced into a bacterial stock BL21 and induced with isopropyl ß-D-thiogalactoside (Sigma) to express the fusion protein. A GST affinity column (Amersham Pharmacia Biotech) was used to purify the fusion protein and its identity was confirmed by SDS-PAGE protein gel electrophoresis and Western blotting.

Cell Culture and Transfection Assays
Murine gonadotrope-derived LßT2 and rat somatolactotrope-derived GGH3-1' cells were grown in DMEM supplemented with 10% fetal bovine serum with high glucose and low glucose, respectively. GGH3-1' cells were generated in our laboratory by stably transfecting GH3 cells with the rat GnRH receptor cDNA (20, 21, 22). LßT2 and GGH3-1' cells were transiently transfected by electroporation. In each experiment, approximately 5 x 106 cells were suspended in 0.4 ml of Dulbecco’s PBS plus 5 mM glucose containing the DNA to be transfected. The cells received a single electrical pulse of 240 V from a total capacitance of 960 microfarads, using a Bio-Rad Laboratories, Inc. gene pulser apparatus (Hercules, CA). After electroporation, cells were plated in 10% fetal bovine serum-containing medium. Forty-eight hours later, cells were treated in the presence or absence of 100 nM of GnRHAg for 4 h and then harvested. Luciferase assays were performed as previously described, using an LB 953 Autolumat (EG&G Berthold, Nashua, NH) by standard protocols (23, 35). ß-Galactosidase activity was assayed colorimetrically by standard protocols (34). Luciferase activity was normalized for expression of RSV-ß-galactosidase.

Northern Blot Analysis
Total RNA was extracted from GH3, GGH3-1', {alpha}T3-1, LßT2 and CV-1 cells according to the TRI REAGENT protocol (Molecular Research Center, Inc., Cincinnati, OH).

Riboprobes were generated using the Strip-EZ RNA kit (Ambion, Inc., Austin, TX). The Ptx1 riboprobe was generated from the mouse Ptx1 cDNA/pcDNA3 plasmid described above. The plasmid was linearized using HindIII. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe was generated using a mouse pTRI-GAPDH template provided with the NorthernMax-Gly kit (Ambion, Inc.). After riboprobe synthesis, the DNA template was digested using the DNA-free kit (Ambion, Inc.) after the manufacturer’s guidelines. Post-synthesis chemical labeling of the riboprobes was performed using the BrightStar Psoralen-Biotin nonisotopic labeling kit (Ambion, Inc.). The riboprobes were then stored at -80 C.

The Northern blot was performed using NorthernMax-Gly, a glyoxal-based system for Northern blots (Ambion, Inc.), after the manufacturer’s guidelines. RNA samples were loaded on a 1% agarose gel, which was run at 100 V for 2 h. The RNA was then transferred to a BrightStar-Plus membrane (Ambion, Inc.), cross-linked using a transilluminator, and prehybridized in ULTRAhyb for 2 h at 68 C. Ptx1 and GAPDH riboprobes were then added at a final concentration of 0.25 nM/probe for an overnight hybridization at 68 C. The membrane was then washed for 10 min at room temperature in low stringency solution, and twice for 15 min each at 68 C in high stringency solution. Detection of the nonisotopic signal was performed using the BrightStar BioDetect detection kit (Ambion, Inc.). The membrane was then exposed to film at room temperature for 2 h.

EMSA
Nuclear protein microextraction from LßT2 cells was performed according to the method of Therrien and Drouin (36). Wild-type and mutant oligonucleotides, P1, P1M, P2, and P2M, corresponding to the putative Ptx1 binding sites in the -140/+15 rFSHß gene promoter, were generated (Table 1Go). Control oligonucleotides encoding the Ptx1 binding site of the mouse LHß gene promoter were also generated and named LH for the intact and LHM for the mutated sequences (Table 1Go). In addition, five overlapping oligonucleotides encoding DNA sequences between -140/-31 of the rFSHß gene promoter were synthesized (Table 1Go). In all cases, the complementary sense and antisense oligonucleotides were annealed, 5'-end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase, and purified over a Nick column (Amersham Pharmacia Biotech). The binding reaction for EMSA was performed by incubating 250,000 cpm of DNA probe with 10 ng of either GST-Ptx1 fusion protein or control GST protein, or 10 µg of LßT2 nuclear extract and 2 µg of salmon sperm DNA in reaction buffer (20 mM HEPES, pH 7.9; 60 mM KCl; 5 mM MgCl2; 10 mM phenylmethylsulfonyl fluoride; 10 mM dithiothreitol; 1 mg/ml BSA; and 5% (vol/vol) glycerol) for 30 min at 4 C. For competition studies, 500-fold excess unlabeled oligonucleotide was added 5 min before the addition of probe. Antibody supershift assays were performed using the Ptx1 specific antibody. The antibody was added to the EMSA reaction samples after 30 min and incubated at 4 C for an additional 2 h prior to gel electrophoresis. Protein-DNA complexes were resolved by 4% low ionic strength nondenaturing polyacrylamide gel electrophoresis in 0.5x Tris borate- EDTA buffer. The gels were then dried and subjected to autoradiography.

Uracil Interference Assay
Uracil interference assays were performed according to Pu and Struhl (37). Oligonucleotides corresponding to the 5' sense (FSH-140) and 3' antisense (3'FSH +15) ends of the -140/+15 bp fragment of the FSHß promoter were used (Table 1Go). The oligonucleotide primers were 5'-end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase, and purified using a Nick column. Two parallel PCRs were performed using the -140/+15 FSHßLuc plasmid as a template with one unlabeled and one 5'-labeled primer in the presence of deoxy (d) GTP, dATP, dCTP, dTTP, and dUTP at 2.5 mM each. Deoxyuracil will be randomly substituted for deoxythymidine on both DNA strands of the PCR products. The PCR products were then subjected to electrophoresis on a 6% native nondenaturing polyacrylamide gel. The wet gel was exposed to an autoradiograph for 2 h, and the radioactive PCR bands were excised and eluted in elution buffer (20 mM Tris, pH 7.4; 1 mM EDTA; 0.5 M Na acetate; 0.1% sodium dodecyl sulfate) overnight at 42 C. EMSAs were then performed as described above, incubating 250 x 103 cpm of radiolabeled PCR probe with 1 µg of either GST or GST-Ptx1 fusion protein. The autoradiograph bands corresponding to the complexed and free probe were excised and purified from gel slices as described above. The DNA was then cleaved at uracil residues by using 1 U of uracil-N-glycosylase and incubating the reaction at 37 C for 60 min. The DNA was then ethanol precipitated, resuspended in 100 µl of 1 M piperidine and incubated at 90 C for 40 min. After piperidine cleavage, the tubes were placed in dry ice, and then the samples were lyophilized in a vacuum evaporator until dry. The freezing lyophilization step was repeated once. The reaction products (3 x 103 cpm/sample) were then separated on a 6% denaturing polyacrylamide gel. Sequencing of -140/+15 rFSHß was performed using the 70770 Sequenase Version 2.0 DNA Sequencing Kit (USB, Cleveland, Ohio). Primers used include the FSH-140 oligonucleotide primer complementary to the 5' end and the 3'FSH+15 oligonucleotide primer reverse complementary to 3' end of the -140/+15 rFSHß construct (Table 1Go). The sequencing reactions were loaded on a 6% denaturing polyacrylamide gel alongside the reaction products of the uracil interference assay. Gels were subjected to electrophoresis, dried and subjected to autoradiography.

Methylation Interference Assay
Methylation interference assays were performed as we have previously described (38), according to the method of Ikeda et al. (39). The sense probe used was generated by linearizing the -140/+15 rFSHßluc construct at the 5'-end of the promoter by BamHI, 3'-end-labeling with [{alpha}32P]-dCTP using Klenow and 4 mM dNTP. After labeling, a second restriction digest was performed at the 3'-end of the promoter with HindIII. The 32P-end-labeled -140/+15 rFSHß probe was then purified on a nondenaturing 6% polyacrylamide gel. The wet gel was exposed to an autoradiograph for 60 sec, a single band was observed and was excised and eluted overnight at 42 C in elution buffer (20 mM Tris-HCl, pH 7.4; 1 mM EDTA; 0.5 M sodium acetate; 0.1% sodium dodecyl sulfate). The probe was then precipitated and counted. Methylation was performed using 4 x 106 cpm of probe and dimethyl sulfate as per protocol. The methylated probe was precipitated using ethanol and sodium acetate. EMSA was then performed as described above using 5 x 105 cpm of the methylated 32P-end-labeled -140/+15 rFSHß probe and 1 µg of GST-Ptx1 fusion protein, or GST alone for control. Complexed and free DNA probes were visualized by autoradiography of the wet gel at 4 C for 2 h and excised. The DNA was eluted, precipitated, cleaved with 1 M piperidine for 30 min at 90 C, lyophilized, and resuspended in formamide loading buffer. Samples (1 x 104 cpm per sample) were loaded on a 6% denaturing polyacrylamide gel and subjected to electrophoresis. Sequencing of the -140/+15 rFSHß construct was performed as described above.

Statistical Analysis
One-way ANOVA with Tukey-Kramer Multiple Comparisons tests was performed using GraphPad inStat version 3.05 for Windows 95 (GraphPad Software, Inc., San Diego, CA; www.graphpad.com). P values less than 0.05 were considered statistically significant.


    ACKNOWLEDGMENTS
 
We thank Dr. Pamela Mellon for generously providing LßT2 and {alpha}T3-1 cells. We thank Ipek Kutlu for technical support.


    FOOTNOTES
 
This work was supported in part by NIH Grants HD-33001 (to U.B.K.), HD-19938 (to U.B.K.), and HD-01383 (to M.M.Z.), The Charles Hood Foundation (to M.M.Z.), as well as by the Lalor Foundation (to K.-H.J.).

Abbreviations: AP-1, Activating protein-1; BMP, bone morphogenetic protein; Egr, early growth response protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRHAg, GnRH agonist; GnRHR, GnRH receptor; GST, glutathione S-transferase; GSU, gonadotropin subunit; oFSHß, ovine FSHß; P1 and P2, putative Ptx1 binding sites; Ptx1, pituitary homeobox 1; rFSHß, rat FSHß; RSV, Rous sarcoma virus.

Received for publication February 28, 2002. Accepted for publication May 1, 2002.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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