| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093-0674
Address all correspondence and requests for reprints to: Pamela L. Mellon, Ph.D., Department of Reproductive Medicine, 0674, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: pmellon{at}ucsd.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of LßT2 and T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied LßT2 cell-specific expression of FSHß using 398 bp of the mouse FSHß regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSHß promoter can direct reporter gene expression specifically to LßT2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in LßT2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSHß gene expression in an LßT2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSHß protein in LßT2 cells and to identify specific elements within the FSHß promoter that contribute to basal and cell-specific expression of the gene. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-subunit [-glycoprotein subunit (-GSU)] and contains a unique ß-subunit that confers the physiological specificity to each hormone. Because the ß-subunit genes of FSH and LH are expressed exclusively in the gonadotrope population of the anterior pituitary, the synthesis and secretion of FSH and LH are restricted to these cells.
Although some success has been achieved in understanding the molecular mechanisms governing basal and cell-specific expression of the -GSU and LHß genes (2, 3, 4, 5, 6, 7), very little is understood about regulation of the FSHß gene. This is due, in part, to the heterogeneity of the anterior pituitary gland in vivo and, until recently, the lack of an FSHß-expressing gonadotrope-derived cell line in which to study its regulation. Use of transgenic mice has allowed large regulatory regions important for hormonal and gonadotrope-specific regulation of FSHß to be defined. These studies demonstrated that a 10-kb region of the human FSHß gene, including 4 kb of 5'-flanking sequence and 2 kb of 3'-flanking sequence, is sufficient to direct gonadotrope-specific expression (8). Similarly, a 5.5-kb fragment of the 5'-flanking sequence of the ovine FSHß gene can confer pituitary-specific expression of a luciferase reporter gene (9). However, specific elements involved in tissue-specific expression have not been identified.
The LßT2 gonadotrope-derived cell line was recently shown to express endogenous FSHß mRNA (10) and secrete substantial levels of FSH in response to activin (11), an important regulator of FSH synthesis in vivo (12, 13). Because LßT2 cells produce FSHß, they must contain all of the factors necessary for its expression. These cells therefore present a model system in which to dissect details of the molecular basis for expression of FSHß in the gonadotrope. Indeed, the molecular mechanisms governing GnRH and activin regulation of the ovine FSHß gene are beginning to be elucidated using these cells (10, 14). In the present study, we examine the basis for cell-specific expression of FSHß in LßT2 gonadotrope cells by comparison to a non-FSH-producing gonadotrope-derived cell line, T3-1, and to a fibroblast-derived cell line, NIH3T3. We demonstrate the presence of endogenous FSHß protein in LßT2 cells, even in the absence of treatment by activin or GnRH. Specific promoter elements that bind the orphan nuclear receptor steroidogenic factor-1 (SF-1), a known regulator of gonadotrope-specific genes, and the ubiquitous transcription factor, nuclear factor Y (NF-Y), are identified within 398 bp of the proximal mouse FSHß regulatory region. These contribute to cell-specific expression but are not involved in repression by follistatin. Finally, NF-Y and SF-1 are shown to interact both physically as well as functionally; this functional interaction occurs in a manner that contributes to LßT2 cell-specific expression of the FSHß gene.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-GSU, and SF-1; the non-FSHß-expressing cell line, T3-1, was used for comparison (Fig. 1
). All gonadotrope markers, including FSHß, are detected in LßT2 cells under these basal conditions. In contrast to LßT2 cells, T3-1 cells have been characterized as committed, but immature, gonadotrope-derived cells (15), expressing high levels of -GSU and SF-1 mRNA but neither of the gonadotropin ß-subunits. Examination of protein expression in these cells confirms their lack of the ß-subunits. Consistent with the previously described translational control of -GSU in T3-1 cells (16), they contain very low levels of -GSU protein despite the high levels of -GSU message.
|
T3-1 cells, we postulated that these cell lines could be used as a model to identify factors involved in the induction of mouse FSHß gene expression during gonadotrope maturation. To test this, a DNA fragment encompassing 398 bp of the mouse FSHß 5'-regulatory region was linked to a luciferase reporter gene (-398FSHßLuc) and used in transient transfection studies. Activity of the FSHß promoter was examined in LßT2 cells and compared with a variety of other pituitary and non-pituitary-derived cell lines (Fig. 2A). A Rous sarcoma virus (RSV) enhancer and promoter driving a ß-galactosidase reporter gene (RSV-ß-gal) was cotransfected as an internal control. Additionally, an RSV-luciferase (RSV-Luc) plasmid was transfected in parallel with FSHßLuc into each cell type to control for differences in transfection efficiency, transcription rates, reporter mRNA half-life, luciferase and ß-galactosidase protein stability, and metabolic rates between distinct cell types. The ratio of RSV-Luc to RSV-ß-gal was set to 100 for each cell line, and the FSHßLuc value was normalized to the RSV-Luc/RSV-ß-gal value. To validate this approach, we tested the activities of the promoterless pUC18-luciferase and a thymidine kinase-Luc plasmid and found each to be expressed at equivalent levels in T3-1 and LßT2 cells relative to the RSV controls (data not shown).
|
). FSHß promoter activity is approximately 4.5-fold higher in LßT2 cells than in the gonadotrope precursor T3-1 cells and up to 80-fold higher than in some non-gonadotrope-derived cell lines such as AtT20 (corticotrope derived), JEG-3 (placental), and HeLa (cervical fibroblast).
Our previous study examining transcriptional regulation of the ovine FSHß 5'-regulatory region demonstrated that activin specifically regulates FSHß gene expression in LßT2 cells but not in T3-1 or NIH3T3 cells (10). This LßT2 cell-specific response to activin includes activation of the ovine FSHß promoter after the addition of exogenous activin as well as inhibition of ovine FSHß promoter activity by neutralizing endogenous activin with follistatin, a potent activin-binding protein that blocks binding of activin to its receptors (17, 18, 19). Similarly, we now show that the mouse FSHß promoter is responsive to both activin and follistatin in LßT2 cells (Fig. 2B). Activity of the 398-bp mouse FSHß promoter in LßT2 cells is reduced approximately 20–25% in follistatin-treated cells when compared with untreated control cells. Treatment with activin stimulates -398FSHßLuc expression approximately 2.5-fold, an effect that is completely blocked by addition of follistatin. We used this ability of follistatin to disrupt autocrine activin activity to determine whether endogenous activin is contributing to LßT2 cell-specific expression of mouse FSHß. LßT2 and T3-1 cells were transiently transfected with mouse -398FSHßLuc and then treated with follistatin (Fig. 2C). The promoterless luciferase vector, pGL3, was used as a negative control. As with our previous work, follistatin treatment inhibits expression of FSHßLuc in LßT2 cells but not T3-1 cells. Although this demonstrates that an endogenous activin autocrine loop contributes some degree to LßT2 cell-specific expression of FSHß, it also reveals that the specificity to LßT2 cells is not solely due to the action of the activin autocrine loop and must reside in additional transcriptional regulators.
To delineate the regions of the mouse FSHß promoter involved in basal expression and LßT2 cell specificity, 5'-truncations of mouse FSHßLuc were transiently transfected into LßT2, T3-1, and NIH3T3 cells (Fig. 3A). Analyses of the transcriptional specificity of these truncations indicate that several regions of the FSHß promoter contribute to LßT2 cell specificity; however, the largest decreases in specificity between the cell types are seen upon truncation of the proximal promoter regions from -127 bp (3.7-fold) to -95 bp (2.4-fold) and from -95 bp to -64 bp (1.7-fold). The only statistically significant decrease in basal expression within LßT2 cells alone is observed upon truncation of the sequences from -95 bp to -64 bp.
|
). In contrast to basal expression, the follistatin responsiveness of the mouse FSHß promoter localizes to more distal sequences of the promoter, between -230 bp and -194 bp. This demonstrates that the contribution of endogenous activin to FSHß promoter activity in LßT2 cells is separate from the downstream factors contributing to basal and cell-specific activity.
The Mouse FSHß Promoter Contains SF-1 and NF-Y Binding Elements
To identify specific elements that may contribute to basal expression of FSHß in LßT2 cells, we analyzed the sequence of the mouse FSHß regulatory region. Because of the known importance of SF-1 for gonadotrope function (20, 21), we began by perusing the sequence for SF-1 binding sites, also known as gonadotrope-specific elements, or GSEs. Two putative GSEs were identified at approximately -341 bp (6/9 match) and -239 bp (8/9 match) relative to the transcriptional start site based on sequence similarity to the GSEs from the LHß and -GSU promoters (Fig. 4A). The -239GSE is conserved in FSHß regulatory regions from rat, human, bovine, and ovine; the -341GSE is conserved in rat. Due to the significant decrease in promoter activity observed upon deletion of proximal promoter regions, the sequence from -127 bp to -64 bp was searched for putative transcription factor binding sites using the TRANSFAC database (22). This identified a consensus-binding element, at approximately -76 bp, for the heterotrimeric nuclear factor-Y (NF-Y) transcription factor (Fig. 4B). Analysis of FSHß promoter sequences from other species indicates that this NF-Y element is conserved in mouse, rat, and human.
|
T3-1 cells, but not NIH3T3 cells, are bound to both the -341SF-1 probe (Fig. 5A) and the -239SF-1 probe (Fig. 5B). This complex is competed by the consensus GSE from the -GSU promoter (lanes 4 and 10, GSEcon), and binding is blocked by addition of a SF-1-specific antibody (lanes 5 and 11). The stronger binding of the SF-1-containing complex to the -239SF-1 probe is consistent with this site being a more highly conserved GSE.
|
T3-1, and NIH3T3 cells by immunocytochemistry and found it to be highly expressed and localized to the nucleus in each cell line (Fig. 6A). Binding of NF-Y to its putative element was examined by EMSA using a probe spanning the mouse FSHß promoter region from -99 bp to -65 bp (FSH 99/65). An antibody directed against the NF-YA subunit supershifts the single major band present in all three cell types (Fig. 6B). Experiments using oligonucleotides containing 2-bp mutations spanning the CCAAT box and surrounding sequences, shown in Fig. 6D, were used in competitions (Fig. 6C) and as probes (not shown) and confirm that NF-Y is interacting with the specific element that was identified (lanes 7–9).
|
. Interestingly, mutation of the SF-1 and NF-Y sites has a cell-dependent effect on activity of the FSHß promoter (Fig. 7A). SF-1 is expressed in LßT2 and T3-1 cells and, although they trend lower, neither the single SF-1 site mutants nor the double SF-1 mutant has a statistically significant effect on promoter activity in either cell line. Mutation of the NF-Y element results in an approximate 30% reduction in activity in LßT2 cells when compared with wild type and a 38% reduction in NIH3T3 cells. Surprisingly, this NF-Y mutation has no effect on promoter activity in T3-1 cells. When the SF-1 and NF-Y mutations are combined, a greater than additive effect is observed only in LßT2 cells (50% reduction); expression is not affected by combining the mutations in either T3-1 or NIH3T3 cells. This results in a corresponding decrease in the specificity between LßT2 and T3-1 cells to 1.7-fold. The cooperative effect of the NF-Y and SF-1 mutations indicates that the proteins acting through these elements are functionally interacting in an LßT2 cell-specific manner.
|
). If the activin system functions through the NF-Y and SF-1 binding sites, then mutation of these elements should eliminate or reduce the contribution of endogenous activin to FSHß promoter activity, and the follistatin effect would be lost. In fact, the NF-Y mutant, SF-1 double mutant, and NF-Y/double SF-1 triple mutant promoters are all inhibited by follistatin equally as well as the wild-type promoter, indicating that these elements are not involved in activin regulation of the FSHß gene. This is consistent with our localization of the follistatin-responsive sequences to an upstream region of the mouse FSHß promoter, which does not encompass the NF-Y and SF-1 binding sites.
The functional interaction between the NF-Y and SF-1 binding sites on basal activity suggests that the proteins may be physically interacting with one another. To test this possibility, glutathione-S-transferase (GST) pull-down assays were performed. As seen in Fig. 8A, GST-SF-1 is capable of interacting with 35S-labeled NF-YA but not with the NF-YB or NF-YC subunits. Pituitary homeobox 1 (Ptx1) has previously been shown to interact with SF-1 and serves here as a positive control, whereas green fluorescent protein (GFP) does not interact with SF-1 and serves here as a negative control. No interactions were observed using GST alone. The converse experiment was also performed using GST-NF-YA and GST-NF-YB proteins with 35S-labeled SF-1 (Fig. 8B). Once again, SF-1 interacts with NF-YA but not with NF-YB. These experiments confirm the ability of NF-Y and SF-1 to physically interact in addition to the functional interaction observed in LßT2 cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
T3-1 cells at the protein level provides further support for classifying these cells as relatively mature and immature gonadotrope cells, respectively. Prior work examining FSHß gene expression in LßT2 cells demonstrated the presence of FSHß mRNA under basal, nonstimulated conditions and showed that it is appropriately regulated by activin and follistatin (10), physiological regulators of FSH synthesis in vivo. A separate study detected low levels of FSH secretion from untreated LßT2 cells and showed that FSH secretion is dramatically increased in response to activin (11). Our current work confirms the findings suggested by the secretion studies, i.e. that LßT2 cells make FSHß protein under nonstimulatory conditions. Although the level of FSHß expression in LßT2 cells may be low, intracellular FSHß protein is both present and detectable. Furthermore, both mouse and ovine transfected FSHß-Luciferase reporter genes are expressed very well in these cells, at an activity level comparable to that of the 1800-bp rat LHß promoter (data not shown). The expression and appropriate hormonal regulation of FSHß in LßT2 cells (10, 11, 14) should help resolve the impasse in our progress in understanding of FSHß gene regulation at the molecular level.
We have previously shown that an endogenous activin autocrine loop is present in LßT2 cells and contributes to cell-specific activity of the ovine FSHß regulatory region (10). In this study, we examine the contribution of activin to cell-specific activity of the mouse FSHß promoter, by neutralizing endogenous activin with follistatin. Whereas activity of the 5.5-kb ovine FSHß regulatory region was reduced approximately 50% by follistatin treatment, activity of the 398-bp mouse FSHß promoter is reduced approximately 25%. The experiments on the ovine FSHß gene were performed in 1% serum (10), while the current experiments on the mouse FSHß gene were performed in 10% serum to allow for direct comparison with the transfections analyzing basal and cell-specific expression. However, we find no significant differences due to serum conditions, having performed the activin and follistatin treatments of the mouse FSHß gene under both conditions in parallel (data not shown). Another difference is the source of follistatin: whereas the previous experiments used follistatin kindly provided by Dr. Shunichi Shimasaki, the current experiments utilize commercially available follistatin. Despite this change, we now show that the current follistatin treatment is sufficient to block induction by exogenous activin treatment as well as the endogenous activin loop. Finally, this difference in magnitude of the follistatin effect could be due to species-specific differences in regulation of the ovine and mouse FSHß genes. Whatever the cause for the difference in magnitude, the contribution of endogenous activin to both ovine and mouse FSHß promoter activity is LßT2 cell specific; neither promoter responds to follistatin in T3-1 or NIH3T3 cells. Taking into account the contribution of activin to mouse FSHß promoter activity in LßT2 cells, an approximate 3- to 3.5-fold basal specificity between LßT2 and T3-1 cells remains. In this report, we identify specific regulatory elements involved in basal expression of the FSHß gene (binding sites for SF-1 and NF-Y) and show that follistatin regulation is acting independently of these elements.
SF-1 is an orphan nuclear receptor that, within the pituitary, is specifically expressed in the gonadotrope cell population (20). SF-1 binding sites have previously been identified in the LHß, -GSU, and GnRH receptor promoters (3, 4, 24); therefore, it is not surprising to find them within the FSHß regulatory region. The importance of SF-1 to gonadotrope function in vivo has been demonstrated by the generation of SF-1-null mice (20). These animals are infertile and have markedly reduced levels of -GSU, LHß, FSHß, and GnRH receptor. Pituitary-specific SF-1 knockout animals indicate that the impaired gonadotropin expression is a primary defect due to the lack of SF-1 in the pituitary and not a secondary defect due to SF-1 deficiency in other tissues (21). Although SF-1 is an important basal regulator of gonadotrope function, it is not sufficient to confer gonadotrope-specific expression since it is made in steroidogenic tissues outside of the pituitary (25), as well as in T3-1 cells, which do not make the FSH or LH ß-subunits. Moreover, its expression commences significantly earlier in pituitary development than the emergence of FSHß gene expression (25). In the context of the LHß promoter, SF-1 regulates gene expression, in part, by interacting with proteins acting through early growth response 1 and homeodomain-binding elements (26). The role of SF-1 in FSHß gene regulation is less clear but appears to be dependent on other regulatory factors. Although not statistically significant, deletion of the -341 SF-1 site or mutation of both SF-1 sites reduces basal expression of FSHßLuc in LßT2 cells approximately 20% compared with the wild-type promoter. However, when combined with a mutation in the NF-Y element, the SF-1 binding site mutations do indeed have a statistically significant effect on FSHß promoter activity, reducing expression an additional 30% from the level of the NF-Y mutant to 50% that of the wild-type promoter. This indicates that the SF-1 elements contribute to FSHß gene expression, in part, through a functional interaction with NF-Y. The necessity for mutating the NF-Y element to observe a role for the SF-1 sites may be due to the ability of these factors to physically interact. For example, if SF-1 is capable of conferring its activity on the FSHß promoter through either binding its own sites or through interacting with NF-Y on the NF-Y site in LßT2 cells, then the result that mutation or deletion of the SF-1 sites alone is insufficient to decrease expression to a significant level would be observed. In addition to NF-Y, SF-1 has been shown to physically interact with many transcription factors including transcription factor IIB, GCN5, c-jun, Ptx1, early growth response 1, SP1, androgen receptor, and GATA-4 (26, 27, 28, 29, 30, 31). It is possible that SF-1 activates transcription of FSHß through interactions with these and other proteins and that these interactions mask the direct role of the SF-1 sites themselves on FSHß promoter activity unless the NF-Y site is mutated.
Consistent with most CCAAT boxes in TATA-containing promoters, the NF-Y site in the mouse FSHß gene is located within the proximal 100 bp of the promoter (32). NF-Y is an important basal regulator of many genes and has been shown to physically interact with several key components of transcriptional machinery, including TATA binding protein (TBP), p300/CREB binding protein (CBP), GCN5, and p300/CBP-associated factor, as well as many other transcription factors, such as SP1, hepatocyte nuclear factor 4, estrogen receptor-, and sterol regulatory element binding proteins (23, 33, 34, 35, 36, 37, 38). These protein-protein interactions can occur through different mechanisms. In many instances, they involve just one of the NF-Y subunits, but in some cases, the interaction requires the NF-Y(BC) dimer or the complete NF-Y trimer. In the case of SF-1, the interaction occurs through the NF-YA subunit. Recently, NF-Y binding sites have been demonstrated to be essential for activity of the Hoxb4 regulatory region (39). Although, an NF-Y site alone is not sufficient to confer tissue-specific expression on a heterologous promoter, inclusion of flanking sequences does lead to a spatially restricted pattern of expression (39). This indicates that NF-Y cooperates with adjacent factors to regulate expression of Hoxb4. The ability of NF-Y to recruit cofactors may be critical to its role as a basal regulator of transcription. It may also represent a mechanism through which NF-Y is involved in cell-specific expression despite its relatively ubiquitous pattern of expression.
Despite the presence of both SF-1 and NF-Y in LßT2 and T3-1 cells and our demonstration that proteins from both cell lines exhibit DNA-binding activity in vitro, neither SF-1 nor NF-Y contributes to activity of the FSHß promoter in T3-1 cells. This specific lack of activity in T3-1 cells can be explained by several possible mechanisms. The activity generated by the interaction of SF-1 with NF-Y may require LßT2 cell-specific posttranslational modification of one or both factors or an LßT2 cell-specific coactivator. Conversely, T3-1 cells may contain a factor that blocks the interaction between NF-Y and SF-1, thus preventing their cooperative activity. The specificity of the SF-1/NF-Y functional interaction to LßT2 cells suggests that additional proteins are interacting with SF-1 and NF-Y and that the components of this complex vary in each cell line. It is likely that additional regulatory elements and transcription factors are also important for FSHß gene expression, which may or may not interact with SF-1 or NF-Y. It is also possible that FSHß gene expression in T3-1 cells may be repressed by a factor binding inside the -64-bp region, preventing activation of any of the promoters used. Furthermore, it is likely that the combination of factors contributing to LßT2 cell specificity when compared with immature gonadotropes (T3-1 cells) differs from the combination of factors contributing to LßT2 cell specificity when compared with a non gonadotrope cell (NIH3T3 cells). Some of these factors may be involved in activin regulation of FSHß, and some may be independent of the activin system, as are SF-1 and NF-Y. One possible regulator of FSHß is the homeodomain-containing transcription factor Ptx1. Ptx1 is important for pituitary development, can physically interact and synergize with SF-1, and has been implicated in LHß gene regulation (26, 40, 41). Recently, a Ptx1 binding site was identified within the proximal region of the rat FSHß promoter and shown to be important for basal expression (42). This site is conserved in the mouse FSHß promoter, located at approximately -54 bp, and may be involved in some of the remaining basal activity present in our -64-bp truncation. However, as with SF-1 and NF-Y, Ptx1 is expressed in both LßT2 and T3-1 cells (7) and is unlikely to be solely responsible for the differential expression of FSHß between the cell lines.
Tissue-specific gene expression is often conferred by complex control regions in which several cell-restricted, but not necessarily cell-specific, factors interact (43, 44). The spatial and temporal pattern in which regulatory factors are expressed is crucial when cell-fate decisions are occurring since it is the repertoire of factors present that ultimately determines cell types. It is likely that a combination of transcriptional activators conferring FSHß gene expression in LßT2 cells and repressors preventing FSHß gene expression in T3-1 cells produces the differential gene expression in these gonadotrope-derived cell lines as well as during gonadotrope maturation. Here we demonstrate that cell specificity can involve the interaction between tissue-restricted factors, such as SF-1, and ubiquitous factors, such as NF-Y. Although NF-Y and SF-1 are not sufficient to confer FSHß gene expression or to fully distinguish between the gonadotrope-derived cell lines, they are clearly required to provide full, basal expression in LßT2 cells. Moreover, the LßT2 cell-specific interaction between these sites contributes to the specific expression of FSHß in these mature gonadotrope cells.
It is of particular interest that functional NF-Y, SF-1, and Ptx1 binding sites have been identified in the FSHß promoter, since they have also been described in the LHß promoter (4, 6, 45, 46). In transgenic mice, the elements through which Ptx1, SF-1, and NF-Y act are essential for basal activity of the LHß promoter (5, 6, 45). This parallel mechanism of gene regulation of LHß and FSHß may be involved in coordinating gonadotrope-specific expression of these genes. SF-1 is clearly involved in gonadotrope determination as it is essential for expression of many gonadotrope markers. Similarly, Ptx1 is important for pituitary development and expression of the gonadotropin genes in vivo (40) and can transactivate numerous pituitary-specific promoters, including FSHß, LHß, -GSU, and GnRH-receptor (41, 47). Additionally, NF-Y can regulate expression of genes involved in similar functions, such as major histocompatibility complex II genes involved in peptide presentation (48) and several genes involved in cholesterol metabolism (23, 38). In the gonadotrope, NF-Y might function to coordinate expression of gonadotrope-specific genes by interacting with SF-1. It will be interesting to determine whether NF-Y binding sites are also present in the -GSU- and GnRH receptor-regulatory regions, whether they are important for cell-specific expression of those genes, and if they interact with the well characterized SF-1 elements and/or the Ptx1 sites. The onset of LHß expression precedes the onset of FSHß expression by 1 d during embryonic development (e16.5 vs. e17.5) (49), indicating that differences in gonadotropin ß-subunit gene activation must also exist. The work presented here is a key step in understanding factors involved in the developmental activation of FSHß gene expression in the maturing gonadotrope cell. Further characterization of the interactions occurring in the regulatory regions of the various gonadotropin genes will likely lead to a greater understanding of the molecular mechanisms, both common and unique, involved in their coordinated and differential expression.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
T3-1), or 20,000 cells per well (NIH3T3). Cell culture conditions have been described previously (7). Immunostaining was performed as follows: Cells were washed twice with 1x PBS containing 1 mM MgCl2 and then fixed for 20 min with 4% formaldehyde and 1 mM MgCl2 in 1x PBS. Cells were blocked and permeabilized with 20% donkey serum in wash buffer (1% BSA, 0.5% NP-40 in 1x PBS) for 1 h. The primary antibodies were diluted in wash buffer and bound for 1 h. The following dilutions were used: FSHß (1:2000), LHß (1:2000), -GSU (1:2000), SF-1 (1:1000), or NF-YA (1:2000). The FSHß, LHß, and -GSU antibodies were purchased from the National Hormone and Peptide Program; the SF-1 antibody was provided by Dr. Bon-Chu Chung, and the NF-YA antibody is from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The secondary antibodies (Texas Red-conjugated donkey -rabbit IgG or fluorescein isothiocyanate-conjugated donkey -mouse IgG from Jackson ImmunoResearch Laboratories, West Grove, PA) were diluted 1:200 in wash buffer and bound for 30 min. After three washes each with wash buffer and 1x PBS, nuclei were stained with 4,6-diamidino-2-phenylindole (1 µg/ml; Roche Biochemical, Indianapolis, IN) for 5 min. Coverslips were mounted in a solution containing 10% gelvatol (polyvinyl alcohol), 33% glycerol, and 0.1 M Tris-Cl, pH 8.5. Images (0.2-µm sections) were captured using a DeltaVision deconvolution microscope system (Applied Precision, Inc., Issaquah, WA) and a Nikon x100 (NA 1.4) lens. The data sets were deconvolved and analyzed using SoftWorx software (Applied Precision, Inc.). Deconvolution microscopy was performed with assistance from Steve McMullen at the UCSD Cancer Center Digital Imaging Shared Resource.
Plasmid Construction and Preparation
The 398-bp mouse FSHß promoter was PCR amplified from a genomic clone (kindly provided by Dr. Malcolm Low) and ligated into the SmaI restriction site of the pGL3 luciferase reporter plasmid (Promega Corp., Madison, WI) to generate -398FSHßLuc. The -304FSHßLuc and -230FSHßLuc truncations were created by digesting the MluI/BglII fragment of -398FSHßLuc with XmnI and BbvI restriction enzymes, respectively. The truncated promoter fragments were then blunt ended with Klenow fragment (New England Biolabs, Beverly, MA) and ligated into the SmaI restriction site of pGL3. The -129FSHßLuc and -64FSHßLuc reporter genes were created by digesting the MluI/BglII fragment of -304FSHßLuc with DpnI and HpaI restriction enzymes, respectively. The fragments were blunt ended with Klenow and ligated into the SmaI restriction site of pGL3. The -194FSHßLuc plasmid was generated by digesting the MluI/BglII fragment of -230FSHßLuc with the RsaI restriction enzyme and cloning the appropriate promoter fragment into the SmaI and BglII sites of pGL3. The -95FSHßLuc plasmid was created by PCR amplifying the promoter from -398FSHßLuc using a forward primer corresponding to the sequence of the mouse FSHß promoter from -95 bp to -77 bp and containing a KpnI linker and a reverse primer spanning the HindIII restriction site from the pGL3 vector. The PCR product was digested with KpnI and BglII restriction enzymes and ligated into the corresponding sites in pGL3. The sequences of all promoter fragments were confirmed by dideoxynucleotide sequencing (50).
Plasmid DNA was prepared from overnight bacterial cultures using a cesium chloride protocol adapted from Sambrook et al. (51).
Transient Transfections, Normalizing Transfection Data, and Statistics
Transient transfections were performed as described previously (7) using 2.5 µg of the reporter plasmids. For the activin and follistatin experiments, cells were treated with vehicle (1x PBS), 100 ng/ml recombinant mouse follistatin 288 (R & D Systems, Minneapolis, MN), 10 ng/ml human recombinant activin A (Calbiochem, San Diego, CA), or a combination of both follistatin and activin 24 h after transfection in DMEM with 10% fetal bovine serum. Twenty-four hours after treatment, cells were harvested and assayed for luciferase and ß-galactosidase activity. All transfection experiments were performed at least three times in triplicate. To control for differences in expression between the different cell types, each experiment was normalized. The RSV enhancer fused to the RSV promoter driving luciferase (RSV-Luc) was transfected in parallel with FSHßLuc in each experiment. The RSV enhancer and promoter fused to ß-galactosidase (RSV-ß-gal, 0.5 µg) was used as an internal control for each transfected plate of cells. The RSV-Luc luciferase values were divided by the RSV-ß-gal ß-galactosidase values, averaged, and set equal to 100. The FSHßLuc/RSV-ß-gal values were normalized to this value in each cell type; thus, the values from the individual cell types can be directly compared. The error bars in all bar graphs represent SEM. Normal or Box Cox Transformed ratios for each promoter construct in each cell type were compared by the ANOVA Factorial test, followed by the Tukey-Kramer HSD post hoc test. In all analyses, P 
0.05 was considered significant.
Nuclear Extracts and EMSAs
Preparation of nuclear extracts and binding conditions for the EMSAs have been described previously (7). Binding reactions were incubated for 5 min at room temperature after addition of probe, loaded onto a 5% nondenaturing polyacrylamide gel, and electrophoresed. For competition and antibody experiments, 100-fold excess unlabeled competitor oligonucleotide or 1 µl antibody was added 20 min before addition of probe. The SF-1 antibody used in EMSA was purchased from Upstate Biotechnology (Lake Placid, NY), and the NF-YA antibody was purchased from Chemicon International (Temecula, CA).
Mutagenesis
Mutagenesis of FSHßLuc was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Oligonucleotides used for mutagenesis were the following: 341SF-1mut [5'-GGATCAATTAAGACATATTTTGGTTTAAATTCGCAATGGAGCCAAAGCAATGTTCAG-3' (top strand)], 239SF-1mut [5'-GTCTAGACTCTAGAGTCACATTTAATTTAGAATTTGAGGGAGTGGGTGTGCTGCC-3' (top strand)], and NF-Ymut [5'-CAGCAGGCTTTATGTTGGTACCGGTCATGTTAACACCC-3' (top strand)]. Mutations (underlined) were confirmed by dideoxynucleotide sequencing performed by the DNA Sequencing Shared Resource, UCSD Cancer Center.
GST-Interaction Assay
The GST-SF1 fusion protein was created by cloning the SF-1 cDNA into the pGEX-4T1 vector. Sequencing confirmed that the SF-1 reading frame was cloned in-frame with the GST protein. The GST-NF-YA and GST-NF-YB vectors were provided by Dr. Sankar Maity and Dr. David Gardner, respectively (37, 52). The SF-1 expression vector was made by cloning the SF-1 cDNA into pcDNA3. The NF-Y and GFP expression vectors were provided by Dr. Roberto Mantovani and Dr. Douglass Forbes, respectively. The Ptx1 vector has been described previously (7). 35S-labeled proteins were produced using the TnT T7 Coupled Reticulocyte Lysate System (Promega Corp.). Bacteria transformed with the pGEX vectors were grown to an OD of 0.5 and then induced with 0.2 mM isopropyl-ß-D-thiogalactoside overnight. Bacterial pellets were sonicated in 0.1% Triton X-100, 5 mM EDTA in 1x PBS and centrifuged, and the supernatant was bound to glutathione sepharose 4B resin (Amersham Pharmacia, Piscataway, NJ). The interaction assay was performed as described previously (53).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Current address for S.B.R.J.: Department of Radiation Oncology, CCSR-South 1255, 269 Campus Drive, Stanford, California 94305-5152.
Abbreviations: GFP, Green fluorescent protein; GSE, gonadotrope-specific element; GST, glutathione-S-transferase; -GSU, -glycoprotein subunit; NF-Y, nuclear factor 1; Ptx1, pituitary homeobox 1; RSV, Rous sarcoma virus; RSV-ß-gal, RSV-ß-galactosidase; RSV-Luc, RSV-luciferase; SF-1, steroidogenic factor 1.
Received for publication August 19, 2002. Accepted for publication April 22, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-subunit gene. Mol Endocrinol 6:893–903-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885-mediated trans-activation of human coagulation factor XII gene by heteromeric transcription factor NF-Y. Endocrinology 142:3380–3388NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  N. A. Ciccone, C. T. Lacza, M. Y. Hou, S. J. Gregory, K.-Y. Kam, S. Xu, and U. B. Kaiser A Composite Element that Binds Basic Helix Loop Helix and Basic Leucine Zipper Transcription Factors Is Important for Gonadotropin-Releasing Hormone Regulation of the Follicle-Stimulating Hormone {beta} Gene Mol. Endocrinol., August 1, 2008; 22(8): 1908 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhu, A. S. Gleiberman, and M. G. Rosenfeld Molecular Physiology of Pituitary Development: Signaling and Transcriptional Networks Physiol Rev, July 1, 2007; 87(3): 933 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zheng, J. Yang, Q. Jiang, Z. He, and L. M Halvorson Liver receptor homologue-1 regulates gonadotrope function J. Mol. Endocrinol., February 1, 2007; 38(2): 207 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B Lee, V. Khivansara, M. M Santos, P. Lamba, T. Yuen, S. C Sealfon, and D. J Bernard Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone {beta} subunit transcription J. Mol. Endocrinol., February 1, 2007; 38(2): 315 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. G. Thackray, S. M. McGillivray, and P. L. Mellon Androgens, Progestins, and Glucocorticoids Induce Follicle-Stimulating Hormone {beta}-Subunit Gene Expression at the Level of the Gonadotrope Mol. Endocrinol., September 1, 2006; 20(9): 2062 - 2079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, Y. L. Zhou, W. Luo, Q.-S. Zhu, D. Levy, O. A. MacDougald, and M. L. Snead NF-Y and CCAAT/Enhancer-binding Protein {alpha} Synergistically Activate the Mouse Amelogenin Gene J. Biol. Chem., June 9, 2006; 281(23): 16090 - 16098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lamba, M. M Santos, D. P Philips, and D. J Bernard Acute regulation of murine follicle-stimulating hormone {beta} subunit transcription by activin A J. Mol. Endocrinol., February 1, 2006; 36(1): 201 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Rave-Harel, N. L. G. Miller, M. L. Givens, and P. L. Mellon The Groucho-related Gene Family Regulates the Gonadotropin-releasing Hormone Gene through Interaction with the Homeodomain Proteins MSX1 and OCT1 J. Biol. Chem., September 2, 2005; 280(35): 30975 - 30983. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-Y. Kam, K.-H. Jeong, E. R. Norwitz, E. M. Jorgensen, and U. B. Kaiser Oct-1 and Nuclear Factor Y Bind to the SURG-1 Element to Direct Basal and Gonadotropin-Releasing Hormone (GnRH)-Stimulated Mouse GnRH Receptor Gene Transcription Mol. Endocrinol., January 1, 2005; 19(1): 148 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. E. West, G. E. Parker, J. J. Savage, P. Kiratipranon, K. S. Toomey, L. R. Beach, S. C. Colvin, K. W. Sloop, and S. J. Rhodes Regulation of the Follicle-Stimulating Hormone {beta} Gene by the LHX3 LIM-Homeodomain Transcription Factor Endocrinology, November 1, 2004; 145(11): 4866 - 4879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q.-S. Zhu, B. Qian, and D. Levy CCAAT/Enhancer-binding Protein {alpha} (C/EBP{alpha}) Activates Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1) through the Interaction with DNA-bound NF-Y J. Biol. Chem., July 16, 2004; 279(29): 29902 - 29910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Spady, R. Shayya, V. G. Thackray, L. Ehrensberger, J. S. Bailey, and P. L. Mellon Androgen Regulates Follicle-Stimulating Hormone {beta} Gene Expression in an Activin-Dependent Manner in Immortalized Gonadotropes Mol. Endocrinol., April 1, 2004; 18(4): 925 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Coss, S. B. R. Jacobs, C. E. Bender, and P. L. Mellon A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone {beta} Gene by Gonadotropin-releasing Hormone J. Biol. Chem., January 2, 2004; 279(1): 152 - 162. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
9). The bar marked with an asterisk (*) (LßT2 cells) is statistically significant from all other bars (P < 0.05). Statistically significant differences between other cell lines are not shown. B, The 398-bp mouse FSHß promoter responds to follistatin and activin. LßT2 cells were transiently transfected with -398FSHßLuc, along with the RSV-ß-gal internal control, and then treated with vehicle (control), 100 ng/ml follistatin (FS), 10 ng/ml activin (Act), or a combination of both follistatin and activin (FS+Act) for 24 h in DMEM with 10% fetal bovine serum. The value of the vehicle-treated sample was set at 1, and the follistatin- and activin-treated samples are shown relative to this control. Results represent the mean ± SEM of three independent experiments, each performed in triplicate (n = 9). The bars marked with 
(FS and FS+Act) and x (Act) are statistically different from the untreated control and are also different from each other (P < 0.05). C, The mouse FSHß promoter responds to follistatin in LßT2 cells, but not 
